U.S. patent application number 15/476849 was filed with the patent office on 2017-10-05 for nucleic acid-polypeptide compositions and uses thereof.
The applicant listed for this patent is AVIDITY BIOSCIENCES LLC. Invention is credited to Palani BALU, Rob BURKE, David Sai-Ho CHU, Michael Caramian COCHRAN, Beatrice Diana DARIMONT, Venkata Ramana DOPPALAPUDI, Andrew John GEALL, Rachel Elizabeth JOHNS.
Application Number | 20170281795 15/476849 |
Document ID | / |
Family ID | 59958453 |
Filed Date | 2017-10-05 |
United States Patent
Application |
20170281795 |
Kind Code |
A1 |
GEALL; Andrew John ; et
al. |
October 5, 2017 |
NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF
Abstract
Disclosed herein are compositions and pharmaceutical
formulations that comprise a binding moiety conjugated to a
polynucleic acid molecule and a polymer. Also described herein
include methods for treating a cancer which utilize a composition
or a pharmaceutical formulation comprising a binding moiety
conjugated to a polynucleic acid molecule and a polymer.
Inventors: |
GEALL; Andrew John;
(Carlsbad, CA) ; DOPPALAPUDI; Venkata Ramana; (San
Diego, CA) ; CHU; David Sai-Ho; (La Jolla, CA)
; COCHRAN; Michael Caramian; (La Jolla, CA) ;
JOHNS; Rachel Elizabeth; (San Diego, CA) ; BALU;
Palani; (Cupertino, CA) ; BURKE; Rob;
(Encinitas, CA) ; DARIMONT; Beatrice Diana; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AVIDITY BIOSCIENCES LLC |
La Jolla |
CA |
US |
|
|
Family ID: |
59958453 |
Appl. No.: |
15/476849 |
Filed: |
March 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62316919 |
Apr 1, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/30 20130101;
C12N 15/88 20130101; A61K 47/6807 20170801; C07K 2317/569 20130101;
A61P 35/00 20180101; C12N 2310/315 20130101; C07K 2317/622
20130101; C12N 2310/321 20130101; A61K 47/60 20170801; C07K 2317/24
20130101; C07K 2317/55 20130101; C07K 2317/21 20130101; A61K 31/713
20130101; C07K 16/2863 20130101; C12N 15/113 20130101; C12N 2310/14
20130101; A61K 31/7088 20130101; C12N 2320/32 20130101; C07K
16/2851 20130101; C12N 2310/3231 20130101; C12N 15/1138 20130101;
A61K 47/6851 20170801; C12N 15/1135 20130101; C12N 2310/332
20130101; C12N 2310/3515 20130101; C07K 16/3061 20130101; A61P
35/02 20180101; A61K 48/005 20130101; A61K 48/0075 20130101; A61K
2039/505 20130101; A61K 47/6889 20170801; A61K 48/0033 20130101;
C12N 2310/3513 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 31/713 20060101 A61K031/713; C07K 19/00 20060101
C07K019/00; A61K 47/48 20060101 A61K047/48; C12N 15/113 20060101
C12N015/113; C07K 16/28 20060101 C07K016/28 |
Claims
1. A molecule of Formula (I): A-X-B-Y-C Formula I wherein, A is an
antibody or its binding fragments thereof; B is a polynucleotide; C
is a polymer; X is a bond or first non-polymeric linker; and Y is a
bond or second linker; wherein the polynucleotide comprises at
least one 2' modified nucleotide, at least one modified
internucleotide linkage, or at least one inverted abasic moiety;
and wherein A and C are not attached to B at the same terminus.
2. The molecule of claim 1, wherein the at least one 2' modified
nucleotide comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide.
3. The molecule of claim 1, wherein the at least one 2' modified
nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic
acid (ENA).
4. The molecule of claim 1, wherein the at least one modified
internucleotide linkage comprises a phosphorothioate linkage or a
phosphorodithioate linkage.
5. The molecule of claim 1, wherein the at least one inverted
abasic moiety is at at least one terminus.
6. The molecule of claim 1, wherein the polynucleotide comprises a
single strand.
7. The molecule of claim 1, wherein the polynucleotide comprises a
first polynucleotide and a second polynucleotide hybridized to the
first polynucleotide to form a double-stranded polynucleic acid
molecule.
8. The molecule of claim 7, wherein the second polynucleotide
comprises at least one modification.
9. The molecule of claim 7, wherein the first polynucleotide and
the second polynucleotide are RNA molecules.
10. The molecule of claim 7, wherein the first polynucleotide
comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955,
1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.
11. The molecule of claim 7, wherein the second polynucleotide
comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955,
1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.
12. The molecule of claim 1, wherein X and Y are independently a
bond.
13. The molecule of claim 1, wherein X and Y are independently a
C.sub.1-C.sub.6 alkyl group.
14. The molecule of claim 1, wherein X is a homobifuctional linker
or a heterobifunctional linker, optionally conjugated to a
C.sub.1-C.sub.6 alkyl group.
15. The molecule of claim 1, wherein Y is a homobifuctional linker
or a heterobifunctional linker.
16. The molecule of claim 1, wherein the antibody or binding
fragment thereof comprises a humanized antibody or binding fragment
thereof, chimeric antibody or binding fragment thereof, monoclonal
antibody or binding fragment thereof, monovalent Fab', divalent
Fab2, single-chain variable fragment (scFv), diabody, minibody,
nanobody, single-domain antibody (sdAb), or camelid antibody or
binding fragment thereof.
17. The molecule of claim 1, wherein C is polyethylene glycol.
18. The molecule of claim 17, wherein C has a molecular weight of
about 1000 Da, 2000 Da, or 5000 Da.
19. The molecule of claim 1, wherein A-X is conjugated to the 5'
end of B and Y-C is conjugated to the 3' end of B, or Y-C is
conjugated to the 5' end of B and A-X is conjugated to the 3' end
of B.
20. The molecule of claim 1, further comprising D, wherein D is an
endosomolytic moiety.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/316,919, filed Apr. 1, 2016, which application
is incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 28, 2017, is named 45532-707_201_SL.txt and is 615,666
bytes in size.
BACKGROUND OF THE DISCLOSURE
[0003] Gene suppression by RNA-induced gene silencing provides
several levels of control: transcription inactivation, small
interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced
transcriptional attenuation. In some instances, RNA interference
(RNAi) provides long lasting effect over multiple cell divisions.
As such, RNAi represents a viable method useful for drug target
validation, gene function analysis, pathway analysis, and disease
therapeutics.
SUMMARY OF THE DISCLOSURE
[0004] Disclosed herein, in certain embodiments, are compositions
and pharmaceutical formulations that comprise a binding moiety
conjugated to a polynucleic acid molecule and a polymer. In some
embodiments, also described herein include methods for treating a
disease or condition (e.g., cancer) that utilize a composition or a
pharmaceutical formulation comprising a binding moiety conjugated
to a polynucleic acid molecule and a polymer.
[0005] Disclosed herein, in certain embodiments, is a molecule of
Formula (I):
A-X-B-Y-C Formula I
[0006] wherein, [0007] A is a binding moiety; [0008] B is a
polynucleotide; [0009] C is a polymer; [0010] X is a bond or first
linker; and [0011] Y is a bond or second linker; and [0012] wherein
the polynucleotide comprises at least one 2' modified nucleotide,
at least one modified internucleotide linkage, or at least one
inverted abasic moiety.
[0013] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA) modified nucleotide. In some
embodiments, the at least one 2' modified nucleotide comprises
locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some
embodiments, the at least one modified internucleotide linkage
comprises a phosphorothioate linkage or a phosphorodithioate
linkage. In some embodiments, the at least one inverted abasic
moiety is at at least one terminus.
[0014] In some embodiments, the polynucleotide comprises a single
strand. In some embodiments, the polynucleotide comprises two or
more strands. In some embodiments, the polynucleotide comprises a
first polynucleotide and a second polynucleotide hybridized to the
first polynucleotide to form a double-stranded polynucleic acid
molecule. In some embodiments, the second polynucleotide comprises
at least one modification.
[0015] In some embodiments, the first polynucleotide and the second
polynucleotide are RNA molecules. In some embodiments, the first
polynucleotide and the second polynucleotide are siRNA
molecules.
[0016] In some embodiments, the first polynucleotide comprises a
sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75,
452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In
some embodiments, the first polynucleotide consists of a sequence
selected from SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,
2013-2032, 2082-2109, or 2117.
[0017] In some embodiments, the second polynucleotide comprises a
sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75,
452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In
some embodiments, the second polynucleotide consists of a sequence
selected from SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,
2013-2032, 2082-2109, or 2117.
[0018] In some embodiments, X and Y are independently a bond or a
non-polymeric linker group. In some embodiments, X is a bond. In
some embodiments, X is a C.sub.1-C.sub.6 alkyl group. In some
embodiments, Y is a C.sub.1-C.sub.6 alkyl group. In some
embodiments, X is a homobifuctional linker or a heterobifunctional
linker, optionally conjugated to a C.sub.1-C.sub.6 alkyl group. In
some embodiments, Y is a homobifuctional linker or a
heterobifunctional linker.
[0019] In some embodiments, the binding moiety is an antibody or
binding fragment thereof. In some embodiments, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some embodiments, the
antibody or binding fragment thereof is an anti-EGFR antibody or
binding fragment thereof.
[0020] In some embodiments, C is polyethylene glycol. In some
embodiments, C has a molecular weight of about 5000 Da.
[0021] In some embodiments, A-X is conjugated to the 5' end of B
and Y-C is conjugated to the 3' end of B. In some embodiments, Y-C
is conjugated to the 5' end of B and A-X is conjugated to the 3'
end of B. In some embodiments, A-X, Y-C or a combination thereof is
conjugated to an internucleotide linkage group.
[0022] In some embodiments, the molecule further comprises D. In
some embodiments, D is conjugated to C or to A.
[0023] In some embodiments, D is conjugated to the molecule of
Formula (I) according to Formula (II):
(A-X-B-Y-C.sub.n)-L-D Formula II
[0024] wherein, [0025] A is a binding moiety; [0026] B is a
polynucleotide; [0027] C is a polymer; [0028] X is a bond or first
linker; [0029] Y is a bond or second linker; [0030] L is a bond or
third linker; [0031] D is an endosomolytic moiety; and [0032] n is
an integer between 0 and 1; and
[0033] wherein the polynucleotide comprises at least one 2'
modified nucleotide, at least one modified internucleotide linkage,
or at least one inverted abasic moiety; and D is conjugated
anywhere on A, B, or C.
[0034] In some embodiments, D is INF7 or melittin.
[0035] In some embodiments, D is an endosomolytic polymer.
[0036] In some embodiments, L is a C.sub.1-C.sub.6 alkyl group. In
some embodiments, L is a homobifuctional linker or a
heterobifunctional linker.
[0037] In some embodiments, the molecule further comprises at least
a second binding moiety A. In some embodiments, the at least second
binding moiety A is conjugated to A, to B, or to C. In some
embodiments, the at least second binding moiety A is
cholesterol.
[0038] In some embodiments, the molecule further comprises at least
an additional polynucleotide B. In some embodiments, the at least
an additional polynucleotide B is conjugated to A, to B, or to
C.
[0039] In some embodiments, the molecule further comprises at least
an additional polymer C. In some embodiments, the at least an
additional polymer C is conjugated to A, to B, or to C.
[0040] Disclosed herein, in certain embodiments, is a molecule of
Formula (I): A-X-B-Y-C (Formula I), wherein A is an antibody or its
binding fragments thereof; B is a polynucleotide; C is a polymer; X
is a bond or first non-polymeric linker; and Y is a bond or second
linker; wherein the polynucleotide comprises at least one 2'
modified nucleotide, at least one modified internucleotide linkage,
or at least one inverted abasic moiety; and wherein A and C are not
attached to B at the same terminus. In some embodiments, the at
least one 2' modified nucleotide comprises 2'-O-methyl,
2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy,
T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
embodiments, the at least one 2' modified nucleotide comprises
locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some
embodiments, the at least one modified internucleotide linkage
comprises a phosphorothioate linkage or a phosphorodithioate
linkage. In some embodiments, the at least one inverted abasic
moiety is at at least one terminus. In some embodiments, the
polynucleotide comprises a single strand. In some embodiments, the
polynucleotide comprises a first polynucleotide and a second
polynucleotide hybridized to the first polynucleotide to form a
double-stranded polynucleic acid molecule. In some embodiments, the
second polynucleotide comprises at least one modification. In some
embodiments, the first polynucleotide and the second polynucleotide
are RNA molecules. In some embodiments, the first polynucleotide
comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955,
1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some
embodiments, the second polynucleotide comprises a sequence having
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,
2013-2032, 2082-2109, or 2117. In some embodiments, Y is a
non-polymeric linker group. In some embodiments, X is a bond. In
some embodiments, X is a C.sub.1-C.sub.6 alkyl group. In some
embodiments, Y is a C.sub.1-C.sub.6 alkyl group. In some
embodiments, X is a homobifuctional linker or a heterobifunctional
linker, optionally conjugated to a C.sub.1-C.sub.6 alkyl group. In
some embodiments, Y is a homobifuctional linker or a
heterobifunctional linker. In some embodiments, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some embodiments, C is
polyethylene glycol. In some embodiments, C has a molecular weight
of about 1000 Da, 2000 Da, or 5000 Da. In some embodiments, A-X is
conjugated to the 5' end of B and Y-C is conjugated to the 3' end
of B. In some embodiments, Y-C is conjugated to the 5' end of B and
A-X is conjugated to the 3' end of B. In some embodiments, the
molecule further comprises D. In some embodiments, D is conjugated
to C or to A. In some embodiments, D is conjugated to the molecule
of Formula (I) according to Formula (II): (A-X-B-Y-C.sub.c)-L-D
(Formula II), wherein A is an antibody or its binding fragments
thereof; B is a polynucleotide; C is a polymer; X is a bond or
first non-polymeric linker; Y is a bond or second linker; L is a
bond or third linker; D is an endosomolytic moiety; and c is an
integer between 0 and 1; wherein the polynucleotide comprises at
least one 2' modified nucleotide, at least one modified
internucleotide linkage, or at least one inverted abasic moiety;
wherein A and C are not attached to B at the same terminus; and
wherein D is conjugated anywhere on A or C or to a terminus of B.
In some embodiments, D is INF7 or melittin. In some embodiments, D
is an endosomolytic polymer. In some embodiments, L is a
C.sub.1-C.sub.6 alkyl group. In some embodiments, L is a
homobifuctional linker or a heterobifunctional linker. In some
embodiments, the molecule further comprises at least a second
binding moiety. In some embodiments, the at least second binding
moiety is conjugated to A, to B, or to C. In some embodiments, the
at least second binding moiety is cholesterol. In some embodiments,
the molecule further comprises at least an additional
polynucleotide B. In some embodiments, the at least an additional
polynucleotide B is conjugated to A, to B, or to C. In some
embodiments, the molecule further comprises at least an additional
polymer C. In some embodiments, the at least an additional polymer
C is conjugated to A, to B, or to C.
[0041] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising a molecule described above,
and a pharmaceutically acceptable excipient. In some embodiments,
the pharmaceutical composition is formulated as a nanoparticle
formulation. In some embodiments, the pharmaceutical composition is
formulated for parenteral, oral, intranasal, buccal, rectal, or
transdermal administration.
[0042] Disclosed herein, in certain embodiments, is a method of
treating a disease or disorder in a patient in need thereof,
comprising administering to the patient a composition comprising a
molecule described above. In some embodiments, the disease or
disorder is a cancer. In some embodiments, the cancer is a solid
tumor. In some embodiments, the cancer is a hematologic malignancy.
In some embodiments, the cancer comprises a KRAS-associated, an
EGFR-associated, an AR-associated cancer, a .beta.-catenin
associated cancer, a PIK3C-associated cancer, or a MYC-associated
cancer. In some embodiments, the cancer comprises bladder cancer,
breast cancer, colorectal cancer, endometrial cancer, esophageal
cancer, glioblastoma multiforme, head and neck cancer, kidney
cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate
cancer, or thyroid cancer. In some embodiments, the cancer
comprises acute myeloid leukemia, CLL, DLBCL, or multiple myeloma.
In some embodiments, the method is an immuno-oncology therapy.
[0043] Disclosed herein, in certain embodiments, is a method of
inhibiting the expression of a target gene in a primary cell of a
patient, comprising administering a molecule described above to the
primary cell. In some embodiments, the method is an in vivo method.
In some embodiments, the patient is a human.
[0044] Disclosed herein, in certain embodiments, is an
immuno-oncology therapy comprising a molecule described above for
the treatment of a disease or disorder in a patient in need
thereof.
[0045] Disclosed herein, in certain embodiments, is a kit
comprising a molecule described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Various aspects of the disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present disclosure will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosure
are utilized, and the accompanying drawings below. The patent
application file contains at least one drawing executed in color.
Copies of this patent application publication with color drawing(s)
will be provided by the Office upon request and payment of the
necessary fee.
[0047] FIG. 1A-FIG. 1C illustrate cartoon representations of
molecules described herein.
[0048] FIG. 2 illustrates a structure of cholesterol conjugate
passenger strand.
[0049] FIG. 3 shows an INF7 peptide sequence (SEQ ID NO: 2055)
described herein.
[0050] FIG. 4 shows a melittin peptide sequence (SEQ ID NO: 2060)
described herein.
[0051] FIG. 5 illustrates an analytical HPLC of EGFR antibody-PEG20
kDa-EGFR.
[0052] FIG. 6 illustrates a SDS-PAGE analysis of EGFR
antibody-PEG20 kDa-EGFR conjugate.
[0053] FIG. 7 illustrates an analytical chromatogram of EGFR
antibody-PEG10 kDa-EGFR siRNA.
[0054] FIG. 8 shows an analytical chromatogram of EGFR
antibody-PEG5 kDa-EGFR siRNA.
[0055] FIG. 9 shows a SDS PAGE analysis of EGFR antibody-PEG10
kDa-EGFR siRNA and EGFR antibody-PEG5 kDa-EGFR siRNA
conjugates.
[0056] FIG. 10 illustrates the overlay of EGFR antibody-PEG1
kDa-EGFR siRNA conjugates with siRNA loading of 1, 2 and 3.
[0057] FIG. 11 shows a HPLC chromatogram of EGFR antibody-KRAS-PEG5
kDa.
[0058] FIG. 12 shows a HPLC chromatogram of Panitumumab-KRAS-PEG5
kDa.
[0059] FIG. 13 shows a HPLC chromatogram of EGFR
antibody-S--S-siRNA-PEG5 kDa (DAR=1).
[0060] FIG. 14 shows a HPLC chromatogram of EGFR
antibody-PEG24-Melittin (loading=.about.1).
[0061] FIG. 15 illustrates a HPLC chromatogram of EGFR
antibody-Melittin (n=.about.1).
[0062] FIG. 16 illustrates a mass spectrum of EGFR
antibody-Melittin (n=1).
[0063] FIG. 17 shows a HIC chromatogram of EGFR
antibody-PEG1kDa-INF7 (Peptide loading=.about.1).
[0064] FIG. 18 shows a HPLC chromatogram of EGFR antibody-INF7
(Peptide Loading=.about.1).
[0065] FIG. 19 shows INF7-PEG1 kDa-(EGFR antibody-KRAS-PEG5
kDa).
[0066] FIG. 20 illustrates Melittin-PEG1 kDa-(EGFR
antibody-KRAS-PEG5 kDa).
[0067] FIG. 21 illustrates plasma concentration-time profiles out
to 96 h post-dose with the siRNA concentration expressed as a
percent of injected dose (% ID).
[0068] FIG. 22 shows plasma concentration-time profiles out to 96 h
post-dose with the siRNA concentration expressed as a percent of
injected dose (% ID).
[0069] FIG. 23 shows plasma concentration-time profiles out to 96 h
post-dose with the siRNA concentration expressed as a percent of
injected dose (% ID).
[0070] FIG. 24 illustrates plasma concentration-time profiles out
to 96 h post-dose with the siRNA concentration expressed as a
percent of injected dose (% ID).
[0071] FIG. 25 illustrates plasma concentration-time profiles out
to 24 h post-dose with the siRNA concentration expressed as a
percent of injected dose (% ID).
[0072] FIG. 26A and FIG. 26B illustrate tissue concentration-time
profiles in tumor or normal livers of mice. FIG. 26A shows tissue
concentration-time profiles out to 168 h post-dose measured in s.c.
flank H358 tumors in a mice model. FIG. 26B shows tissue
concentration-time profiles out to 168 h post-dose measured in
normal livers of mice.
[0073] FIG. 27 shows tissue concentration-time profiles out to 168
h post-dose measured in s.c. flank H358 tumors and normal livers of
mice.
[0074] FIG. 28 illustrates tissue concentration-time profiles out
to 168 h post-dose measured in s.c. flank H358 tumors and normal
livers of mice.
[0075] FIG. 29 illustrates tissue concentration-time profiles out
to 168 h post-dose measured in s.c. flank H358 tumors and normal
livers of mice.
[0076] FIG. 30 shows tissue concentration-time profiles out to 168
h post-dose measured in s.c. flank H358 tumors and normal livers of
mice.
[0077] FIG. 31A and FIG. 31B illustrate siRNA-mediated mRNA
knockdown of human KRAS in human s.c. flank H358 tumors (FIG. 31A)
or mouse KRAS in normal mouse liver (FIG. 31B).
[0078] FIG. 32 illustrates siRNA-mediated mRNA knockdown of human
EGFR in human s.c. flank H358 tumors.
[0079] FIG. 33 illustrates siRNA-mediated mRNA knockdown of human
KRAS in human s.c. flank H358 tumors.
[0080] FIG. 34 illustrates siRNA-mediated mRNA knockdown of human
EGFR in human s.c. flank H358 tumors.
[0081] FIG. 35 shows siRNA-mediated mRNA knockdown of mouse KRAS in
mouse liver.
[0082] FIG. 36 illustrates plasma concentration-time profiles out
to 96 h post-dose with the siRNA concentration expressed as a
percent of injected dose (% ID).
[0083] FIG. 37 illustrates tissue concentration-time profiles out
to 144 h post-dose measured in liver, kidneys, and lungs of
wild-type CD-1 mice.
[0084] FIG. 38A and FIG. 38B illustrate tissue concentration-time
profiles out to 144 h post-dose measured in human s.c. flank H358
tumors for chol-KRAS mixed with either chol-INF7 peptide (FIG. 38A)
or chol-melittin peptide (FIG. 38B).
[0085] FIG. 39A and FIG. 39B illustrate tissue concentration-time
profiles out to 144 h post-dose measured in mouse liver for
chol-KRAS mixed with either chol-INF7 peptide (FIG. 39A) or
chol-melittin peptide (FIG. 39B).
[0086] FIG. 40A and FIG. 40B illustrate tissue concentration-time
profiles out to 144 h post-dose measured in mouse kidneys for
chol-KRAS mixed with either chol-INF7 peptide (FIG. 40A) or
chol-melittin peptide (FIG. 40B).
[0087] FIG. 41A and FIG. 41B illustrate tissue concentration-time
profiles out to 144 h post-dose measured in mouse lungs for
chol-KRAS mixed with either chol-INF7 peptide (FIG. 41A) or
chol-melittin peptide (FIG. 41B).
[0088] FIG. 42 illustrates siRNA-mediated mRNA knockdown of mouse
KRAS in mouse liver.
[0089] FIG. 43A and FIG. 43B illustrate tissue concentration-time
profiles out to 96 h post-dose measured in human s.c. flank H358
tumors (FIG. 43A) or mouse liver (FIG. 43B).
[0090] FIG. 44A and FIG. 44B show tissue concentration-time
profiles out to 96 h post-dose measured in mouse kidneys (FIG. 44A)
or mouse lungs (FIG. 44B).
[0091] FIG. 45 shows siRNA-mediated mRNA knockdown of mouse KRAS in
human s.c. flank H358 tumors.
[0092] FIG. 46 shows tissue concentrations of siRNA at 96 h
post-dose measured in human s.c. flank H358 tumors and mouse liver,
kidneys, and lungs.
[0093] FIG. 47A and FIG. 47B show siRNA-mediated mRNA knockdown in
human s.c. flank H358 tumors of EGFR (FIG. 47A) or KRAS (FIG.
47B).
[0094] FIG. 48 shows siRNA-mediated mRNA knockdown of human CTNNB1
in Hep3B orthotopic liver tumors.
[0095] FIG. 49 shows human alpha-Fetoprotein in serum from mice
with Hep3B orthotopic liver tumors.
[0096] FIG. 50A shows siRNA-mediated mRNA knockdown of human EGFR
in LNCaP tumor.
[0097] FIG. 50B shows siRNA concentration in tumor or liver tissues
at 96 hour post-dose.
[0098] FIG. 51A illustrates siRNA-mediated mRNA knockdown of human
EGFR in LNCaP tumor at 96 hour.
[0099] FIG. 51B shows siRNA concentration in tumor or liver tissues
at 96 hour post-dose.
[0100] FIG. 52 shows plasma siRNA concentration of exemplary
molecules described herein.
[0101] FIG. 53A illustrates siRNA concentration of exemplary
molecules described herein in HCC827 tumor or liver tissue.
[0102] FIG. 53B shows EGFR EGFR mRNA expression level of exemplary
molecules described herein.
[0103] FIG. 54 illustrates exemplary As and Bs to generate
molecules encompassed by Formula (I).
[0104] FIG. 55 illustrates EGFR mRNA expression level of exemplary
molecules described herein.
[0105] FIG. 56A illustrates siRNA concentration of exemplary
molecules described herein in HCC827 tumor or liver tissue.
[0106] FIG. 56B shows EGFR mRNA expression level of exemplary
molecules described herein.
[0107] FIG. 57A-FIG. 57B illustrate siRNA concentration of
exemplary molecules described herein in liver (FIG. 57A) and tumor
(FIG. 57B).
[0108] FIG. 57C shows KRAS mRNA expression level of exemplary
molecules described herein.
[0109] FIG. 58A illustrates plasma siRNA concentration of exemplary
molecules described herein.
[0110] FIG. 58B shows plasma antibody concentration of exemplary
molecules described herein.
[0111] FIG. 59A illustrates siRNA concentration of exemplary
molecules described herein in tumor or liver tissue.
[0112] FIG. 59B shows mRNA expression level of exemplary molecules
described herein in Hep3B tumor.
[0113] FIG. 60 shows CTNNB1 mRNA expression level of an exemplary
molecule described herein in liver.
[0114] FIG. 61 shows KRAS mRNA expression level of an exemplary
molecule described herein in liver.
[0115] FIG. 62 illustrates plasma siRNA or monoclonal antibody
(mAb) concentration of exemplary molecules described herein.
[0116] FIG. 63A illustrates siRNA concentration of exemplary
molecules described herein in tumor or liver tissue.
[0117] FIG. 63B shows EGFR mRNA expression level of exemplary
molecules described herein in LNCaP tumor.
[0118] FIG. 64A-FIG. 64E illustrate HPRT mRNA expression level in
heart (FIG. 64A), HPRT mRNA expression level in gastrointestinal
tissue (FIG. 64B), HPRT mRNA expression level in liver (FIG. 64C),
HPRT mRNA expression level in lung (FIG. 64D), and siRNA
concentration in tissue (FIG. 64E) of exemplary molecules described
herein.
[0119] FIG. 65A-FIG. 65E illustrate mRNA expression level in heart
(FIG. 65A), mRNA expression level in gastrointestinal tissue (FIG.
65B), mRNA expression level in liver (FIG. 65C), mRNA expression
level in lung (FIG. 65D), and siRNA concentration in tissue (FIG.
65E) of exemplary molecules described herein.
[0120] FIG. 66A-FIG. 66D illustrate siRNA concentration in heart
(FIG. 66A), mRNA expression level in heart (FIG. 66B), mRNA
expression level in gastrointestinal tissue (FIG. 66C), and siRNA
concentration in muscle (FIG. 66D).
[0121] FIG. 67A illustrate mRNA expression level of exemplary
molecules described herein.
[0122] FIG. 67B shows siRNA concentration of exemplary molecules
described herein in tumor or liver tissues.
[0123] FIG. 68A-FIG. 68B illustrate anti-B cell antibody-siRNA
conjugates which activate primary mouse B cells. FIG. 68A
illustrates an anti-B cell Fab-siRNA conjugate. FIG. 68B shows an
anti-B cell monoclonal antibody-siRNA conjugate.
[0124] FIG. 69A illustrates plasma siRNA concentration of exemplary
molecules described herein.
[0125] FIG. 69B shows antibody zalutumumab concentration of
exemplary molecules described herein in the plasma at a 5 mg/kg
dose.
[0126] FIG. 70A shows mRNA expression level of exemplary molecules
described herein.
[0127] FIG. 70B shows siRNA concentration of exemplary molecules
described herein in tumor or liver tissues.
[0128] FIG. 70C shows plasma siRNA concentration of exemplary
molecules described herein.
[0129] FIG. 71A illustrates siRNA concentration of exemplary
molecules described herein in LNCaP tumor.
[0130] FIG. 71B-FIG. 71C illustrate mRNA expression level of
exemplary molecules described herein in LNCaP tumor.
[0131] FIG. 72A illustrates siRNA concentration of exemplary
molecules described herein in tissue.
[0132] FIG. 72B shows mRNA expression level of exemplary molecules
described herein in HCC827 tumors at 96 h post-treatment.
[0133] FIG. 73A illustrates siRNA concentration of exemplary
molecules described herein in the plasma at a 0.5 mg/kg dose.
[0134] FIG. 73B shows antibody zalutumumab concentration of
exemplary molecules described herein in the plasma at a 5 mg/kg
dose.
[0135] FIG. 74 illustrates plasma clearance of exemplary molecules
encompassed by Formula (I) which contains different linkers.
[0136] FIG. 75A illustrates the mRNA expression level of exemplary
molecules described herein in HCC827 tumor at a 0.5 mg/kg dose.
[0137] FIG. 75B-FIG. 75D illustrate siRNA concentration in tumor
(FIG. 75B), liver (FIG. 75C), and plasma (FIG. 75D).
[0138] FIG. 76A-FIG. 76D illustrate mRNA expression levels of
exemplary molecules described herein targeting HPRT. FIG. 76A shows
the mRNA expression level in heart. FIG. 76B shows the mRNA
expression level in muscle. FIG. 76C shows the mRNA expression
level in liver. FIG. 76D shows the mRNA expression level in
lung.
[0139] FIG. 77A-FIG. 77D illustrate siRNA concentrations of
exemplary molecules encompassed by Formula (I) in muscle (FIG.
77A), heart (FIG. 77B), liver (FIG. 77C), and lung (FIG. 77D).
[0140] FIG. 78A-FIG. 78D illustrate mRNA expression levels of
exemplary molecules encompassed by Formula (I) in heart (FIG. 78A),
gastrointestinal tissue (FIG. 78B), liver (FIG. 78C), and lung
(FIG. 78D) at 96 h post-treatment.
[0141] FIG. 79 illustrates plasma siRNA concentration of exemplary
molecules encompassed by Formula (I).
[0142] FIG. 80A shows mRNA expression level of exemplary molecules
encompassed by Formula (I) in LNCaP tumor at 96 h
post-treatment.
[0143] FIG. 80B shows siRNA concentration of exemplary molecules
encompassed by Formula (I) in LNCaP tumor, liver, kidney, lung, and
spleen tissue samples.
[0144] FIG. 81A shows mRNA expression level of exemplary molecules
encompassed by Formula (I) in HCC827 tumor at 96 h
post-treatment.
[0145] FIG. 81B illustrates siRNA concentrations of exemplary
molecules encompassed by Formula (I) in tumor, liver, kidney, lung,
and spleen tissue samples.
[0146] FIG. 82 illustrates plasma siRNA concentration of exemplary
molecules encompassed by Formula (I).
[0147] FIG. 83 illustrates plasma siRNA concentration of exemplary
molecules encompassed by Formula (I).
[0148] FIG. 84 illustrates mRNA expression levels of exemplary
molecules encompassed by Formula (I) in HCC827 tumor at 96 h post
treatment.
[0149] FIG. 85 illustrates siRNA concentration in HCC827 tumor or
liver tissues at 96 hour post-dose.
[0150] FIG. 86 illustrates the relative mRNA expression levels of
exemplary molecules encompassed by Formula (I) in mouse splenic B
cells 48 h post treatment. Each exemplary molecule is further
denoted with a number.
[0151] FIG. 87 illustrates stability of exemplary molecules
encompassed by Formula (I) (or ASCs) in mouse plasma.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0152] Nucleic acid (e.g., RNAi) therapy is a targeted therapy with
high selectivity and specificity. However, in some instances,
nucleic acid therapy is also hindered by poor intracellular uptake,
limited blood stability and non-specific immune stimulation. To
address these issues, various modifications of the nucleic acid
composition are explored, such as for example, novel linkers for
better stabilizing and/or lower toxicity, optimization of binding
moiety for increased target specificity and/or target delivery, and
nucleic acid polymer modifications for increased stability and/or
reduced off-target effect.
[0153] In some embodiments, the arrangement or order of the
different components that make-up the nucleic acid composition
further effects intracellular uptake, stability, toxicity,
efficacy, and/or non-specific immune stimulation. For example, if
the nucleic acid component includes a binding moiety, a polymer,
and a polynucleic acid molecule (or polynucleotide), the order or
arrangement of the binding moiety, the polymer, and/or the
polynucleic acid molecule (or polynucleotide) (e.g., binding
moiety-polynucleic acid molecule-polymer, binding
moiety-polymer-polynucleic acid molecule, or polymer-binding
moiety-polynucleic acid molecule) further effects intracellular
uptake, stability, toxicity, efficacy, and/or non-specific immune
stimulation.
[0154] In some embodiments, described herein include a molecule
those arrangement of the nucleic acid components effects
intracellular uptake, stability, toxicity, efficacy, and/or
non-specific immune stimulation. In some instances, the molecule
comprises a binding moiety conjugated to a polynucleic acid
molecule and a polymer. In some embodiments, the molecule comprises
a molecule according to Formula (I): A-X-B-Y-C; in which A is a
binding moiety, B is a polynucleotide, C is a polymer, X is a bond
or first linker, and Y is a bond or second linker. In some
instances, the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety. In some instances, the molecule
of Formula (I) further comprises D, an endosomolytic moiety.
[0155] In some embodiments, a molecule comprising a binding moiety
conjugated to a polynucleic acid molecule and a polymer arranged as
described herein enhances intracellular uptake, stability, and/or
efficacy. In some instances, a molecule comprising a binding moiety
conjugated to a polynucleic acid molecule and a polymer arranged as
described herein reduces toxicity and/or non-specific immune
stimulation. In some cases, the molecule comprises a molecule
according to Formula (I): A-X-B-Y-C; in which A is a binding
moiety, B is a polynucleotide, C is a polymer, X is a bond or first
linker, and Y is a bond or second linker. In some instances, the
polynucleotide comprises at least one 2' modified nucleotide, at
least one modified internucleotide linkage, or at least one
inverted abasic moiety. In some instances, the molecule of Formula
(I) further comprises D, an endosomolytic moiety.
[0156] In some embodiments, a molecule described herein is further
used to treat a disease or disorder. In some instances, a molecule
for the treatment of a disease or disorder is a molecule according
to Formula (I): A-X-B-Y-C; in which A is a binding moiety, B is a
polynucleotide, C is a polymer, X is a bond or first linker, and Y
is a bond or second linker. In some instances, the polynucleotide
comprises at least one 2' modified nucleotide, at least one
modified internucleotide linkage, or at least one inverted abasic
moiety. In some instances, the molecule of Formula (I) further
comprises D, an endosomolytic moiety.
[0157] In some embodiments, a molecule described herein is also
used for inhibiting the expression of a target gene in a primary
cell of a patient in need thereof. In such instances, a molecule
for such use is a molecule according to Formula (I): A-X-B-Y-C; in
which A is a binding moiety, B is a polynucleotide, C is a polymer,
X is a bond or first linker, and Y is a bond or second linker. In
some instances, the polynucleotide comprises at least one 2'
modified nucleotide, at least one modified internucleotide linkage,
or at least one inverted abasic moiety. In some instances, the
molecule of Formula (I) further comprises D, an endosomolytic
moiety.
[0158] In some embodiments, a molecule described herein is
additionally used as an immuno-oncology therapy for the treatment
of a disease or disorder. In some instance, the molecule is a
molecule according to Formula (I): A-X-B-Y-C; in which A is a
binding moiety, B is a polynucleotide, C is a polymer, X is a bond
or first linker, and Y is a bond or second linker. In some
instances, the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety. In some instances, the molecule
of Formula (I) further comprises D, an endosomolytic moiety.
[0159] In additional embodiments, described herein include a kit,
which comprises one or more of the molecules described herein.
Therapeutic Molecule Platform
[0160] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a binding moiety conjugated to
a polynucleic acid molecule and a polymer. In some embodiments, a
molecule (e.g., a therapeutic molecule) comprises a molecule
according to Formula (I):
A-X-B-Y-C Formula I
wherein,
[0161] A is a binding moiety;
[0162] B is a polynucleotide;
[0163] C is a polymer;
[0164] X is a bond or first linker; and
[0165] Y is a bond or second linker; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety.
[0166] In some instances, the molecule of Formula (I) further
comprises D, an endosomolytic moiety.
[0167] In some embodiments, at least one A and/or at least one C
are conjugated to the 5' terminus of B, the 3' terminus of B, an
internal site on B, or in any combinations thereof. In some
instances, at least one A is conjugated at one terminus of B while
at least one C is conjugated at the opposite terminus of B. In some
instances, at least one of A is conjugated at one terminus of B
while at least one of C is conjugated at an internal site on B.
[0168] In some cases, A and C are not conjugated or attached to B
at the same terminus. In some cases, A is attached or conjugated to
B at a first terminus of B. In some cases, C is attached or
conjugated to B at a second terminus of B, and the second terminus
of B is different than the first terminus. In some cases, A is
attached or conjugated to B at the 5' terminus of B, and C is
attached or conjugated to B at the 3' terminus of B. In other
cases, A is attached or conjugated to B at the 3' terminus of B,
and C is attached or conjugated to B at the 5' terminus of B.
[0169] In some embodiments, A is an antibody or binding fragment
thereof. In some cases, C is a polymer. In some cases, A and C are
not conjugated or attached to B at the same terminus. In some
cases, A is attached or conjugated to B at a first terminus of B.
In some cases, C is attached or conjugated to B at a second
terminus of B, and the second terminus of B is different than the
first terminus. In some cases, A is attached or conjugated to B at
the 5' terminus of B, and C is attached or conjugated to B at the
3' terminus of B. In other cases, A is attached or conjugated to B
at the 3' terminus of B, and C is attached or conjugated to B at
the 5' terminus of B. In some cases, X which connects A to B is a
bond or a non-polymeric linker. In some cases, X is a non-peptide
linker (or a linker that does not comprise an amino acid residue).
In some cases, Y which connects B to C is a bond or a second
linker. In some instances, X connects A to the 5' terminus of B,
and Y connects C to the 3' terminus of B. In other instances, X
connects A to the 3' terminus of B, and Y connects C to the 5'
terminus of B.
[0170] In some embodiments, X-B is conjugated or attached to the
N-terminus, C-terminus, a constant region, a hinge region, or a Fc
region of A. In some instances, X-B is conjugated or attached to
the N-terminus of A. In some instances, X-B is conjugated or
attached to the C-terminus of A. In some instances, X-B is
conjugated or attached to a hinge region of A. In some instances,
X-B is conjugated or attached to a constant region of A. In some
instances, X-B is conjugated or attached to the Fc region of A.
[0171] In some instances, at least one B and/or at least one C, and
optionally at least one D are conjugated to a first A. In some
instances, the at least one B is conjugated at a terminus (e.g., a
5' terminus or a 3' terminus) to the first A or are conjugated via
an internal site to the first A. In some cases, the at least one C
is conjugated either directly to the first A or indirectly via the
two or more Bs. If indirectly via the two or more Bs, the two or
more Cs are conjugated either at the same terminus as the first A
on B, at opposing terminus from the first A, or independently at an
internal site. In some instances, at least one additional A is
further conjugated to the first A, to B, or to C. In additional
instances, the at least one D is optionally conjugated either
directly or indirectly to the first A, to the at least one B, or to
the at least one C. If directly to the first A, the at least one D
is also optionally conjugated to the at least one B to form a A-D-B
conjugate or is optionally conjugated to the at least one B and the
at least one C to form a A-D-B-C conjugate. In some cases, the at
least one additional A is different than the first A.
[0172] In some cases, two or more Bs and/or two or more Cs are
conjugated to a first A. In some instances, the two or more Bs are
conjugated at a terminus (e.g., a 5' terminus or a 3' terminus) to
the first A or are conjugated via an internal site to the first A.
In some instances, the two or more Cs are conjugated either
directly to the first A or indirectly via the two or more Bs. If
indirectly via the two or more Bs, the two or more Cs are
conjugated either at the same terminus as the first A on B, at
opposing terminus from the first A, or independently at an internal
site. In some instances, at least one additional A is further
conjugated to the first A, to two or more Bs, or to two or more Cs.
In additional instances, at least one D is optionally conjugated
either directly or indirectly to the first A, to the two or more
Bs, or to the two or more Cs. If indirectly to the first A, the at
least one D is conjugated to the first A through the two or more
Bs, through the two or more Cs, through a B-C orientation to form a
A-B-C-D type conjugate, or through a C-B orientation to form a
A-C-B-D type conjugate. In some cases, the at least one additional
A is different than the first A. In some cases, the two or more Bs
are different. In other cases, the two or more Bs are the same. In
some instances, the two or more Cs are different. In other
instances, the two or more Cs are the same. In additional
instances, the two or more Ds are different. In additional
instances, the two or more Ds are the same.
[0173] In other cases, two or more Bs and/or two or more Ds,
optionally two or more Cs are conjugated to a first A. In some
instances, the two or more Bs are conjugated at a terminus (e.g., a
5' terminus or a 3' terminus) to the first A or are conjugated via
an internal site to the first A. In some instances, the two or more
Ds are conjugated either directly to the first A or indirectly via
the two or more Bs. If indirectly via the two or more Bs, the two
or more Ds are conjugated either at the same terminus as the first
A on B, at opposing terminus from the first A, or independently at
an internal site. In some instances, at least one additional A is
further conjugated to the first A, to the two or more Bs, or to the
two or more Ds. In additional instances, the two or more Cs are
optionally conjugated either directly or indirectly to the first A,
to the two or more Bs, or to the two or more Ds. In some cases, the
at least one additional A is different than the first A. In some
cases, the two or more Bs are different. In other cases, the two or
more Bs are the same. In some instances, the two or more Cs are
different. In other instances, the two or more Cs are the same. In
additional instances, the two or more Ds are different. In
additional instances, the two or more Ds are the same.
[0174] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (II):
(A-X-B-Y-C.sub.c)-L-D Formula II
wherein,
[0175] A is a binding moiety;
[0176] B is a polynucleotide;
[0177] C is a polymer;
[0178] X is a bond or first linker;
[0179] Y is a bond or second linker;
[0180] L is a bond or third linker;
[0181] D is an endosomolytic moiety; and
[0182] c is an integer between 0 and 1; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety; and D is conjugated anywhere on
A, B, or C.
[0183] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (III):
A.sub.a-X-B.sub.b-Y-C.sub.c-L-D.sub.n Formula III
wherein,
[0184] A is a binding moiety;
[0185] B is a polynucleotide;
[0186] C is a polymer;
[0187] D is an endosomolytic moiety;
[0188] X is a bond or first linker;
[0189] Y is a bond or second linker;
[0190] L is a bond or third linker;
[0191] a and b are independently an integer between 1-3;
[0192] c is an integer between 0 and 3; and
[0193] n is an integer between 0 and 10; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety; A is conjugated anywhere on B, C,
or D; B is conjugated anywhere on A, C, or D; C is conjugated
anywhere on A, B, or D; and D is conjugated anywhere on A, B, or
C.
[0194] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (Ma): A-X-B-L-D-Y-C.
[0195] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (Mb): A.sub.a-X-B.sub.b-L-D.sub.n.
[0196] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (IV):
A-X--(B.sub.b-Y-C.sub.c-L-D.sub.n).sub.m
wherein,
[0197] A is a binding moiety;
[0198] B is a polynucleotide;
[0199] C is a polymer;
[0200] D is an endosomolytic moiety;
[0201] X is a bond or first linker;
[0202] Y is a bond or second linker;
[0203] L is a bond or third linker;
[0204] a and b are independently an integer between 1-3;
[0205] c is an integer between 0 and 3;
[0206] n is an integer between 0 and 10; and
[0207] m is an integer between 1-3; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety; C is conjugated anywhere on B or
D; and D is conjugated anywhere on B or C.
[0208] In some embodiments, a molecule (e.g., a therapeutic
molecule) described herein comprises a molecule according to
Formula (IVa): A-X-(B.sub.b-L-D.sub.n-Y-C.sub.c).sub.m.
[0209] In some embodiment, a molecule (e.g., a therapeutic
molecule) described herein is a molecule as illustrated in FIG. 1.
In some instances, a molecule (e.g., a therapeutic molecule)
described herein is a molecule as illustrated in FIG. 1A. In some
cases, a molecule (e.g., a therapeutic molecule) described herein
is a molecule as illustrated in FIG. 1B. In additional cases, a
molecule (e.g., a therapeutic molecule) described herein is a
molecule as illustrated in FIG. 1C.
[0210] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0211] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0212] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0213] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0214] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0215] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0216] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0217] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0218] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0219] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0220] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0221] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0222] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0223] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0224] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0225] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0226] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0227] In some embodiments, a molecule (e.g., a therapeutic
molecule) is a molecule as illustrated:
[0228] The
as illustrated above is for representation purposes only and
encompasses a humanized antibody or binding fragment thereof,
chimeric antibody or binding fragment thereof, monoclonal antibody
or binding fragment thereof, monovalent Fab', divalent Fab2,
single-chain variable fragment (scFv), diabody, minibody, nanobody,
single-domain antibody (sdAb), or camelid antibody or binding
fragment thereof.
Polynucleic Acid Molecule Targets
[0229] In some embodiments, the polynucleic acid molecule B is a
polynucleic acid molecule (or polynucleotide) that hybridizes to a
target region on an oncogene. In some instances, oncogenes are
further classified into several categories: growth factors or
mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases,
cytoplasmic serine/threonine kinases, regulatory GTPases, and
transcription factors. Exemplary growth factors include c-Sis.
Exemplary receptor tyrosine kinases include epidermal growth factor
receptor (EGFR), platelet-derived growth factor receptor (PDGFR),
vascular endothelial growth factor receptor (VEGFR), and HER2/neu.
Exemplary cytoplasmic tyrosine kinases include Src-family tyrosine
kinases, Syk-ZAP-70 family of tyrosine kinases, BTK family of
tyrosine kinases, and Abl gene in CML. Exemplary cytoplasmic
serine/threonine kinases include Raf kinase and cyclin-dependent
kinases. Exemplary regulatory GTPases include Ras family of
proteins such as KRAS. Exemplary transcription factors include MYC
gene. In some instances, an oncogene described herein comprises an
oncogene selected from growth factors or mitogens, receptor
tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic
serine/threonine kinases, regulatory GTPases, or transcription
factors. In some embodiments, the polynucleic acid molecule is a
polynucleic acid molecule that hybridizes to a target region of an
oncogene selected from growth factors or mitogens, receptor
tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic
serine/threonine kinases, regulatory GTPases, or transcription
factors.
[0230] In some embodiments, an oncogene described herein comprises
Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF,
c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3,
INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1,
MAS1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11/CBFB, NOTCH1 (TAN1),
NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR/PML, HRAS,
KRAS, NRAS, REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some
embodiments, the polynucleic acid molecule is a polynucleic acid
molecule that hybridizes to a target region of Abl, AKT-2, ALK,
AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2
(Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3,
K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS1, MDM2, MET, MLL
(KMT2A), MOS, MYB, MYH11/CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST
(SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR/PML, HRAS, KRAS, NRAS,
REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2.
[0231] In some embodiments, an oncogene described herein comprises
KRAS, EGFR, AR, HPRT1, CNNTB1 (.beta.-catenin), or .beta.-catenin
associated genes. In some embodiments, the polynucleic acid
molecule B is a polynucleic acid molecule that hybridizes to a
target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (.beta.-catenin), or
.beta.-catenin associated genes. In some embodiments, the
polynucleic acid molecule B is a polynucleic acid molecule that
hybridizes to a target region of KRAS. In some embodiments, the
polynucleic acid molecule B is a polynucleic acid molecule that
hybridizes to a target region of EGFR. In some embodiments, the
polynucleic acid molecule B is a polynucleic acid molecule that
hybridizes to a target region of AR. In some embodiments, the
polynucleic acid molecule B is a polynucleic acid molecule that
hybridizes to a target region of CNNTB1 (.beta.-catenin). In some
embodiments, the polynucleic acid molecule B is a polynucleic acid
molecule that hybridizes to a target region of CNNTB1
(.beta.-catenin) associated genes. In some instances, the
.beta.-catenin associated genes comprise PIK3CA, PIK3CB, and Myc.
In some instances, the polynucleic acid molecule B is a polynucleic
acid molecule that hybridizes to a target region of HPRT1.
Polynucleic Acid Molecules that Target Kirsten Rat Sarcoma Viral
Oncogene Homolog (KRAS)
[0232] Kirsten Rat Sarcoma Viral Oncogene Homolog (also known as
GTPase KRas, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog,
or KRAS) is involved in regulating cell division. The K-Ras protein
is a GTPase belonging to the Ras superfamily. In some instances,
K-Ras modulates cell cycle progression, as well as induces growth
arrest, apoptosis, and replicative senescence under different
environmental triggers (e.g., cellular stress, ultraviolet, heat
shock, or ionizing irradiation). In some cases, wild type KRAS gene
has been shown to be frequently lost during tumor progression in
different types of cancer, while mutations of KRAS gene have been
linked to cancer development. In some instances, KRAS amplification
has also been implicated in cancer development (see, for example,
Valtorta et al. "KRAS gene amplification in colorectal cancer and
impact on response to EGFR-targeted therapy," Int. J. Cancer 133:
1259-1266 (2013)). In such cases, the cancer pertains to a
refractory cancer in which the patient has acquired resistance to a
particular inhibitor or class of inhibitors.
[0233] In some embodiments, the KRAS gene is wild type or comprises
a mutation. In some instances, KRAS mRNA is wild type or comprises
a mutation. In some instances, the polynucleic acid molecule is a
polynucleic acid molecule that hybridizes to a target region of
wild type KRAS DNA or RNA. In some instances, the polynucleic acid
molecule is a polynucleic acid molecule that hybridizes to a target
region of KRAS DNA or RNA comprising a mutation (e.g., a
substitution, a deletion, or an addition).
[0234] In some embodiments, KRAS DNA or RNA comprises one or more
mutations. In some embodiments, KRAS DNA or RNA comprises one or
more mutations at codons 12 or 13 in exon 1. In some instances,
KRAS DNA or RNA comprises one or more mutations at codons 61, 63,
117, 119, or 146. In some instances, KRAS DNA or RNA comprises one
or more mutations at positions corresponding to amino acid residues
12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116,
117, 119, 146, 147, 158, 164, 176, or a combination thereof of the
KRAS polypeptide. In some embodiments, KRAS DNA or RNA comprises
one or more mutations at positions corresponding to amino acid
residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C,
G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G,
A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N,
C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a
combination thereof of the KRAS polypeptide.
[0235] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of KRAS DNA or RNA comprising one or
more mutations. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of KRAS DNA or RNA comprising one or
more mutations at codons 12 or 13 in exon 1. In some embodiments,
the polynucleic acid molecule hybridizes to a target region of KRAS
DNA or RNA comprising one or more mutations at codons 61, 63, 117,
119, or 146. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of KRAS DNA or RNA comprising one or
more mutations at positions corresponding to amino acid residues
12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116,
117, 119, 146, 147, 158, 164, 176, or a combination thereof of the
KRAS polypeptide. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of KRAS DNA or RNA
comprising one or more mutations corresponding to amino acid
residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C,
G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G,
A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N,
C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a
combination thereof of the KRAS polypeptide.
Polynucleic Acid Molecules that Target Epidermal Growth Factor
Receptor (EGFR)
[0236] Epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is
a transmembrane tyrosine kinase receptor and a member of the ErbB
family of receptors, which also include HER2/c-neu (ErbB-2), Her3
(ErbB-3) and Her4 (ErbB-4). In some instances, EGFR mutations drive
the downstream activation of RAS/RAF/MAPK, PI3K/AKT, and/or
JAK/STAT pathways, leading to mitosis, cell proliferation, and
suppression of apoptosis. In addition, amplification of wild-type
EGFR gene has been implicated in the development of cancers such as
glioblastomas and non-small cell lung cancer (Talasila, et al.,
"EGFR Wild-type Amplification and Activation Promote Invasion and
Development of Glioblastoma Independent of Angiogenesis," Acta
Neuropathol. 125(5): 683-698 (2013); Bell et al., "Epidermal Growth
Factor Receptor Mutations and Gene Amplification in Non-Small-Cell
Lung Cancer: Molecular Analysis of the IDEAL/INTACT Gefitinib
Trials," J. Clinical Oncology 23(31): 8081-8092 (2005)).
[0237] In some embodiments, EGFR DNA or RNA is wild type EGFR or
EGFR comprising a mutation. In some instances, EGFR is wild type
EGFR. In some instances, EGFR DNA or RNA comprises a mutation. In
some instances, the polynucleic acid molecule hybridizes to a
target region of wild type EGFR DNA or RNA. In some instances, the
polynucleic acid molecule hybridizes to a target region of EGFR DNA
or RNA comprising a mutation (e.g., a substitution, a deletion, or
an addition).
[0238] In some instances, EGFR DNA or RNA comprises one or more
mutations. In some embodiments, EGFR DNA or RNA comprises one or
more mutations within one or more exons. In some instances, the one
or more exons comprise exon 18, exon 19, exon 20, exon 21 or exon
22. In some instances, EGFR DNA or RNA comprises one or more
mutations in exon 18, exon 19, exon 20, exon 21, exon 22 or a
combination thereof.
[0239] In some instances, EGFR DNA or RNA comprises one or more
mutations at positions corresponding to amino acid residues 34, 38,
45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178,
189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255,
256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314,
326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441,
447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589,
596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689,
694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733,
739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751,
747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765,
767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773,
774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803,
805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842,
843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967,
1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a
combination thereof of the EGFR polypeptide. In some embodiments,
EGFR DNA or RNA comprises one or more mutations at positions
corresponding to amino acid residues 747, 761, 790, 854, 858, or a
combination thereof of the EGFR polypeptide. In some embodiments,
EGFR DNA or RNA comprises one or more mutations at positions
corresponding to amino acid residues 761, 790, 858, or a
combination thereof of the EGFR polypeptide. In some embodiments,
EGFR DNA or RNA comprises a mutation at a position corresponding to
amino acid residue 747 of the EGFR polypeptide. In some
embodiments, EGFR DNA or RNA comprises a mutation at a position
corresponding to amino acid residue 761 of the EGFR polypeptide. In
some embodiments, EGFR DNA or RNA comprises a mutation at a
position corresponding to amino acid residue 790 of the EGFR
polypeptide. In some embodiments, EGFR DNA or RNA comprises a
mutation at a position corresponding to amino acid residue 854 of
the EGFR polypeptide. In some embodiments, EGFR DNA or RNA
comprises a mutation at a position corresponding to amino acid
residue 858 of the EGFR polypeptide.
[0240] In some embodiments, EGFR DNA or RNA comprises one or more
mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F,
F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K,
S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y,
S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense
mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288
(frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion),
D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T3631, P373Q,
R337S, S380 (frame shift), T384S, D393Y, R427L, G428S, S437Y,
V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense),
V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R,
P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F,
C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D,
E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A,
G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L,
K739-1744 (insertion), V742I, V742A, E746-A750 (deletion), E746K,
L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753
(deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion),
S7524759 (deletion), A750P, D761-E762 (e.g., residues EAFQ
insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A, A767-5768
(e.g., residues TLA insertion), A767-V769 (e.g., residues ASV
insertion), 57681, S768T, V769L, V769M, V769-D770 (e.g., residue Y
insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g.,
residue G insertion), 770-771 (e.g., residues CV insertion),
770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g.,
residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g.,
residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A,
V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S,
N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L,
D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N,
V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L,
G917A, E967A, D1006Y, P1019L, 51042N, R1100S, H1129Y, T1141S,
511531, Q1164R, L1167M, or a combination thereof of the EGFR
polypeptide.
[0241] In some instances, the polynucleic acid molecule hybridizes
to a target region of EGFR DNA or RNA comprising one or more
mutations. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising one or
more mutations in exon 18, exon 19, exon 20, exon 21, exon 22 or a
combination thereof.
[0242] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising one or
more mutations at positions corresponding to amino acid residues
34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177,
178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254,
255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309,
314, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437,
441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588,
589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671,
689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733,
739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751,
747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765,
767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773,
774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803,
805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842,
843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967,
1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, ora
combination thereof of the EGFR polypeptide. In some embodiments,
the polynucleic acid molecule hybridizes to a target region of EGFR
DNA or RNA comprising one or more mutations at positions
corresponding to amino acid residues 747, 761, 790, 854, 858, or a
combination thereof of the EGFR polypeptide. In some embodiments,
the polynucleic acid molecule hybridizes to a target region of EGFR
DNA or RNA comprising one or more mutations at positions
corresponding to amino acid residues 761, 790, 858, or a
combination thereof of the EGFR polypeptide. In some embodiments,
the polynucleic acid molecule hybridizes to a target region of EGFR
DNA or RNA comprising a mutation at a position corresponding to
amino acid residue 747 of the EGFR polypeptide. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of EGFR DNA or RNA comprising a mutation at a position
corresponding to amino acid residue 761 of the EGFR polypeptide. In
some embodiments, the polynucleic acid molecule hybridizes to a
target region of EGFR DNA or RNA comprising a mutation at a
position corresponding to amino acid residue 790 of the EGFR
polypeptide. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising a
mutation at a position corresponding to amino acid residue 854 of
the EGFR polypeptide. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of EGFR DNA or RNA
comprising a mutation at a position corresponding to amino acid
residue 858 of the EGFR polypeptide.
[0243] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising one or
more mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K,
S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K,
S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y,
S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense
mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288
(frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion),
D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T3631, P373Q,
R337S, 5380 (frame shift), T384S, D393Y, R427L, G428S, S437Y,
V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense),
V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R,
P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F,
C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D,
E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A,
G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L,
K739-1744 (insertion), V742I, V742A, E746-A750 (deletion), E746K,
L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753
(deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion),
S7524759 (deletion), A750P, D761-E762 (e.g., residues EAFQ
insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A, A767-S768
(e.g., residues TLA insertion), A767-V769 (e.g., residues ASV
insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y
insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g.,
residue G insertion), 770-771 (e.g., residues CV insertion),
770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g.,
residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g.,
residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A,
V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S,
N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L,
D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N,
V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L,
G917A, E967A, D1006Y, P1019L, 51042N, R1100S, H1129Y, T1141S,
S1153I, Q1164R, L1167M, or a combination thereof of the EGFR
polypeptide. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising one or
more mutations selected from L747S, D761Y, T790M, T854A, L858R, or
a combination thereof of the EGFR polypeptide. In some embodiments,
the polynucleic acid molecule hybridizes to a target region of EGFR
DNA or RNA comprising one or more mutations selected from D761Y,
T790M, L858R, or a combination thereof of the EGFR polypeptide. In
some embodiments, the polynucleic acid molecule hybridizes to a
target region of EGFR DNA or RNA comprising mutation L747S of the
EGFR polypeptide. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of EGFR DNA or RNA
comprising mutation D761Y of the EGFR polypeptide. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of EGFR DNA or RNA comprising mutation T790M of the EGFR
polypeptide. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of EGFR DNA or RNA comprising
mutation T854A of the EGFR polypeptide. In some embodiments, the
polynucleic acid molecule hybridizes to a target region of EGFR DNA
or RNA comprising mutation L858R of the EGFR polypeptide.
Polynucleic Acid Molecules that Target Androgen Receptor (AR)
[0244] Androgen receptor (AR) (also known as NR3C4, nuclear
receptor subfamily 3, group C, gene 4) belongs to the steroid
hormone group of nuclear receptor superfamily along with related
members: estrogen receptor (ER), glucocorticoid receptor (GR),
progesterone receptor (PR), and mineralocorticoid receptor (MR).
Androgens, or steroid hormones, modulate protein synthesis and
tissue remodeling through the androgen receptor. The AR protein is
a ligand-inducible zinc finger transcription factor that regulates
target gene expression. The presence of mutations in the AR gene
has been observed in several types of cancers (e.g., prostate
cancer, breast cancer, bladder cancer, or esophageal cancer), and
in some instances, has been linked to metastatic progression.
[0245] In some embodiments, AR DNA or RNA is wild type or comprises
one or more mutations and/or splice variants. In some instances, AR
DNA or RNA comprises one or more mutations. In some instances, AR
DNA or RNA comprises one or more splice variants selected from AR
splice variants including but not limited to AR1/2/2b, ARV2, ARV3,
ARV4, AR1/2/3/2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12,
ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some instances, the
polynucleic acid molecule hybridizes to a target region of AR DNA
or RNA comprising a mutation (e.g., a substitution, a deletion, or
an addition) or a splice variant.
[0246] In some embodiments, AR DNA or RNA comprises one or more
mutations. In some embodiments, AR DNA or RNA comprises one or more
mutations within one or more exons. In some instances, the one or
more exons comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6,
exon 7, or exon 8. In some embodiments, AR DNA or RNA comprises one
or more mutations within exon 1, exon 2, exon 3, exon 4, exon 5,
exon 6, exon 7, exon 8 or a combination thereof. In some instances,
AR DNA or RNA comprises one or more mutations at positions
corresponding to amino acid residues 2, 14, 16, 29, 45, 54, 57, 64,
106, 112, 176, 180, 184, 194, 198, 204, 214, 221, 222, 233, 243,
252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 368, 369, 390,
403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548, 564, 567,
568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580,
581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609,
610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653,
662, 664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688,
689, 690, 695, 700, 701, 702, 703, 705, 706, 707, 708, 710, 711,
712, 715, 717, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730,
732, 733, 737, 739, 741, 742, 743, 744, 745, 746, 748, 749, 750,
751, 752, 754, 755, 756, 757, 758, 759, 762, 763, 764, 765, 766,
767, 768, 771, 772, 774, 777, 779, 786, 795, 780, 782, 784, 787,
788, 790, 791, 793, 794, 798, 802, 803, 804, 806, 807, 812, 813,
814, 819, 820, 821, 824, 827, 828, 830, 831, 834, 840, 841, 842,
846, 854, 855, 856, 863, 864, 866, 869, 870, 871, 874, 875, 877,
879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897, 898, 902,
903, 904, 907, 909, 910, 911, 913, 916, 919, or a combination
thereof of the AR polypeptide. In some embodiments, AR DNA or RNA
comprises one or more mutations at positions corresponding to amino
acid residues selected from E2K, P14Q, K16N, V29M, S45T, L54S,
L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S,
G214R, K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S,
A287D, E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390R,
P390S, P390L, A403V, Q443R, G491S, G505D, P513S, G524D, G524S,
D528G, P533S, L547F, P548S, D564Y, S567F, G568W, L574P, L547F,
C559Y, G568W, G568V, Y571C, Y571H, A573D, T575A, C576R, C576F,
G577R, S578T, C579Y, C579F, K580R, V581F, F582Y, F582S, R585K,
A586V, A587S, A596T, A596S, S597G, 55971, N599Y, C601F, D604Y,
R607Q, R608K, K609N, D610T, C611Y, R615H, R615P, R615G, R616C,
L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T, L638M,
A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H,
I672T, L674P, L677P, E681L, P682T, G683A, V6841, V684A, A687V,
G688Q, H689P, D690V, D695N, D695V, D695H, L700M, L701P, L7011,
H701H, S702A, S703G, N705S, N705Y, E706 (nonsense), L707R, G708A,
R710T, Q711E, L712F, V715M, K717Q, K720E, A721T, L722F, P723S,
G724S, G724D, G724N, F725L, R726L, N727K, L728S, L728I, V730M,
D732N, D732Y, D732E, Q733H, I737T, Y739D, W741R, M742V, M7421,
G743R, G743V, L744F, M745T, V746M, A748D, A748V, A748T, M749V,
M7491, G750S, G750D, W751R, R752Q, F754V, F754L, T755A, N756S,
N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C, F764L,
A765T, A765V, P766A, P766S, D767E, L768P, L768M, N771H, E772G,
E772A, R774H, R774C, K777T, R779W, R786Q, G795V, M7801, S782N,
C784Y, M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R,
G803L, F804L, C806Y, M807V, M807R, M8071, L812P, F813V, S814N,
N819Q, G820A, L821V, Q824L, Q824R, F827L, F827V, D828H, L830V,
L830P, R831Q, R831L, Y834C, R840C, R840H, I841S, I842T, R846G,
R854K, R855C, R855H, F856L, L863R, D864N, D864E, D864G, V866L,
V866M, V866E, I869M, A870G, A870V, R871G, H874Y, H874R, Q875K,
T877S, T877A, D879T, D879G, L880Q, L881V, M886V, S888L, V889M,
F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M, P904S,
P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or a
combination thereof of the AR polypeptide.
[0247] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of AR DNA or RNA comprising one or
more mutations. In some embodiments the polynucleic acid hybridizes
to one or more AR splice variants. In some embodiments the
polynucleic acid hybridizes to AR DNA or RNA comprising one or more
AR splice variants including but not limited to AR1/2/2b, ARV2,
ARV3, ARV4, AR1/2/3/2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11,
ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of AR DNA or RNA comprising one or more mutations within
exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or a
combination thereof. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of AR DNA or RNA comprising
one or more mutations at positions corresponding to amino acid
residues 2, 14, 16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184,
194, 198, 204, 214, 221, 222, 233, 243, 252, 255, 266, 269, 287,
288, 334, 335, 340, 363, 368, 369, 390, 403, 443, 491, 505, 513,
524, 524, 528, 533, 547, 548, 564, 567, 568, 574, 547, 559, 568,
571, 573, 575, 576, 577, 578, 579, 580, 581, 582, 585, 586, 587,
596, 597, 599, 601, 604, 607, 608, 609, 610, 611, 615, 616, 617,
619, 622, 629, 630, 638, 645, 647, 653, 662, 664, 670, 671, 672,
674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695, 700, 701,
702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720, 721,
722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 741,
742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756,
757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774,
777, 779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794,
798, 802, 803, 804, 806, 807, 812, 813, 814, 819, 820, 821, 824,
827, 828, 830, 831, 834, 840, 841, 842, 846, 854, 855, 856, 863,
864, 866, 869, 870, 871, 874, 875, 877, 879, 880, 881, 886, 888,
889, 891, 892, 895, 896, 897, 898, 902, 903, 904, 907, 909, 910,
911, 913, 916, 919, or a combination thereof of the AR polypeptide.
In some embodiments, the polynucleic acid molecule hybridizes to a
target region of AR DNA or RNA comprising one or more mutations
selected from E2K, P14Q, K16N, V29M, S45T, L54S, L57Q, Q64R, Y106C,
Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R, K221N,
N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D, E288K,
S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L,
A403V, Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S,
L547F, P548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W,
G568V, Y571C, Y571H, A573D, T575A, C576R, C576F, G577R, S578T,
C579Y, C579F, K580R, V581F, F582Y, F582S, R585K, A586V, A587S,
A596T, A596S, S597G, 55971, N599Y, C601F, D604Y, R607Q, R608K,
K609N, D610T, C611Y, R615H, R615P, R615G, R616C, L616R, L616P,
R617P, C619Y, A622V, R629W, R629Q, K630T, L638M, A645D, S647N,
E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T, L674P,
L677P, E681L, P682T, G683A, V6841, V684A, A687V, G688Q, H689P,
D690V, D695N, D695V, D695H, L700M, L701P, L7011, H701H, S702A,
S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E,
L712F, V715M, K717Q, K720E, A721T, L722F, P723S, G724S, G724D,
G724N, F725L, R726L, N727K, L728S, L728I, V730M, D732N, D732Y,
D732E, Q733H, I737T, Y739D, W741R, M742V, M7421, G743R, G743V,
L744F, M745T, V746M, A748D, A748V, A748T, M749V, M7491, G750S,
G750D, W751R, R752Q, F754V, F754L, T755A, N756S, N756D, V757A,
N758T, S759F, S759P, L762F, Y763H, Y763C, F764L, A765T, A765V,
P766A, P766S, D767E, L768P, L768M, N771H, E772G, E772A, R774H,
R774C, K777T, R779W, R786Q, G795V, M7801, S782N, C784Y, M787V,
R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L,
C806Y, M807V, M807R, M8071, L812P, F813V, S814N, N819Q, G820A,
L821V, Q824L, Q824R, F827L, F827V, D828H, L830V, L830P, R831Q,
R831L, Y834C, R840C, R840H, I841S, I842T, R846G, R854K, R855C,
R855H, F856L, L863R, D864N, D864E, D864G, V866L, V866M, V866E,
I869M, A870G, A870V, R871G, H874Y, H874R, Q875K, T877S, T877A,
D879T, D879G, L880Q, L881V, M886V, S888L, V889M, F891L, P892L,
M895T, A896T, E897D, I898T, Q902R, V903M, P904S, P904H, L907F,
G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or a combination
thereof of the AR polypeptide.
Polynucleic Acid Molecules that Target B-Catenin and
B-Catenin-Associated Genes
[0248] Catenin beta-1 (also known as CTNNB1, .beta.-catenin, or
beta-catenin) is a member of the catenin protein family. In humans,
it is encoded by the CTNNB1 gene and is known for its dual
functions--cell-cell adhesion and gene transcription. Beta-catenin
is an integral structural component of cadherin-based adherens
junctions and regulates cell growth and adhesion between cells and
anchors the actin cytoskeleton. In some instance, beta-catenin is
responsible for transmitting the contact inhibition signal that
causes the cells to stop dividing once the epithelial sheet is
complete. Beta-catenin is also a key nuclear effector of the Wnt
signaling pathway. In some instances, imbalance in the structural
and signaling properties of beta-catenin results in diseases and
deregulated growth connected to malignancies such as cancer. For
example, overexpression of beta-catenin has been linked to cancers
such as gastric cancer (Suriano, et al., "Beta-catenin (CTNNB1)
gene amplification: a new mechanism of protein overexpression in
cancer," Genes Chromosomes Cancer 42(3): 238-246 (2005)). In some
cases, mutations in CTNNB1 gene have been linked to cancer
development (e.g., colon cancer, melanoma, hepatocellular
carcinoma, ovarian cancer, endometrial cancer, medulloblastoma
pilomatricomas, or prostrate cancer), and in some instances, has
been linked to metastatic progression. In additional cases,
mutations in the CTNNB1 gene cause beta-catenin to translocate to
the nucleus without any external stimulus and drive the
transcription of its target genes continuously. In some cases, the
potential of beta-catenin to change the previously epithelial
phenotype of affected cells into an invasive, mesenchyme-like type
contributes to metastasis formation.
[0249] In some embodiments, CTNNB1 gene is wild type CTNNB1 or
CTNNB1 comprising one or more mutations. In some instances, CTNNB1
is wild type CTNNB1. In some instances, CTNNB1 is CTNNB1 comprising
one or more mutations. In some instances, the polynucleic acid
molecule is a polynucleic acid molecule that hybridizes to a target
region of wild type CTNNB1. In some instances, the polynucleic acid
molecule is a polynucleic acid molecule that hybridizes to a target
region of CTNNB1 comprising a mutation (e.g., a substitution, a
deletion, or an addition).
[0250] In some embodiments, CTNNB1 DNA or RNA comprises one or more
mutations. In some embodiments, CTNNB1 DNA or RNA comprises one or
more mutations within one or more exons. In some instances, the one
or more exons comprise exon 3. In some instances, CTNNB1 DNA or RNA
comprises one or more mutations at codons 32, 33, 34, 37, 41, 45,
183, 245, 287 or a combination thereof. In some instances, CTNNB1
DNA or RNA comprises one or more mutations at positions
corresponding to amino acid residues 25, 31, 32, 33, 34, 35, 36,
37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354,
367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553,
555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724,
738, 777, or a combination thereof of the CTNNB1 polypeptide. In
some embodiments, CTNNB1 DNA or RNA comprises one or more mutations
at positions corresponding to amino acid residues selected from W25
(nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C,
S33Y, S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C,
S37A, T41N, T41A, T41I, S45Y, S45F, S45C, 1140T, D162E, K170M,
V199I, C213F, A215T, T257I, 1303M, Q322K, E334K, K354T, G367V,
P373S, W383G, N387K, L402F, N426D, R453L, R453Q, R474 (nonsense
mutation), R486C, R515Q, L517F, R535 (nonsense mutation), R535Q,
M553V, G555A, R582Q, R587Q, C619Y, Q623E, T641 (frame shift),
S646F, M688T, Q703H, R710H, D712N, P714R, Y724H, E738K, F777S, or a
combination thereof of the CTNNB1 polypeptide.
[0251] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of CTNNB1 DNA or RNA comprising one
or more mutations. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of CTNNB1 DNA or RNA
comprising one or more mutations within exon 3. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of CTNNB1 DNA or RNA comprising one or more mutations at
codons 32, 33, 34, 37, 41, 45, 183, 245, 287 or a combination
thereof. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of CTNNB1 DNA or RNA comprising one
or more mutations at positions corresponding to amino acid residues
25, 31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213,
215, 257, 303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453,
474, 486, 515, 517, 535, 553, 555, 582, 587, 619, 623, 641, 646,
688, 703, 710, 712, 714, 724, 738, 777, or a combination thereof of
the CTNNB1 polypeptide. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of CTNNB1 DNA or RNA
comprising one or more mutations selected from W25 (nonsense
mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F,
S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N,
T41A, T41I, S45Y, S45F, S45C, 1140T, D162E, K170M, V199I, C213F,
A215T, T257I, 1303M, Q322K, E334K, K354T, G367V, P373S, W383G,
N387K, L402F, N426D, R453L, R453Q, R474 (nonsense mutation), R486C,
R515Q, L517F, R535 (nonsense mutation), R535Q, M553V, G555A, R582Q,
R587Q, C619Y, Q623E, T641 (frame shift), S646F, M688T, Q703H,
R710H, D712N, P714R, Y724H, E738K, F777S, or a combination thereof
of the CTNNB1 polypeptide.
[0252] In some embodiments, beta-catenin associated genes further
comprise PIK3CA, PIK3CB, and MYC. In some embodiments, beta-catenin
associated genes further comprise PIK3CA DNA or RNA. PIK3CA
(phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit
alpha or p110.alpha. protein) is a class i PI 3-kinase catalytic
subunit that uses ATP to phosphorylate phosphatidylinositols. In
some embodiments, PIK3CA gene is wild type PIK3CA or PIK3CA
comprising one or more mutations. In some instances, PIK3CA DNA or
RNA is wild type PIK3CA. In some instances, PIK3CA DNA or RNA
comprises one or more mutations. In some instances, the polynucleic
acid molecule hybridizes to a target region of wild type PIK3CA DNA
or RNA. In some instances, the polynucleic acid molecule hybridizes
to a target region of PIK3CA DNA or RNA comprising a mutation
(e.g., a substitution, a deletion, or an addition).
[0253] In some embodiments, PIK3CA DNA or RNA comprises one or more
mutations. In some embodiments, PIK3CA DNA or RNA comprises one or
more mutation within one or more exons. In some instances, PIK3CA
DNA or RNA comprises one or more mutation within exons 9 and/or 20.
In some instances, PIK3CA DNA or RNA comprises one or more
mutations at positions corresponding to amino acid residues 1, 4,
10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79, 81, 83, 88, 90, 93,
102, 103, 103-104, 103-106, 104, 105-108, 106, 106-107, 106-108,
107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279,
320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366,
378, 398, 401, 417, 420, 447-455, 449, 449-457, 451, 453, 454, 455,
455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576,
604, 614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866,
901, 909, 939, 951, 958, 970, 971, 975, 992, 1004, 1007, 1016,
1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048,
1049, 1052, 1065, 1069, or a combination thereof of the PIK3CA
polypeptide. In some embodiments, PIK3CA DNA or RNA comprises one
or more mutations at positions corresponding to amino acid residues
selected from M1V, R4 (nonsense mutation), L10-M16 (deletion),
W11-P18 (deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G,
E65K, S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y,
C90G, R93Q, R93W, 1102 (deletion), E103G, E103-P104 (deletion),
E103-G106 (deletion), P104L, V105-R108 (deletion), G106V, G106-N107
(deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H,
E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111
(deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272
(nonsense mutation), L279I, G320V, W328S, R335G, T342S, V344G,
V344M, V344A, N345K, N345I, N345T, D350N, D350G, R357Q, G359R,
G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q,
E417K, C420R, C420G, P447-L455 (deletion), P449L, P449-N457
(deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455 (frame
shift insertion), L455-G460 (deletion), G463-N465 (deletion),
P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V,
E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D, Q546K,
Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H,
I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W,
C901F, F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P,
M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S,
D1029H, E1037K, M1040V, M1043V, M10431, N1044K, N1044Y, D1045V,
H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L,
1069W (nonstop mutation), or a combination thereof of the PIK3CA
polypeptide.
[0254] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of PIK3CA DNA or RNA comprising one
or more mutations. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of PIK3CA DNA or RNA
comprising one or more mutations within an exon. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of PIK3CA DNA or RNA comprising one or more mutations within
exon 9 or exon 20. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of PIK3CA DNA or RNA
comprising one or more mutations at positions corresponding to
amino acid residues 1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75,
79, 81, 83, 88, 90, 93, 102, 103, 103-104, 103-106, 104, 105-108,
106, 106-107, 106-108, 107, 108, 109-112, 110, 111, 113, 115, 137,
170, 258, 272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359,
363, 364, 365, 366, 378, 398, 401, 417, 420, 447-455, 449, 449-457,
451, 453, 454, 455, 455-460, 463-465, 471, 495, 522, 538, 539, 542,
545, 546, 547, 576, 604, 614, 617, 629, 643, 663, 682, 725, 726,
777, 791, 818, 866, 901, 909, 939, 951, 958, 970, 971, 975, 992,
1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044,
1045, 1047, 1048, 1049, 1052, 1065, 1069, or a combination thereof
of the PIK3CA polypeptide. In some embodiments, the polynucleic
acid molecule is a polynucleic acid molecule that hybridizes to a
target region of PIK3CA DNA or RNA comprising one or more mutations
at positions corresponding to amino acid residues selected from
M1V, R4 (nonsense mutation), L10-M16 (deletion), W11-P18
(deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G, E65K,
S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y, C90G,
R93Q, R93W, 1102 (deletion), E103G, E103-P104 (deletion), E103-G106
(deletion), P104L, V105-R108 (deletion), G106V, G106-N107
(deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H,
E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111
(deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272
(nonsense mutation), L279I, G320V, W328S, R335G, T342S, V344G,
V344M, V344A, N345K, N345I, N345T, D350N, D350G, R357Q, G359R,
G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q,
E417K, C420R, C420G, P447-L455 (deletion), P449L, P449-N457
(deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455 (frame
shift insertion), L455-G460 (deletion), G463-N465 (deletion),
P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V,
E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D, Q546K,
Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H,
I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W,
C901F, F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P,
M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S,
D1029H, E1037K, M1040V, M1043V, M10431, N1044K, N1044Y, D1045V,
H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L,
1069W (nonstop mutation), or a combination thereof of the PIK3CB
polypeptide.
[0255] In some embodiments, beta-catenin associated genes further
comprise PIK3CB. In some embodiments, PIK3CB gene is wild type or
comprises one or more mutations. In some instances, PIK3CB DNA or
RNA is wild type PIK3CB DNA or RNA. In some instances, PIK3CB DNA
or RNA comprises one or more mutations. In some instances, the
polynucleic acid molecule hybridizes to a target region of wild
type PIK3CB DNA or RNA. In some instances, the polynucleic acid
molecule hybridizes to a target region of PIK3CB DNA or RNA
comprising a mutation (e.g., a substitution, a deletion, or an
addition).
[0256] In some embodiments, PIK3CB DNA or RNA comprises one or more
mutations. In some embodiments, PIK3CB DNA or RNA comprises one or
more mutations within one or more exons. In some instances, PIK3CB
DNA or RNA comprises one or more mutations at positions
corresponding to amino acid residues 18, 19, 21, 28, 50, 61, 68,
103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442,
470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619,
628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036,
1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereof
of the PIK3CB polypeptide. In some embodiments, PIK3CB DNA or RNA
comprises one or more mutations at positions corresponding to amino
acid residues selected from W18 (nonsense mutation), A19V, D21H,
G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W,
K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K, E497D,
P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K, R562
(nonsense mutation), E567D, A593V, L595P, V619A, R628 (nonsense
mutation), R668W, L768F, K805E, D824E, A830T, E887 (nonsense
mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N, E1047K,
A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or a combination
thereof of the PIK3CB polypeptide.
[0257] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of PIK3CB DNA or RNA comprising one
or more mutations. In some embodiments, the polynucleic acid
molecule hybridizes to a target region of PIK3CB DNA or RNA
comprising one or more mutations within an exon. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of PIK3CB DNA or RNA comprising one or more mutations at
positions corresponding to amino acid residues 18, 19, 21, 28, 50,
61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417,
442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595,
619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036,
1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereof
of the PIK3CB polypeptide. In some embodiments, the polynucleic
acid molecule hybridizes to a target region of PIK3CB DNA or RNA
comprising one or more mutations at positions corresponding to
amino acid residues selected from W18 (nonsense mutation), A19V,
D21H, G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W,
R270W, K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K,
E497D, P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K,
R562 (nonsense mutation), E567D, A593V, L595P, V619A, R628
(nonsense mutation), R668W, L768F, K805E, D824E, A830T, E887
(nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N,
E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or a
combination thereof of the PIK3CB polypeptide.
[0258] In some embodiments, beta-catenin associated genes further
comprise MYC. In some embodiments, MYC gene is wild type MYC or MYC
comprising one or more mutations. In some instances, MYC is wild
type MYC DNA or RNA. In some instances, MYC DNA or RNA comprisesone
or more mutations. In some instances, the polynucleic acid molecule
hybridizes to a target region of wild type MYC DNA or RNA. In some
instances, the polynucleic acid molecule is a polynucleic acid
molecule that hybridizes to a target region of MYC DNA or RNA
comprising a mutation (e.g., a substitution, a deletion, or an
addition).
[0259] In some embodiments, MYC DNA or RNA comprises one or more
mutations. In some embodiments, MYC DNA or RNA comprises one or
more mutation within one or more exons. In some instances, MYC DNA
or RNA comprises one or more mutations within exon 2 or exon 3. In
some instances, MYC DNA or RNA comprises one or more mutations at
positions corresponding to amino acid residues 2, 7, 17, 20, 32,
44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202,
203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419,
431, 439, or a combination thereof. In some embodiments, MYC DNA or
RNA comprises one or more mutations at positions corresponding to
amino acid residues selected from P2L, F7L, D17N, Q20E, Y32N, A44V,
A44T, T58I, P59L, A76V, F115L, F138S, A141S, V145I, S146L, S169C,
S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E,
V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K,
R439Q, or a combination thereof of the MYC polypeptide.
[0260] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of MYC DNA or RNA comprising one or
more mutations. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of MYC DNA or RNA comprising one or
more mutations within an exon. In some embodiments, the polynucleic
acid molecule hybridizes to a target region of MYC DNA or RNA
comprising one or more mutations within exon 2 or exon 3. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of MYC DNA or RNA comprising one or more mutations at
positions corresponding to amino acid residues 2, 7, 17, 20, 32,
44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202,
203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419,
431, 439, or a combination thereof of the MYC polypeptide. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of MYC DNA or RNA comprising one or more mutations at
positions corresponding to amino acid residues selected from P2L,
F7L, D17N, Q20E, Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S,
A141S, V145I, S146L, S169C, S175N, C188F, N200S, S202N, S203T,
T248S, D251E, S298Y, Q321E, V340D, V369D, T373K, H374R, F389L,
Q395H, K404N, L419M, E431K, R439Q, or a combination thereof of the
MYC polypeptide.
Polynucleic Acid Molecules that Target Hypoxanthine
Phosphoribosyltransferase 1 (HPRT1)
[0261] Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a
transferase that catalyzes the conversion of hypoxanthine to
inosine monophosphate and guanine to guanosine monophosphate. HGPRT
is encoded by the hypoxanthine Phosphoribosyltransferase 1 (HPRT1)
gene.
[0262] In some embodiments, HPRT1 DNA or RNA is wild type or
comprises one or more mutations. In some instances, HPRT1 DNA or
RNA comprises one or more mutations within one or more exons. In
some instances, the one or more exons comprise exon 2, exon 3, exon
4, exon 6, exon 8, or exon 9. In some instances, HPRT1 DNA or RNA
comprises one or more mutations at positions corresponding to amino
acid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or
a combination thereof of the HPRT1 polypeptide. In some
embodiments, the polynucleic acid molecule hybridizes to a target
region of HPRT1 DNA or RNA comprising one or more mutations
selected from V35M, R48H, E56D, F74L, R87I, N129 (splice-site
mutation), N154H, 5162 (splice-site mutation), Y195C, Y195N, R200M,
E210K, or a combination thereof of the HPRT1 polypeptide.
[0263] In some embodiments, the polynucleic acid molecule
hybridizes to a target region of HPRT1 DNA or RNA comprising one or
more mutations. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of HPRT1 DNA or RNA comprising one or
more mutations within exon 2, exon 3, exon 4, exon 6, exon 8, or
exon 9. In some embodiments, the polynucleic acid molecule
hybridizes to a target region of HPRT1 DNA or RNA comprising one or
more mutations at positions corresponding to amino acid residues
35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or a combination
thereof of the HPRT1 polypeptide. In some embodiments, the
polynucleic acid molecule hybridizes to a target region of HPRT1
DNA or RNA comprising one or more mutations selected from V35M,
R48H, E56D, F74L, R87I, N129 (splice-site mutation), N154H, 5162
(splice-site mutation), Y195C, Y195N, R200M, E210K, or a
combination thereof of the HPRT1 polypeptide.
Polynucleic Acid Molecule Sequences
[0264] In some embodiments, the polynucleic acid molecule comprises
a sequence that hybridizes to a target sequence illustrated in
Tables 1, 4, 7, 8, or 10. In some instances, the polynucleic acid
molecule is B. In some instances, the polynucleic acid molecule B
comprises a sequence that hybridizes to a target sequence
illustrated in Table 1 (KRAS target sequences). In some instances,
the polynucleic acid molecule B comprises a sequence that
hybridizes to a target sequence illustrated in Table 4 (EGFR target
sequences). In some cases, the polynucleic acid molecule B
comprises a sequence that hybridizes to a target sequence
illustrated in Table 7 (AR target sequences). In some cases, the
polynucleic acid molecule B comprises a sequence that hybridizes to
a target sequence illustrated in Table 8 (.beta.-catenin target
sequences). In additional cases, the polynucleic acid molecule B
comprises a sequence that hybridizes to a target sequence
illustrated in Table 10 (PIK3CA and PIK3CB target sequences).
[0265] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 2 or Table 3. In some embodiments,
the polynucleic acid molecule comprises a sequence having at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 50% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 60% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 70% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 75% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 80% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 85% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 90% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 95% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 96% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 97% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 98% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 99% sequence identity to SEQ ID NOs: 16-75. In some
embodiments, the polynucleic acid molecule consists of SEQ ID NOs:
16-75.
[0266] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75.
In some cases, the second polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75.
In some cases, the polynucleic acid molecule comprises a first
polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ
ID NOs: 16-75 and a second polynucleotide having at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 16-75.
[0267] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 5 or Table 6. In some embodiments,
the polynucleic acid molecule comprises a sequence having at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% sequence identity to SEQ ID NOs: 452-1955. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 50% sequence identity to SEQ ID NOs: 452-1955. In
some embodiments, the polynucleic acid molecule comprises a
sequence having at least 60% sequence identity to SEQ ID NOs:
452-1955. In some embodiments, the polynucleic acid molecule
comprises a sequence having at least 70% sequence identity to SEQ
ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 75% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 80% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 85% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 90% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 95% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 96% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 97% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 98% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 99% sequence identity
to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid
molecule consists of SEQ ID NOs: 452-1955.
[0268] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
452-1955. In some cases, the second polynucleotide comprises a
sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 452-1955. In some cases, the polynucleic acid molecule
comprises a first polynucleotide having at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 452-1955 and a second
polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ
ID NOs: 452-1955.
[0269] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 7. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 1956-1962. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 50% sequence identity to SEQ ID NOs: 1956-1962. In
some embodiments, the polynucleic acid molecule comprises a
sequence having at least 60% sequence identity to SEQ ID NOs:
1956-1962. In some embodiments, the polynucleic acid molecule
comprises a sequence having at least 70% sequence identity to SEQ
ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 75% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 80% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 85% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 90% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 95% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 96% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 97% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 98% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 99% sequence identity
to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid
molecule consists of SEQ ID NOs: 1956-1962.
[0270] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
1956-1962. In some cases, the second polynucleotide comprises a
sequence that is complementary to a sequence having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 1956-1962. In some instances,
the polynucleic acid molecule comprises a first polynucleotide
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
1956-1962, and a second polynucleotide that is complementary to a
sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 1956-1962.
[0271] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 9. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 1967-2002. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 50% sequence identity to SEQ ID NOs: 1967-2002. In
some embodiments, the polynucleic acid molecule comprises a
sequence having at least 60% sequence identity to SEQ ID NOs:
1967-2002. In some embodiments, the polynucleic acid molecule
comprises a sequence having at least 70% sequence identity to SEQ
ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 75% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 80% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 85% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 90% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 95% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 96% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 97% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 98% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 99% sequence identity
to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid
molecule consists of SEQ ID NOs: 1967-2002.
[0272] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
1967-2002. In some cases, the second polynucleotide comprises a
sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 1967-2002. In some cases, the polynucleic acid molecule
comprises a first polynucleotide having at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 1967-2002 and a second
polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ
ID NOs: 1967-2002.
[0273] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 11. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 2013-2032. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 50% sequence identity to SEQ ID NOs: 2013-2032. In
some embodiments, the polynucleic acid molecule comprises a
sequence having at least 60% sequence identity to SEQ ID NOs:
2013-2032. In some embodiments, the polynucleic acid molecule
comprises a sequence having at least 70% sequence identity to SEQ
ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 75% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 80% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 85% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 90% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 95% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 96% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 97% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 98% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 99% sequence identity
to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid
molecule consists of SEQ ID NOs: 2013-2032.
[0274] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
2013-2032. In some cases, the second polynucleotide comprises a
sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 2013-2032. In some cases, the polynucleic acid molecule
comprises a first polynucleotide having at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 2013-2032 and a second
polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ
ID NOs: 2013-2032.
[0275] In some embodiments, the polynucleic acid molecule B
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to a sequence listed in Table 12.
[0276] In some embodiments, the polynucleic acid molecule comprises
a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 50% sequence identity
to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 60%
sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 70% sequence identity to SEQ ID NOs: 2082-2109 or
2117. In some embodiments, the polynucleic acid molecule comprises
a sequence having at least 75% sequence identity to SEQ ID NOs:
2082-2109 or 2117. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 80% sequence identity
to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 85%
sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 90% sequence identity to SEQ ID NOs: 2082-2109 or
2117. In some embodiments, the polynucleic acid molecule comprises
a sequence having at least 95% sequence identity to SEQ ID NOs:
2082-2109 or 2117. In some embodiments, the polynucleic acid
molecule comprises a sequence having at least 96% sequence identity
to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the
polynucleic acid molecule comprises a sequence having at least 97%
sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some
embodiments, the polynucleic acid molecule comprises a sequence
having at least 98% sequence identity to SEQ ID NOs: 2082-2109 or
2117. In some embodiments, the polynucleic acid molecule comprises
a sequence having at least 99% sequence identity to SEQ ID NOs:
2082-2109 or 2117. In some embodiments, the polynucleic acid
molecule consists of SEQ ID NOs: 2082-2109 or 2117.
[0277] In some embodiments, the polynucleic acid molecule B
comprises a first polynucleotide and a second polynucleotide. In
some instances, the first polynucleotide comprises a sequence
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
2082-2109 or 2117. In some cases, the second polynucleotide
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to SEQ ID NOs: 2082-2109 or 2117. In some cases, the polynucleic
acid molecule comprises a first polynucleotide having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 2082-2109 or 2117 and a
second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to SEQ ID NOs: 2082-2109 or 2117.
Polynucleic Acid Molecules
[0278] In some embodiments, the polynucleic acid molecule described
herein comprises RNA or DNA. In some cases, the polynucleic acid
molecule comprises RNA. In some instances, RNA comprises short
interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA
(miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA),
ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some
instances, RNA comprises shRNA. In some instances, RNA comprises
miRNA. In some instances, RNA comprises dsRNA. In some instances,
RNA comprises tRNA. In some instances, RNA comprises rRNA. In some
instances, RNA comprises hnRNA. In some instances, the RNA
comprises siRNA. In some instances, the polynucleic acid molecule
comprises siRNA. In some cases, B comprises siRNA.
[0279] In some embodiments, the polynucleic acid molecule is from
about 10 to about 50 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 10 to about 30, from about
15 to about 30, from about 18 to about 25, from about 18 to about
24, from about 19 to about 23, or from about 20 to about 22
nucleotides in length.
[0280] In some embodiments, the polynucleic acid molecule is about
50 nucleotides in length. In some instances, the polynucleic acid
molecule is about 45 nucleotides in length. In some instances, the
polynucleic acid molecule is about 40 nucleotides in length. In
some instances, the polynucleic acid molecule is about 35
nucleotides in length. In some instances, the polynucleic acid
molecule is about 30 nucleotides in length. In some instances, the
polynucleic acid molecule is about 25 nucleotides in length. In
some instances, the polynucleic acid molecule is about 20
nucleotides in length. In some instances, the polynucleic acid
molecule is about 19 nucleotides in length. In some instances, the
polynucleic acid molecule is about 18 nucleotides in length. In
some instances, the polynucleic acid molecule is about 17
nucleotides in length. In some instances, the polynucleic acid
molecule is about 16 nucleotides in length. In some instances, the
polynucleic acid molecule is about 15 nucleotides in length. In
some instances, the polynucleic acid molecule is about 14
nucleotides in length. In some instances, the polynucleic acid
molecule is about 13 nucleotides in length. In some instances, the
polynucleic acid molecule is about 12 nucleotides in length. In
some instances, the polynucleic acid molecule is about 11
nucleotides in length. In some instances, the polynucleic acid
molecule is about 10 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 10 to about 50 nucleotides
in length. In some instances, the polynucleic acid molecule is from
about 10 to about 45 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 10 to about 40 nucleotides
in length. In some instances, the polynucleic acid molecule is from
about 10 to about 35 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 10 to about 30 nucleotides
in length. In some instances, the polynucleic acid molecule is from
about 10 to about 25 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 10 to about 20 nucleotides
in length. In some instances, the polynucleic acid molecule is from
about 15 to about 25 nucleotides in length. In some instances, the
polynucleic acid molecule is from about 15 to about 30 nucleotides
in length. In some instances, the polynucleic acid molecule is from
about 12 to about 30 nucleotides in length.
[0281] In some embodiments, the polynucleic acid molecule comprises
a first polynucleotide. In some instances, the polynucleic acid
molecule comprises a second polynucleotide. In some instances, the
polynucleic acid molecule comprises a first polynucleotide and a
second polynucleotide. In some instances, the first polynucleotide
is a sense strand or passenger strand. In some instances, the
second polynucleotide is an antisense strand or guide strand.
[0282] In some embodiments, the polynucleic acid molecule is a
first polynucleotide. In some embodiments, the first polynucleotide
is from about 10 to about 50 nucleotides in length. In some
instances, the first polynucleotide is from about 10 to about 30,
from about 15 to about 30, from about 18 to about 25, from about 18
to about 24, from about 19 to about 23, or from about 20 to about
22 nucleotides in length.
[0283] In some instances, the first polynucleotide is about 50
nucleotides in length. In some instances, the first polynucleotide
is about 45 nucleotides in length. In some instances, the first
polynucleotide is about 40 nucleotides in length. In some
instances, the first polynucleotide is about 35 nucleotides in
length. In some instances, the first polynucleotide is about 30
nucleotides in length. In some instances, the first polynucleotide
is about 25 nucleotides in length. In some instances, the first
polynucleotide is about 20 nucleotides in length. In some
instances, the first polynucleotide is about 19 nucleotides in
length. In some instances, the first polynucleotide is about 18
nucleotides in length. In some instances, the first polynucleotide
is about 17 nucleotides in length. In some instances, the first
polynucleotide is about 16 nucleotides in length. In some
instances, the first polynucleotide is about 15 nucleotides in
length. In some instances, the first polynucleotide is about 14
nucleotides in length. In some instances, the first polynucleotide
is about 13 nucleotides in length. In some instances, the first
polynucleotide is about 12 nucleotides in length. In some
instances, the first polynucleotide is about 11 nucleotides in
length. In some instances, the first polynucleotide is about 10
nucleotides in length. In some instances, the first polynucleotide
is from about 10 to about 50 nucleotides in length. In some
instances, the first polynucleotide is from about 10 to about 45
nucleotides in length. In some instances, the first polynucleotide
is from about 10 to about 40 nucleotides in length. In some
instances, the first polynucleotide is from about 10 to about 35
nucleotides in length. In some instances, the first polynucleotide
is from about 10 to about 30 nucleotides in length. In some
instances, the first polynucleotide is from about 10 to about 25
nucleotides in length. In some instances, the first polynucleotide
is from about 10 to about 20 nucleotides in length. In some
instances, the first polynucleotide is from about 15 to about 25
nucleotides in length. In some instances, the first polynucleotide
is from about 15 to about 30 nucleotides in length. In some
instances, the first polynucleotide is from about 12 to about 30
nucleotides in length.
[0284] In some embodiments, the polynucleic acid molecule is a
second polynucleotide. In some embodiments, the second
polynucleotide is from about 10 to about 50 nucleotides in length.
In some instances, the second polynucleotide is from about 10 to
about 30, from about 15 to about 30, from about 18 to about 25,
from about 18 to about 24, from about 19 to about 23, or from about
20 to about 22 nucleotides in length.
[0285] In some instances, the second polynucleotide is about 50
nucleotides in length. In some instances, the second polynucleotide
is about 45 nucleotides in length. In some instances, the second
polynucleotide is about 40 nucleotides in length. In some
instances, the second polynucleotide is about 35 nucleotides in
length. In some instances, the second polynucleotide is about 30
nucleotides in length. In some instances, the second polynucleotide
is about 25 nucleotides in length. In some instances, the second
polynucleotide is about 20 nucleotides in length. In some
instances, the second polynucleotide is about 19 nucleotides in
length. In some instances, the second polynucleotide is about 18
nucleotides in length. In some instances, the second polynucleotide
is about 17 nucleotides in length. In some instances, the second
polynucleotide is about 16 nucleotides in length. In some
instances, the second polynucleotide is about 15 nucleotides in
length. In some instances, the second polynucleotide is about 14
nucleotides in length. In some instances, the second polynucleotide
is about 13 nucleotides in length. In some instances, the second
polynucleotide is about 12 nucleotides in length. In some
instances, the second polynucleotide is about 11 nucleotides in
length. In some instances, the second polynucleotide is about 10
nucleotides in length. In some instances, the second polynucleotide
is from about 10 to about 50 nucleotides in length. In some
instances, the second polynucleotide is from about 10 to about 45
nucleotides in length. In some instances, the second polynucleotide
is from about 10 to about 40 nucleotides in length. In some
instances, the second polynucleotide is from about 10 to about 35
nucleotides in length. In some instances, the second polynucleotide
is from about 10 to about 30 nucleotides in length. In some
instances, the second polynucleotide is from about 10 to about 25
nucleotides in length. In some instances, the second polynucleotide
is from about 10 to about 20 nucleotides in length. In some
instances, the second polynucleotide is from about 15 to about 25
nucleotides in length. In some instances, the second polynucleotide
is from about 15 to about 30 nucleotides in length. In some
instances, the second polynucleotide is from about 12 to about 30
nucleotides in length.
[0286] In some embodiments, the polynucleic acid molecule comprises
a first polynucleotide and a second polynucleotide. In some
instances, the polynucleic acid molecule further comprises a blunt
terminus, an overhang, or a combination thereof. In some instances,
the blunt terminus is a 5' blunt terminus, a 3' blunt terminus, or
both. In some cases, the overhang is a 5' overhang, 3' overhang, or
both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 non-base pairing nucleotides. In some cases, the overhang
comprises 1, 2, 3, 4, 5, or 6 non-base pairing nucleotides. In some
cases, the overhang comprises 1, 2, 3, or 4 non-base pairing
nucleotides. In some cases, the overhang comprises 1 non-base
pairing nucleotide. In some cases, the overhang comprises 2
non-base pairing nucleotides. In some cases, the overhang comprises
3 non-base pairing nucleotides. In some cases, the overhang
comprises 4 non-base pairing nucleotides.
[0287] In some embodiments, the sequence of the polynucleic acid
molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence
described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 50% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 60% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 70% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 80% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 90% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 95% complementary to a target
sequence described herein. In some embodiments, the sequence of the
polynucleic acid molecule is at least 99% complementary to a target
sequence described herein. In some instances, the sequence of the
polynucleic acid molecule is 100% complementary to a target
sequence described herein.
[0288] In some embodiments, the sequence of the polynucleic acid
molecule has 5 or less mismatches to a target sequence described
herein. In some embodiments, the sequence of the polynucleic acid
molecule has 4 or less mismatches to a target sequence described
herein. In some instances, the sequence of the polynucleic acid
molecule may has 3 or less mismatches to a target sequence
described herein. In some cases, the sequence of the polynucleic
acid molecule may has 2 or less mismatches to a target sequence
described herein. In some cases, the sequence of the polynucleic
acid molecule may has 1 or less mismatches to a target sequence
described herein.
[0289] In some embodiments, the specificity of the polynucleic acid
molecule that hybridizes to a target sequence described herein is a
95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the
polynucleic acid molecule to a target sequence. In some instances,
the hybridization is a high stringent hybridization condition.
[0290] In some embodiments, the polynucleic acid molecule
hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or more contiguous bases of a target sequence described
herein. In some embodiments, the polynucleic acid molecule
hybridizes to at least 8 contiguous bases of a target sequence
described herein. In some embodiments, the polynucleic acid
molecule hybridizes to at least 9 contiguous bases of a target
sequence described herein. In some embodiments, the polynucleic
acid molecule hybridizes to at least 10 contiguous bases of a
target sequence described herein. In some embodiments, the
polynucleic acid molecule hybridizes to at least 11 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 12 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 13 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 14 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 15 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 16 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 17 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 18 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 19 contiguous
bases of a target sequence described herein. In some embodiments,
the polynucleic acid molecule hybridizes to at least 20 contiguous
bases of a target sequence described herein.
[0291] In some embodiments, the polynucleic acid molecule has
reduced off-target effect. In some instances, "off-target" or
"off-target effects" refer to any instance in which a polynucleic
acid polymer directed against a given target causes an unintended
effect by interacting either directly or indirectly with another
mRNA sequence, a DNA sequence or a cellular protein or other
moiety. In some instances, an "off-target effect" occurs when there
is a simultaneous degradation of other transcripts due to partial
homology or complementarity between that other transcript and the
sense and/or antisense strand of the polynucleic acid molecule.
[0292] In some embodiments, the polynucleic acid molecule comprises
natural, synthetic, or artificial nucleotide analogues or bases. In
some cases, the polynucleic acid molecule comprises combinations of
DNA, RNA and/or nucleotide analogues. In some instances, the
synthetic or artificial nucleotide analogues or bases comprise
modifications at one or more of ribose moiety, phosphate moiety,
nucleoside moiety, or a combination thereof.
[0293] In some embodiments, a nucleotide analogue or artificial
nucleotide base described above comprises a nucleic acid with a
modification at a 2' hydroxyl group of the ribose moiety. In some
instances, the modification includes an H, OR, R, halo, SH, SR,
NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl
moiety includes, but is not limited to, halogens, sulfurs, thiols,
thioethers, thioesters, amines (primary, secondary, or tertiary),
amides, ethers, esters, alcohols and oxygen. In some instances, the
alkyl moiety further comprises a modification. In some instances,
the modification comprises an azo group, a keto group, an aldehyde
group, a carboxyl group, a nitro group, a nitroso, group, a nitrile
group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino)
group, an isocyanate or cyanate group, or a sulfur containing group
(e.g., sulfoxide, sulfone, sulfide, or disulfide). In some
instances, the alkyl moiety further comprises a hetero
substitution. In some instances, the carbon of the heterocyclic
group is substituted by a nitrogen, oxygen or sulfur. In some
instances, the heterocyclic substitution includes but is not
limited to, morpholino, imidazole, and pyrrolidino.
[0294] In some instances, the modification at the 2' hydroxyl group
is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE)
modification. In some cases, the 2'-O-methyl modification adds a
methyl group to the 2' hydroxyl group of the ribose moiety whereas
the 2'O-methoxyethyl modification adds a methoxyethyl group to the
2' hydroxyl group of the ribose moiety. Exemplary chemical
structures of a 2'-O-methyl modification of an adenosine molecule
and 2'O-methoxyethyl modification of an uridine are illustrated
below.
##STR00001##
[0295] In some instances, the modification at the 2' hydroxyl group
is a 2'-O-aminopropyl modification in which an extended amine group
comprising a propyl linker binds the amine group to the 2' oxygen.
In some instances, this modification neutralizes the
phosphate-derived overall negative charge of the oligonucleotide
molecule by introducing one positive charge from the amine group
per sugar and thereby improves cellular uptake properties due to
its zwitterionic properties. An exemplary chemical structure of a
2'-O-aminopropyl nucleoside phosphoramidite is illustrated
below.
##STR00002##
[0296] In some instances, the modification at the 2' hydroxyl group
is a locked or bridged ribose modification (e.g., locked nucleic
acid or LNA) in which the oxygen molecule bound at the 2' carbon is
linked to the 4' carbon by a methylene group, thus forming a
2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer.
Exemplary representations of the chemical structure of LNA are
illustrated below. The representation shown to the left highlights
the chemical connectivities of an LNA monomer. The representation
shown to the right highlights the locked 3'-endo (.sup.3E)
conformation of the furanose ring of an LNA monomer.
##STR00003##
[0297] In some instances, the modification at the 2' hydroxyl group
comprises ethylene nucleic acids (ENA) such as for example
2'-4'-ethylene-bridged nucleic acid, which locks the sugar
conformation into a C.sub.3'-endo sugar puckering conformation. ENA
are part of the bridged nucleic acids class of modified nucleic
acids that also comprises LNA. Exemplary chemical structures of the
ENA and bridged nucleic acids are illustrated below.
##STR00004##
[0298] In some embodiments, additional modifications at the 2'
hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro,
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA).
[0299] In some embodiments, a nucleotide analogue comprises a
modified base such as, but not limited to, 5-propynyluridine,
5-propynylcytidine, 6-methyladenine, 6-methylguanine,
N,N,-dimethyladenine, 2-propyladenine, 2propylguanine,
2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine,
5-methyluridine and other nucleotides having a modification at the
5 position, 5-(2-amino) propyl uridine, 5-halocytidine,
5-halouridine, 4-acetylcytidine, 1-methyladenosine,
2-methyladenosine, 3-methylcytidine, 6-methyluridine,
2-methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine,
5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
(such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or
6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as
2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine,
pseudouridine, queuosine, archaeosine, naphthyl and substituted
naphthyl groups, any O- and N-alkylated purines and pyrimidines
(such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine
5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and
modified phenyl groups such as aminophenol or 2,4,6-trimethoxy
benzene, modified cytosines that act as G-clamp nucleotides,
8-substituted adenines and guanines, 5-substituted uracils and
thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,
carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated
nucleotides. Modified nucleotides also include those nucleotides
that are modified with respect to the sugar moiety, as well as
nucleotides having sugars or analogs thereof that are not ribosyl.
For example, the sugar moieties, in some cases are or are based on,
mannoses, arabinoses, glucopyranoses, galactopyranoses,
4'-thioribose, and other sugars, heterocycles, or carbocycles. The
term nucleotide also includes what are known in the art as
universal bases. By way of example, universal bases include but are
not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.
[0300] In some embodiments, a nucleotide analogue further comprises
a morpholino, a peptide nucleic acid (PNA), a methylphosphonate
nucleotide, a thiolphosphonate nucleotide, a 2'-fluoro
N3-P5'-phosphoramidite, or a 1', 5'-anhydrohexitol nucleic acid
(HNA). Morpholino or phosphorodiamidate morpholino oligo (PMO)
comprises synthetic molecules whose structure mimics natural
nucleic acid structure but deviates from the normal sugar and
phosphate structures. In some instances, the five member ribose
ring is substituted with a six member morpholino ring containing
four carbons, one nitrogen, and one oxygen. In some cases, the
ribose monomers are linked by a phosphordiamidate group instead of
a phosphate group. In such cases, the backbone alterations remove
all positive and negative charges making morpholinos neutral
molecules capable of crossing cellular membranes without the aid of
cellular delivery agents such as those used by charged
oligonucleotides.
##STR00005##
[0301] In some embodiments, a morpholino or PMO described above is
a PMO comprising a positive or cationic charge. In some instances,
the PMO is PMOplus (Sarepta). PMOplus refers to phosphorodiamidate
morpholino oligomers comprising any number of
(1-piperazino)phosphinylideneoxy,
(1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy
linkages (e.g., as such those described in PCT Publication No.
WO2008/036127. In some cases, the PMO is a PMO described in U.S.
Pat. No. 7,943,762.
[0302] In some embodiments, a morpholino or PMO described above is
a PMO-X (Sarepta). In some cases, PMO-X refers to
phosphorodiamidate morpholino oligomers comprising at least one
linkage or at least one of the disclosed terminal modifications,
such as those disclosed in PCT Publication No. WO2011/150408 and
U.S. Publication No. 2012/0065169.
[0303] In some embodiments, a morpholino or PMO described above is
a PMO as described in Table 5 of U.S. Publication No.
2014/0296321.
[0304] In some embodiments, peptide nucleic acid (PNA) does not
contain sugar ring or phosphate linkage and the bases are attached
and appropriately spaced by oligoglycine-like molecules, therefore,
eliminating a backbone charge.
##STR00006##
[0305] In some embodiments, one or more modifications optionally
occur at the internucleotide linkage. In some instances, modified
internucleotide linkage includes, but is not limited to,
phosphorothioates; phosphorodithioates; methylphosphonates;
5'-alkylenephosphonates; 5'-methylphosphonate; 3'-alkylene
phosphonates; borontrifluoridates; borano phosphate esters and
selenophosphates of 3'-5'linkage or 2'-5'linkage; phosphotriesters;
thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl
phosphonates; alkylphosphonothioates; arylphosphonothioates;
phosphoroselenoates; phosphorodiselenoates; phosphinates;
phosphoramidates; 3'-alkylphosphoramidates;
aminoalkylphosphoramidates; thionophosphoramidates;
phosphoropiperazidates; phosphoroanilothioates;
phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates;
carbamates; methylenehydrazos; methylenedimethylhydrazos;
formacetals; thioformacetals; oximes; methyleneiminos;
methylenemethyliminos; thioamidates; linkages with riboacetyl
groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or
cycloalkyl linkages with or without heteroatoms of, for example, 1
to 10 carbons that are saturated or unsaturated and/or substituted
and/or contain heteroatoms; linkages with morpholino structures,
amides, or polyamides wherein the bases are attached to the aza
nitrogens of the backbone directly or indirectly; and combinations
thereof.
[0306] In some instances, the modification is a methyl or thiol
modification such as methylphosphonate or thiolphosphonate
modification. Exemplary thiolphosphonate nucleotide (left) and
methylphosphonate nucleotide (right) are illustrated below.
##STR00007##
[0307] In some instances, a modified nucleotide includes, but is
not limited to, 2'-fluoro N3-P5'-phosphoramidites illustrated
as:
##STR00008##
[0308] In some instances, a modified nucleotide includes, but is
not limited to, hexitol nucleic acid (or 1', 5'-anhydrohexitol
nucleic acids (HNA)) illustrated as:
##STR00009##
[0309] In some embodiments, one or more modifications comprise a
modified phosphate backbone in which the modification generates a
neutral or uncharged backbone. In some instances, the phosphate
backbone is modified by alkylation to generate an uncharged or
neutral phosphate backbone. As used herein, alkylation includes
methylation, ethylation, and propylation. In some cases, an alkyl
group, as used herein in the context of alkylation, refers to a
linear or branched saturated hydrocarbon group containing from 1 to
6 carbon atoms. In some instances, exemplary alkyl groups include,
but are not limited to, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,
neopentyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl,
3.3-dimethylbutyl, and 2-ethylbutyl groups. In some cases, a
modified phosphate is a phosphate group as described in U.S. Pat.
No. 9,481,905.
[0310] In some embodiments, additional modified phosphate backbones
comprise methylphosphonate, ethylphosphonate,
methylthiophosphonate, or methoxyphosphonate. In some cases, the
modified phosphate is methylphosphonate. In some cases, the
modified phosphate is ethylphosphonate. In some cases, the modified
phosphate is methylthiophosphonate. In some cases, the modified
phosphate is methoxyphosphonate.
[0311] In some embodiments, one or more modifications further
optionally include modifications of the ribose moiety, phosphate
backbone and the nucleoside, or modifications of the nucleotide
analogues at the 3' or the 5' terminus. For example, the 3'
terminus optionally include a 3' cationic group, or by inverting
the nucleoside at the 3'-terminus with a 3'-3' linkage. In another
alternative, the 3'-terminus is optionally conjugated with an
aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. In an additional
alternative, the 3'-terminus is optionally conjugated with an
abasic site, e.g., with an apurinic or apyrimidinic site. In some
instances, the 5'-terminus is conjugated with an aminoalkyl group,
e.g., a 5-O-alkylamino substituent. In some cases, the 5'-terminus
is conjugated with an abasic site. e.g., with an apurinic or
apyrimidinic site.
[0312] In some embodiments, the polynucleic acid molecule comprises
one or more of the artificial nucleotide analogues described
herein. In some instances, the polynucleic acid molecule comprises
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
25, or more of the artificial nucleotide analogues described
herein. In some embodiments, the artificial nucleotide analogues
include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA,
morpholino, methylphosphonate nucleotides, thiolphosphonate
nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination
thereof. In some instances, the polynucleic acid molecule comprises
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
25, or more of the artificial nucleotide analogues selected from
2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl,
2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA,
morpholino, methylphosphonate nucleotides, thiolphosphonate
nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination
thereof. In some instances, the polynucleic acid molecule comprises
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
25, or more of 2'-O-methyl modified nucleotides. In some instances,
the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of
2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some
instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of
thiolphosphonate nucleotides.
[0313] In some embodiments, the polynucleic acid molecule comprises
at least about 1, about 2, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, about 15, about 16, about 17, about 18, about 19, about
20, about 21, about 22, or more modifications. In some instances,
the polynucleic acid molecule is a polynucleic acid molecule of SEQ
ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032,
2082-2109, or 2117.
[0314] In some instances, the polynucleic acid molecule comprises
at least about 1, about 2, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, about 15, about 16, about 17, about 18, about 19, about
20, about 21, about 22, or more modified nucleotides. In some
instances, the polynucleic acid molecule is a polynucleic acid
molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,
2013-2032, 2082-2109, or 2117.
[0315] In some instances, the polynucleic acid molecule comprises
at least one of: from about 5% to about 100% modification, from
about 10% to about 100% modification, from about 20% to about 100%
modification, from about 30% to about 100% modification, from about
40% to about 100% modification, from about 50% to about 100%
modification, from about 60% to about 100% modification, from about
70% to about 100% modification, from about 80% to about 100%
modification, and from about 90% to about 100% modification. In
some instances, the polynucleic acid molecule is a polynucleic acid
molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,
2013-2032, 2082-2109, or 2117.
[0316] In some instances, about 5 to about 100% of the polynucleic
acid molecule comprise the artificial nucleotide analogues
described herein. In some instances, about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% of the polynucleic acid molecule comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955,
1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 16-45 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 452-1203 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 1956-1962 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 1967-2002 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 2013-2032 comprise the
artificial nucleotide analogues described herein. In some
instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a
polynucleic acid molecule of SEQ ID NOs: 2082-2109 or 2117 comprise
the artificial nucleotide analogues described herein. In some
embodiments, the artificial nucleotide analogues include
2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl,
2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA,
morpholino, methylphosphonate nucleotides, thiolphosphonate
nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination
thereof.
[0317] In some instances, the polynucleic acid molecule that
comprises an artificial nucleotide analogue comprises SEQ ID NOs:
46-75. In some instances, the polynucleic acid molecule that
comprises an artificial nucleotide analogue comprises SEQ ID NOs:
1204-1955. In some instances, the polynucleic acid molecule that
comprises an artificial nucleotide analogue comprises SEQ ID NOs:
1967-2002. In some instances, the polynucleic acid molecule that
comprises an artificial nucleotide analogue comprises SEQ ID NOs:
2013-2032. In some instances, the polynucleic acid molecule that
comprises an artificial nucleotide analogue comprises SEQ ID NOs:
2082-2109 or 2117.
[0318] In some cases, one or more of the artificial nucleotide
analogues described herein are resistant toward nucleases such as
for example ribonuclease such as RNase H, deoxyribunuclease such as
DNase, or exonuclease such as 5'-3' exonuclease and 3'-5'
exonuclease when compared to natural polynucleic acid molecules. In
some instances, artificial nucleotide analogues comprising
2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl,
2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA,
morpholino, methylphosphonate nucleotides, thiolphosphonate
nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations
thereof are resistant toward nucleases such as for example
ribonuclease such as RNase H, deoxyribunuclease such as DNase, or
exonuclease such as 5'-3' exonuclease and 3'-5' exonuclease. In
some instances, 2'-O-methyl modified polynucleic acid molecule is
nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or
3'-5' exonuclease resistant). In some instances, 2'O-methoxyethyl
(2'-O-MOE) modified polynucleic acid molecule is nuclease resistant
(e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some instances, 2'-O-aminopropyl modified
polynucleic acid molecule is nuclease resistant (e.g., RNase H,
DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some
instances, 2'-deoxy modified polynucleic acid molecule is nuclease
resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5'
exonuclease resistant). In some instances, T-deoxy-2'-fluoro
modified polynucleic acid molecule is nuclease resistant (e.g.,
RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).
In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic
acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid
molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid
molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic
acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
2'-O-N-methylacetamido (2'-O-NMA) modified polynucleic acid
molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
LNA-modified polynucleic acid molecule is nuclease resistant (e.g.,
RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).
In some instances, ENA-modified polynucleic acid molecule is
nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or
3'-5' exonuclease resistant). In some instances, HNA-modified
polynucleic acid molecule is nuclease resistant (e.g., RNase H,
DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).
Morpholinos may be nuclease resistant (e.g., RNase H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances,
PNA-modified polynucleic acid molecule is resistant to nucleases
(e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some instances, methylphosphonate
nucleotide-modified polynucleic acid molecule is nuclease resistant
(e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some instances, thiolphosphonate nucleotide-modified
polynucleic acid molecule is nuclease resistant (e.g., RNase H,
DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some
instances, polynucleic acid molecule comprising 2'-fluoro
N3-P5'-phosphoramidites is nuclease resistant (e.g., RNase H,
DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some
instances, the 5' conjugates described herein inhibit 5'-3'
exonucleolytic cleavage. In some instances, the 3' conjugates
described herein inhibit 3'-5' exonucleolytic cleavage.
[0319] In some embodiments, one or more of the artificial
nucleotide analogues described herein have increased binding
affinity toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. The one or more of the artificial
nucleotide analogues comprising 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro,
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA,
morpholino, methylphosphonate nucleotides, thiolphosphonate
nucleotides, or 2'-fluoro N3-P5'-phosphoramidites can have
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
2'-O-methyl modified polynucleic acid molecule has increased
binding affinity toward their mRNA target relative to an equivalent
natural polynucleic acid molecule. In some instances,
2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecule has
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
2'-O-aminopropyl modified polynucleic acid molecule has increased
binding affinity toward their mRNA target relative to an equivalent
natural polynucleic acid molecule. In some instances, 2'-deoxy
modified polynucleic acid molecule has increased binding affinity
toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. In some instances, T-deoxy-2'-fluoro
modified polynucleic acid molecule has increased binding affinity
toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. In some instances, 2'-O-aminopropyl
(2'-O-AP) modified polynucleic acid molecule has increased binding
affinity toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. In some instances,
2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid
molecule has increased binding affinity toward their mRNA target
relative to an equivalent natural polynucleic acid molecule. In
some instances, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified
polynucleic acid molecule has increased binding affinity toward
their mRNA target relative to an equivalent natural polynucleic
acid molecule. In some instances, T-O-dimethylaminoethyloxyethyl
(2'-O-DMAEOE) modified polynucleic acid molecule has increased
binding affinity toward their mRNA target relative to an equivalent
natural polynucleic acid molecule. In some instances,
2'-O-N-methylacetamido (2'-O-NMA) modified polynucleic acid
molecule has increased binding affinity toward their mRNA target
relative to an equivalent natural polynucleic acid molecule. In
some instances, LNA-modified polynucleic acid molecule has
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
ENA-modified polynucleic acid molecule has increased binding
affinity toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. In some instances, PNA-modified
polynucleic acid molecule has increased binding affinity toward
their mRNA target relative to an equivalent natural polynucleic
acid molecule. In some instances, HNA-modified polynucleic acid
molecule has increased binding affinity toward their mRNA target
relative to an equivalent natural polynucleic acid molecule. In
some instances, morpholino-modified polynucleic acid molecule has
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
methylphosphonate nucleotide-modified polynucleic acid molecule has
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
thiolphosphonate nucleotide-modified polynucleic acid molecule has
increased binding affinity toward their mRNA target relative to an
equivalent natural polynucleic acid molecule. In some instances,
polynucleic acid molecule comprising 2'-fluoro
N3-P5'-phosphoramidites has increased binding affinity toward their
mRNA target relative to an equivalent natural polynucleic acid
molecule. In some cases, the increased affinity is illustrated with
a lower Kd, a higher melt temperature (Tm), or a combination
thereof.
[0320] In some embodiments, a polynucleic acid molecule described
herein is a chirally pure (or stereo pure) polynucleic acid
molecule, or a polynucleic acid molecule comprising a single
enantiomer. In some instances, the polynucleic acid molecule
comprises L-nucleotide. In some instances, the polynucleic acid
molecule comprises D-nucleotides. In some instance, a polynucleic
acid molecule composition comprises less than 30%, 25%, 20%, 15%,
10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some
cases, a polynucleic acid molecule composition comprises less than
30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic
mixture. In some instances, the polynucleic acid molecule is a
polynucleic acid molecule described in: U.S. Patent Publication
Nos: 2014/194610 and 2015/211006; and PCT Publication No.:
WO2015107425.
[0321] In some embodiments, a polynucleic acid molecule described
herein is further modified to include an aptamer-conjugating
moiety. In some instances, the aptamer conjugating moiety is a DNA
aptamer-conjugating moiety. In some instances, the
aptamer-conjugating moiety is Alphamer (Centauri Therapeutics),
which comprises an aptamer portion that recognizes a specific
cell-surface target and a portion that presents a specific epitopes
for attaching to circulating antibodies. In some instance, a
polynucleic acid molecule described herein is further modified to
include an aptamer-conjugating moiety as described in: U.S. Pat.
Nos. 8,604,184, 8,591,910, and 7,850,975.
[0322] In additional embodiments, a polynucleic acid molecule
described herein is modified to increase its stability. In some
embodiment, the polynucleic acid molecule is RNA (e.g., siRNA), the
polynucleic acid molecule is modified to increase its stability. In
some instances, the polynucleic acid molecule is modified by one or
more of the modifications described above to increase its
stability. In some cases, the polynucleic acid molecule is modified
at the 2' hydroxyl position, such as by 2'-O-methyl,
2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy,
T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modification or by a locked or
bridged ribose conformation (e.g., LNA or ENA). In some cases, the
polynucleic acid molecule is modified by 2'-O-methyl and/or
2'-O-methoxyethyl ribose. In some cases, the polynucleic acid
molecule also includes morpholinos, PNAs, HNA, methylphosphonate
nucleotides, thiolphosphonate nucleotides, and/or 2'-fluoro
N3-P5'-phosphoramidites to increase its stability. In some
instances, the polynucleic acid molecule is a chirally pure (or
stereo pure) polynucleic acid molecule. In some instances, the
chirally pure (or stereo pure) polynucleic acid molecule is
modified to increase its stability. Suitable modifications to the
RNA to increase stability for delivery will be apparent to the
skilled person.
[0323] In some embodiments, a polynucleic acid molecule described
herein has RNAi activity that modulates expression of RNA encoded
by a gene described supra. In some instances, a polynucleic acid
molecule described herein is a double-stranded siRNA molecule that
down-regulates expression of a gene, wherein one of the strands of
the double-stranded siRNA molecule comprises a nucleotide sequence
that is complementary to a nucleotide sequence of the gene or RNA
encoded by the gene or a portion thereof, and wherein the second
strand of the double-stranded siRNA molecule comprises a nucleotide
sequence substantially similar to the nucleotide sequence of the
gene or RNA encoded by the gene or a portion thereof. In some
cases, a polynucleic acid molecule described herein is a
double-stranded siRNA molecule that down-regulates expression of a
gene, wherein each strand of the siRNA molecule comprises about 15
to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each
strand comprises at least about 14, 17, or 19 nucleotides that are
complementary to the nucleotides of the other strand. In some
cases, a polynucleic acid molecule described herein is a
double-stranded siRNA molecule that down-regulates expression of a
gene, wherein each strand of the siRNA molecule comprises about 19
to about 23 nucleotides, and wherein each strand comprises at least
about 19 nucleotides that are complementary to the nucleotides of
the other strand. In some instances, the gene is KRAS, EGFR, AR,
HPRT1, CNNTB1 (.beta.-catenin), or .beta.-catenin associated
genes.
[0324] In some embodiments, a polynucleic acid molecule described
herein is constructed using chemical synthesis and/or enzymatic
ligation reactions using procedures known in the art. For example,
a polynucleic acid molecule is chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
polynucleic acid molecule and target nucleic acids. Exemplary
methods include those described in: U.S. Pat. Nos. 5,142,047;
5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No.
WO2009099942; or European Publication No. 1579015. Additional
exemplary methods include those described in: Griffey et al.,
"2'-O-aminopropyl ribonucleotides: a zwitterionic modification that
enhances the exonuclease resistance and biological activity of
antisense oligonucleotides," J. Med. Chem. 39(26):5100-5109
(1997)); Obika, et al. "Synthesis of 2'-O,4'-C-methyleneuridine and
-cytidine. Novel bicyclic nucleosides having a fixed C3, -endo
sugar puckering". Tetrahedron Letters 38 (50): 8735 (1997);
Koizumi, M. "ENA oligonucleotides as therapeutics". Current opinion
in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et
al., "Novel oligonucleotide analogues based on morpholino
nucleoside subunits-antisense technologies: new chemical
possibilities," Indian Journal of Chemistry 48B:1721-1726 (2009).
Alternatively, the polynucleic acid molecule is produced
biologically using an expression vector into which a polynucleic
acid molecule has been subcloned in an antisense orientation (i.e
RNA transcribed from the inserted polynucleic acid molecule will be
of an antisense orientation to a target polynucleic acid molecule
of interest).
Conjugation Chemistry
[0325] In some embodiments, a polynucleic acid molecule is
conjugated to a binding moiety. In some instances, the binding
moiety comprises amino acids, peptides, polypeptides, proteins,
antibodies, antigens, toxins, hormones, lipids, nucleotides,
nucleosides, sugars, carbohydrates, polymers such as polyethylene
glycol and polypropylene glycol, as well as analogs or derivatives
of all of these classes of substances. Additional examples of
binding moiety also include steroids, such as cholesterol,
phospholipids, di- and triacylglycerols, fatty acids, hydrocarbons
(e.g., saturated, unsaturated, or contains substitutions), enzyme
substrates, biotin, digoxigenin, and polysaccharides. In some
instances, the binding moiety is an antibody or binding fragment
thereof. In some instances, the polynucleic acid molecule is
further conjugated to a polymer, and optionally an endosomolytic
moiety.
[0326] In some embodiments, the polynucleic acid molecule is
conjugated to the binding moiety by a chemical ligation process. In
some instances, the polynucleic acid molecule is conjugated to the
binding moiety by a native ligation. In some instances, the
conjugation is as described in: Dawson, et al. "Synthesis of
proteins by native chemical ligation," Science 1994, 266, 776-779;
Dawson, et al. "Modulation of Reactivity in Native Chemical
Ligation through the Use of Thiol Additives," J. Am. Chem. Soc.
1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native
chemical ligation: Expanded scope by using straightforward
methodology," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or
Wu, et al. "Building complex glycopeptides: Development of a
cysteine-free native chemical ligation protocol," Angew. Chem. Int.
Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as
described in U.S. Pat. No. 8,936,910. In some embodiments, the
polynucleic acid molecule is conjugated to the binding moiety
either site-specifically or non-specifically via native ligation
chemistry.
[0327] In some instances, the polynucleic acid molecule is
conjugated to the binding moiety by a site-directed method
utilizing a "traceless" coupling technology (Philochem). In some
instances, the "traceless" coupling technology utilizes an
N-terminal 1,2-aminothiol group on the binding moiety which is then
conjugate with a polynucleic acid molecule containing an aldehyde
group. (see Casi et al., "Site-specific traceless coupling of
potent cytotoxic drugs to recombinant antibodies for
pharmacodelivery," JACS 134(13): 5887-5892 (2012))
[0328] In some instances, the polynucleic acid molecule is
conjugated to the binding moiety by a site-directed method
utilizing an unnatural amino acid incorporated into the binding
moiety. In some instances, the unnatural amino acid comprises
p-acetylphenylalanine (pAcPhe). In some instances, the keto group
of pAcPhe is selectively coupled to an alkoxy-amine derivatived
conjugating moiety to form an oxime bond. (see Axup et al.,
"Synthesis of site-specific antibody-drug conjugates using
unnatural amino acids," PNAS 109(40): 16101-16106 (2012)).
[0329] In some instances, the polynucleic acid molecule is
conjugated to the binding moiety by a site-directed method
utilizing an enzyme-catalyzed process. In some instances, the
site-directed method utilizes SMARTag.TM. technology (Redwood). In
some instances, the SMARTag.TM. technology comprises generation of
a formylglycine (FGly) residue from cysteine by
formylglycine-generating enzyme (FGE) through an oxidation process
under the presence of an aldehyde tag and the subsequent
conjugation of FGly to an alkylhydraine-functionalized polynucleic
acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (see
Wu et al., "Site-specific chemical modification of recombinant
proteins produced in mammalian cells by using the genetically
encoded aldehyde tag," PNAS 106(9): 3000-3005 (2009); Agarwal, et
al., "A Pictet-Spengler ligation for protein chemical
modification," PNAS 110(1): 46-51 (2013))
[0330] In some instances, the enzyme-catalyzed process comprises
microbial transglutaminase (mTG). In some cases, the polynucleic
acid molecule is conjugated to the binding moiety utilizing a
microbial transglutaminze catalyzed process. In some instances, mTG
catalyzes the formation of a covalent bond between the amide side
chain of a glutamine within the recognition sequence and a primary
amine of a functionalized polynucleic acid molecule. In some
instances, mTG is produced from Streptomyces mobarensis. (see Strop
et al., "Location matters: site of conjugation modulates stability
and pharmacokinetics of antibody drug conjugates," Chemistry and
Biology 20(2) 161-167 (2013))
[0331] In some instances, the polynucleic acid molecule is
conjugated to the binding moiety by a method as described in PCT
Publication No. WO2014/140317, which utilizes a sequence-specific
transpeptidase.
[0332] In some instances, the polynucleic acid molecule is
conjugated to the binding moiety by a method as described in U.S.
Patent Publication Nos. 2015/0105539 and 2015/0105540.
Binding Moiety
[0333] In some embodiments, the binding moiety A is a polypeptide.
In some instances, the polypeptide is an antibody or its fragment
thereof. In some cases, the fragment is a binding fragment. In some
instances, the antibody or binding fragment thereof comprises a
humanized antibody or binding fragment thereof, murine antibody or
binding fragment thereof, chimeric antibody or binding fragment
thereof, monoclonal antibody or binding fragment thereof,
monovalent Fab', divalent Fab.sub.2, F(ab)'.sub.3 fragments,
single-chain variable fragment (scFv), bis-scFv, (scFv).sub.2,
diabody, minibody, nanobody, triabody, tetrabody, disulfide
stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig
NAR, camelid antibody or binding fragment thereof, bispecific
antibody or biding fragment thereof, or a chemically modified
derivative thereof.
[0334] In some instances, A is an antibody or binding fragment
thereof. In some instances, A is a humanized antibody or binding
fragment thereof, murine antibody or binding fragment thereof,
chimeric antibody or binding fragment thereof, monoclonal antibody
or binding fragment thereof, monovalent Fab', divalent Fab.sub.2,
F(ab)'.sub.3 fragments, single-chain variable fragment (scFv),
bis-scFv, (scFv).sub.2, diabody, minibody, nanobody, triabody,
tetrabody, disulfide stabilized Fv protein ("dsFv"), single-domain
antibody (sdAb), Ig NAR, camelid antibody or binding fragment
thereof, bispecific antibody or biding fragment thereof, or a
chemically modified derivative thereof. In some instances, A is a
humanized antibody or binding fragment thereof. In some instances,
A is a murine antibody or binding fragment thereof. In some
instances, A is a chimeric antibody or binding fragment thereof. In
some instances, A is a monoclonal antibody or binding fragment
thereof. In some instances, A is a monovalent Fab'. In some
instances, A is a diavalent Fab.sub.2. In some instances, A is a
single-chain variable fragment (scFv).
[0335] In some embodiments, the binding moiety A is a bispecific
antibody or binding fragment thereof. In some instances, the
bispecific antibody is a trifunctional antibody or a bispecific
mini-antibody. In some cases, the bispecific antibody is a
trifunctional antibody. In some instances, the trifunctional
antibody is a full length monoclonal antibody comprising binding
sites for two different antigens. Exemplary trifunctional
antibodies include catumaxomab (which targets EpCAM and CD3;
Fresenius Biotech/Trion Pharma), ertumaxomab (targets HER2/neu/CD3;
Fresenius Biotech/Trion Pharma), lymphomun FBTA05 (targets
CD20/CD3; Fresenius Biotech/Trion Pharma), RG7221 (R05520985;
targets Angiopoietin 2/VEGF; Roche), RG7597 (targets Her1/Her3;
Genentech/Roche), MM141 (targets IGF1R/Her3; Merrimack), ABT122
(targets TNF.alpha./IL17; Abbvie), ABT981 (targets
IL1.alpha./IL1.beta.; Abbott), LY3164530 (targets Her1/cMET; Eli
Lilly), and TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh).
Additional exemplary trifunctional antibodies include mAb.sup.2
from F-star Biotechnology Ltd. In some instances, A is a bispecific
trifunctional antibody. In some embodiments, A is a bispecific
trifunctional antibody selected from: catumaxomab (which targets
EpCAM and CD3; Fresenius Biotech/Trion Pharma), ertumaxomab
(targets HER2/neu/CD3; Fresenius Biotech/Trion Pharma), lymphomun
FBTA05 (targets CD20/CD3; Fresenius Biotech/Trion Pharma), RG7221
(R05520985; targets Angiopoietin 2/VEGF; Roche), RG7597 (targets
Her1l/Her3; Genentech/Roche), MM141 (targets IGF1R/Her3;
Merrimack), ABT122 (targets TNF.alpha./IL17; Abbvie), ABT981
(targets ILla/IL1(3; Abbott), LY3164530 (targets Her1l/cMET; Eli
Lilly), TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh), and
a mAb.sup.2 from F-star Biotechnology Ltd.
[0336] In some cases, the bispecific antibody is a bispecific
mini-antibody. In some instances, the bispecific mini-antibody
comprises divalent Fab.sub.2, F(ab)'.sub.3 fragments, bis-scFv,
(scFv).sub.2, diabody, minibody, triabody, tetrabody or a
bi-specific T-cell engager (BiTE). In some embodiments, the
bi-specific T-cell engager is a fusion protein that contains two
single-chain variable fragments (scFvs) in which the two scFvs
target epitopes of two different antigens. Exemplary bispecific
mini-antibodies include, but are not limited to, DART
(dual-affinity re-targeting platform; MacroGenics), blinatumomab
(MT103 or AMG103; which targets CD19/CD3; Micromet), MT111 (targets
CEA/CD3; Micromet/Amegen), MT112 (BAY2010112; targets PSMA/CD3;
Micromet/Bayer), MT110 (AMG 110; targets EPCAM/CD3;
Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics), MGD007
(targets GPA33/CD3; MacroGenics), BI1034020 (targets two different
epitopes on .beta.-amyloid; Ablynx), ALX0761 (targets IL17A/IL17F;
Ablynx), TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb
(BMS), AFM13 (targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3;
Affimed), and domain antibodies (dAbs from Domantis/GSK).
[0337] In some embodiments, the binding moiety A is a bispecific
mini-antibody. In some instances, A is a bispecific Fab.sub.2. In
some instances, A is a bispecific F(ab)'.sub.3 fragment. In some
cases, A is a bispecific bis-scFv. In some cases, A is a bispecific
(scFv).sub.2. In some embodiments, A is a bispecific diabody. In
some embodiments, A is a bispecific minibody. In some embodiments,
A is a bispecific triabody. In other embodiments, A is a bispecific
tetrabody. In other embodiments, A is a bi-specific T-cell engager
(BiTE). In additional embodiments, A is a bispecific mini-antibody
selected from: DART (dual-affinity re-targeting platform;
MacroGenics), blinatumomab (MT103 or AMG103; which targets
CD19/CD3; Micromet), MT111 (targets CEA/CD3; Micromet/Amegen),
MT112 (BAY2010112; targets PSMA/CD3; Micromet/Bayer), MT110 (AMG
110; targets EPCAM/CD3; Amgen/Micromet), MGD006 (targets CD123/CD3;
MacroGenics), MGD007 (targets GPA33/CD3; MacroGenics), BI1034020
(targets two different epitopes on .beta.-amyloid; Ablynx), ALX0761
(targets IL17A/IL17F; Ablynx), TF2 (targets CEA/hepten;
Immunomedics), IL-17/IL-34 biAb (BMS), AFM13 (targets CD30/CD16;
Affimed), AFM11 (targets CD19/CD3; Affimed), and domain antibodies
(dAbs from Domantis/GSK).
[0338] In some embodiments, the binding moiety A is a trispecific
antibody. In some instances, the trispecific antibody comprises
F(ab)'.sub.3 fragments or a triabody. In some instances, A is a
trispecific F(ab)'.sub.3 fragment. In some cases, A is a triabody.
In some embodiments, A is a trispecific antibody as described in
Dimas, et al., "Development of a trispecific antibody designed to
simultaneously and efficiently target three different antigens on
tumor cells," Mol. Pharmaceutics, 12(9): 3490-3501 (2015).
[0339] In some embodiments, the binding moiety A is an antibody or
binding fragment thereof that recognizes a cell surface protein. In
some instances, the cell surface protein is an antigen expressed by
a cancerous cell. Exemplary cancer antigens include, but are not
limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican,
CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA),
CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1,
SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth
factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB
(Beta subunit of MHC class II molecule (Ia antigen), human
chorionic gonadotropin, ICOS, IL-2 receptor, IL20R.alpha.,
Immunoglobulin superfamily receptor translocation associated 2
(IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4,
MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage
inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b
(SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline
phosphatase, prostate specific membrane antigen (PMSA), prostatic
acid phosphatase, PSCA hlg, p97, Purinergic receptor P2.times.
ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64
(RP105), gp100, P21, six transmembrane epithelial antigen of
prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein
72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4) and the
like.
[0340] In some instances, the cell surface protein comprises
clusters of differentiation (CD) cell surface markers. Exemplary CD
cell surface markers include, but are not limited to, CD1, CD2,
CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c,
CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19,
CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30,
CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41,
CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48,
CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53,
CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L
(L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d,
CD66e, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104,
CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221,
CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the
like.
[0341] In some instances, the binding moiety A is an antibody or
binding fragment thereof that recognizes a cancer antigen. In some
instances, the binding moiety A is an antibody or binding fragment
thereof that recognizes alpha fetoprotein, ASLG659, B7-H3, BAFF-R,
Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen
(CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1,
SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth
factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB
(Beta subunit of MHC class II molecule (Ia antigen), human
chorionic gonadotropin, ICOS, IL-2 receptor, IL20R.alpha.,
Immunoglobulin superfamily receptor translocation associated 2
(IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4,
MART1, mesothelin, MCP1 (CCL2), MDP, macrophage inhibitory factor
(MIF), MPF (SMR, MSLN), MPG, MSG783, mucin, MUC1-KLH, Napi3b
(SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline
phosphatase, prostate specific membrane antigen (PMSA), prostatic
acid phosphatase, PSCA hlg, p97, Purinergic receptor P2.times.
ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64
(RP105), gp100, P21, six transmembrane epithelial antigen of
prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein
72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4) or a
combination thereof.
[0342] In some instances, the binding moiety A is an antibody or
binding fragment thereof that recognizes a CD cell surface marker.
In some instances, the binding moiety A is an antibody or binding
fragment thereof that recognizes CD1, CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14,
CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,
CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35,
CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO,
CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d,
CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58,
CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64,
CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD79 (e.g., CD79a, CD79b),
CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137
(4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319
(SLAMF7), CD326 (EpCAM), or a combination thereof.
[0343] In some embodiments, the antibody or binding fragment
thereof comprises zalutumumab (HuMax-EFGr, Genmab), abagovomab
(Menarini), abituzumab (Merck), adecatumumab (MT201), alacizumab
pegol, alemtuzumab (Campath.RTM., MabCampath, or Campath-1H;
Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek, Inc.),
anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10),
ascrinvacumab (Pfizer Inc.), atezolizumab (MPDL3280A;
Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab
(Simulect.RTM., Novartis), belimumab (Benlysta.RTM.,
GlaxoSmithKline), bevacizumab (Avastin.RTM., Genentech),
blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems
Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion
Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux.RTM., ImClone),
citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12, ImClone
Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40,
huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex.RTM.,
Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab
(MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A,
Panorex, Glaxo Wellcome), elotuzumab (Empliciti.TM., Bristol-Myers
Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech
Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab
(MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.),
epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab
(Rexomun.RTM., Trion Pharma), etaracizumab (Abegrin, MedImmune),
farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion
Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab
(CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab
(GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen),
girentuximab (Rencarex.RTM., Wilex AG), IMAB362 (Claudiximab,
Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone
Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab
(Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab
(Yervoy.RTM., Bristol-Myers Squibb), iratumumab (Medarex, Inc.),
isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE,
Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody
Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab
(Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome
Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1,
Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab
(ImClone Systems), necitumumab (Portrazza.TM., Eli Lilly),
nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb
EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.),
nivolumab (Opdivo.RTM., Bristol-Myers Squibb), obinutuzumab (Gazyva
or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298;
Mentrik Biotech, LLC), ofatumumab (Arzerra.RTM., Genmab),
onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab
(OvaRex.RTM., AltaRex Corp.), otlertuzumab (Emergent BioSolutions),
panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH),
parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda.RTM.,
Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta,
Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin
(Genentech/Roche), pritumumab, racotumomab (Vaxira.RTM., Recombio),
ramucirumab (Cyramza.RTM., ImClone Systems Inc.), rituximab
(Rituxan.RTM., Genentech), robatumumab (Schering-Plough),
Seribantumab (Sanofi/Merrimack Pharmaceuticals, Inc.),
sibrotuzumab, siltuximab (Sylvant.TM., Janssen Biotech), Smart MI95
(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs,
Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox,
tenatumomab, teprotumumab (Roche), tetulomab, TGN1412
(CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo),
tositumomab, trastuzumab (Herceptin.RTM.), tremelimumab
(CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD
Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers
Squibb), volociximab (M200, Biogen Idec), zatuximab, and the
like.
[0344] In some embodiments, the binding moiety A comprises
zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab
(Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab
(Campath.RTM., MabCampath, or Campath-1H; Leukosite), AlloMune
(BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF
(Genetech), anatumomab mafenatox, apolizumab (hu1D10),
ascrinvacumab (Pfizer Inc.), atezolizumab (MPDL3280A;
Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab
(Simulect.RTM., Novartis), belimumab (Benlysta.RTM.,
GlaxoSmithKline), bevacizumab (Avastin.RTM., Genentech),
blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems
Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion
Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux.RTM., ImClone),
citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12, ImClone
Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40,
huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex.RTM.,
Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab
(MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A,
Panorex, Glaxo Wellcome), elotuzumab (Empliciti.TM., Bristol-Myers
Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech
Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab
(MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.),
epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab
(Rexomun.RTM., Trion Pharma), etaracizumab (Abegrin, MedImmune),
farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion
Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab
(CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab
(GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen),
girentuximab (Rencarex.RTM., Wilex AG), IMAB362 (Claudiximab,
Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone
Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab
(Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab
(Yervoy.RTM., Bristol-Myers Squibb), iratumumab (Medarex, Inc.),
isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE,
Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody
Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab
(Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome
Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1,
Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab
(ImClone Systems), necitumumab (Portrazza.TM., Eli Lilly),
nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb
EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.),
nivolumab (Opdivo.RTM., Bristol-Myers Squibb), obinutuzumab (Gazyva
or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298;
Mentrik Biotech, LLC), ofatumumab (Arzerra.RTM., Genmab),
onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab
(OvaRex.RTM., AltaRex Corp.), otlertuzumab (Emergent BioSolutions),
panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH),
parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda.RTM.,
Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta,
Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin
(Genentech/Roche), pritumumab, racotumomab (Vaxira.RTM., Recombio),
ramucirumab (Cyramza.RTM., ImClone Systems Inc.), rituximab
(Rituxan.RTM., Genentech), robatumumab (Schering-Plough),
Seribantumab (S anofi/Merrimack Pharmaceuticals, Inc.),
sibrotuzumab, siltuximab (Sylvant.TM., Janssen Biotech), Smart MI95
(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs,
Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox,
tenatumomab, teprotumumab (Roche), tetulomab, TGN1412
(CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo),
tositumomab, trastuzumab (Herceptin.RTM.), tremelimumab
(CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD
Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers
Squibb), volociximab (M200, Biogen Idec), or zatuximab. In some
embodiments, the binding moiety A is zalutumumab (HuMax-EFGr, by
Genmab).
[0345] In some embodiments, the binding moiety A is conjugated
according to Formula (I) to a polynucleic acid molecule (B), and a
polymer (C), and optionally an endosomolytic moiety (D) according
to Formula (II) described herein. In some instances, the
polynucleic acid molecule comprises a sequence having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to a sequence listed in Tables 2, 3, 5, 6,
7, 9, or 11. In some embodiments, the polynucleic acid molecule
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032,
2082-2109, or 2117. In some instances, the polynucleic acid
molecule comprises a sequence selected from SEQ ID NOs: 16-75,
452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In
some instances, the polymer C comprises polyalkylen oxide (e.g.,
polyethylene glycol). In some embodiments, the endosomolytic moiety
D comprises INF7 or melittin, or their respective derivatives.
[0346] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid molecule (B), and a polymer (C), and optionally an
endosomolytic moiety (D) as illustrated in FIG. 1. In some
instances, the binding moiety A is an antibody or binding fragment
thereof.
[0347] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid molecule (B) non-specifically. In some instances,
the binding moiety A is conjugated to a polynucleic acid molecule
(B) via a lysine residue or a cysteine residue, in a non-site
specific manner. In some instances, the binding moiety A is
conjugated to a polynucleic acid molecule (B) via a lysine residue
in a non-site specific manner. In some cases, the binding moiety A
is conjugated to a polynucleic acid molecule (B) via a cysteine
residue in a non-site specific manner. In some instances, the
binding moiety A is an antibody or binding fragment thereof.
[0348] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid molecule (B) in a site-specific manner. In some
instances, the binding moiety A is conjugated to a polynucleic acid
molecule (B) through a lysine residue, a cysteine residue, at the
5'-terminus, at the 3'-terminus, an unnatural amino acid, or an
enzyme-modified or enzyme-catalyzed residue, via a site-specific
manner. In some instances, the binding moiety A is conjugated to a
polynucleic acid molecule (B) through a lysine residue via a
site-specific manner. In some instances, the binding moiety A is
conjugated to a polynucleic acid molecule (B) through a cysteine
residue via a site-specific manner. In some instances, the binding
moiety A is conjugated to a polynucleic acid molecule (B) at the
5'-terminus via a site-specific manner. In some instances, the
binding moiety A is conjugated to a polynucleic acid molecule (B)
at the 3'-terminus via a site-specific manner. In some instances,
the binding moiety A is conjugated to a polynucleic acid molecule
(B) through an unnatural amino acid via a site-specific manner. In
some instances, the binding moiety A is conjugated to a polynucleic
acid molecule (B) through an enzyme-modified or enzyme-catalyzed
residue via a site-specific manner. In some instances, the binding
moiety A is an antibody or binding fragment thereof.
[0349] In some embodiments, one or more regions of a binding moiety
A (e.g., an antibody or binding fragment thereof) is conjugated to
a polynucleic acid molecule (B). In some instances, the one or more
regions of a binding moiety A comprise the N-terminus, the
C-terminus, in the constant region, at the hinge region, or the Fc
region of the binding moiety A. In some instances, the polynucleic
acid molecule (B) is conjugated to the N-terminus of the binding
moiety A (e.g., the N-terminus of an antibody or binding fragment
thereof). In some instances, the polynucleic acid molecule (B) is
conjugated to the C-terminus of the binding moiety A (e.g., the
N-terminus of an antibody or binding fragment thereof). In some
instances, the polynucleic acid molecule (B) is conjugated to the
constant region of the binding moiety A (e.g., the constant region
of an antibody or binding fragment thereof). In some instances, the
polynucleic acid molecule (B) is conjugated to the hinge region of
the binding moiety A (e.g., the constant region of an antibody or
binding fragment thereof). In some instances, the polynucleic acid
molecule (B) is conjugated to the Fc region of the binding moiety A
(e.g., the constant region of an antibody or binding fragment
thereof).
[0350] In some embodiments, one or more polynucleic acid molecule
(B) is conjugated to a binding moiety A. In some instances, about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more
polynucleic acid molecules are conjugated to one binding moiety A.
In some instances, about 1 polynucleic acid molecule is conjugated
to one binding moiety A. In some instances, about 2 polynucleic
acid molecules are conjugated to one binding moiety A. In some
instances, about 3 polynucleic acid molecules are conjugated to one
binding moiety A. In some instances, about 4 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 5 polynucleic acid molecules are conjugated to one
binding moiety A. In some instances, about 6 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 7 polynucleic acid molecules are conjugated to one
binding moiety A. In some instances, about 8 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 9 polynucleic acid molecules are conjugated to one
binding moiety A. In some instances, about 10 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 11 polynucleic acid molecules are conjugated to
one binding moiety A. In some instances, about 12 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 13 polynucleic acid molecules are conjugated to
one binding moiety A. In some instances, about 14 polynucleic acid
molecules are conjugated to one binding moiety A. In some
instances, about 15 polynucleic acid molecules are conjugated to
one binding moiety A. In some instances, about 16 polynucleic acid
molecules are conjugated to one binding moiety A. In some cases,
the one or more polynucleic acid molecules are the same. In other
cases, the one or more polynucleic acid molecules are different. In
some instances, the binding moiety A is an antibody or binding
fragment thereof.
[0351] In some embodiments, the number of polynucleic acid molecule
(B) conjugated to a binding moiety A (e.g., an antibody or binding
fragment thereof) forms a ratio. In some instances, the ratio is
referred to as a DAR (drug-to-antibody) ratio, in which the drug as
referred to herein is the polynucleic acid molecule (B). In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, or greater. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 1 or
greater. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is about 2 or greater. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is about 3 or greater. In some instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is
about 4 or greater. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 5 or
greater. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is about 6 or greater. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is about 7 or greater. In some instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is
about 8 or greater. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 9 or
greater. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is about 10 or greater. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is about 11 or greater. In some instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is
about 12 or greater.
[0352] In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A (e.g., an antibody or binding
fragment thereof) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, or 16. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 1. In
some instances, the DAR ratio of the polynucleic acid molecule (B)
to binding moiety A is about 2. In some instances, the DAR ratio of
the polynucleic acid molecule (B) to binding moiety A is about 3.
In some instances, the DAR ratio of the polynucleic acid molecule
(B) to binding moiety A is about 4. In some instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is
about 5. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is about 6. In some instances, the
DAR ratio of the polynucleic acid molecule (B) to binding moiety A
is about 7. In some instances, the DAR ratio of the polynucleic
acid molecule (B) to binding moiety A is about 8. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is about 9. In some instances, the DAR ratio of
the polynucleic acid molecule (B) to binding moiety A is about 10.
In some instances, the DAR ratio of the polynucleic acid molecule
(B) to binding moiety A is about 11. In some instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is
about 12. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is about 13. In some instances,
the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about 14. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 15. In
some instances, the DAR ratio of the polynucleic acid molecule (B)
to binding moiety A is about 16.
[0353] In some instances, the DAR ratio of the polynucleic acid
molecule (B) to binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is 1. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is 2. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is 4. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is 6. In some instances, the DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is 8. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is 12.
[0354] In some embodiments, an antibody or its binding fragment is
further modified using conventional techniques known in the art,
for example, by using amino acid deletion, insertion, substitution,
addition, and/or by recombination and/or any other modification
(e.g. posttranslational and chemical modifications, such as
glycosylation and phosphorylation) known in the art either alone or
in combination. In some instances, the modification further
comprises a modification for modulating interaction with Fc
receptors. In some instances, the one or more modifications include
those described in, for example, International Publication No.
WO97/34631, which discloses amino acid residues involved in the
interaction between the Fc domain and the FcRn receptor. Methods
for introducing such modifications in the nucleic acid sequence
underlying the amino acid sequence of an antibody or its binding
fragment is well known to the person skilled in the art.
[0355] In some instances, an antibody binding fragment further
encompasses its derivatives and includes polypeptide sequences
containing at least one CDR.
[0356] In some instances, the term "single-chain" as used herein
means that the first and second domains of a bi-specific single
chain construct are covalently linked, preferably in the form of a
co-linear amino acid sequence encodable by a single nucleic acid
molecule.
[0357] In some instances, a bispecific single chain antibody
construct relates to a construct comprising two antibody derived
binding domains. In such embodiments, bi-specific single chain
antibody construct is tandem bi-scFv or diabody. In some instances,
a scFv contains a VH and VL domain connected by a linker peptide.
In some instances, linkers are of a length and sequence sufficient
to ensure that each of the first and second domains can,
independently from one another, retain their differential binding
specificities.
[0358] In some embodiments, binding to or interacting with as used
herein defines a binding/interaction of at least two
antigen-interaction-sites with each other. In some instances,
antigen-interaction-site defines a motif of a polypeptide that
shows the capacity of specific interaction with a specific antigen
or a specific group of antigens. In some cases, the
binding/interaction is also understood to define a specific
recognition. In such cases, specific recognition refers to that the
antibody or its binding fragment is capable of specifically
interacting with and/or binding to at least two amino acids of each
of a target molecule. For example, specific recognition relates to
the specificity of the antibody molecule, or to its ability to
discriminate between the specific regions of a target molecule. In
additional instances, the specific interaction of the
antigen-interaction-site with its specific antigen results in an
initiation of a signal, e.g. due to the induction of a change of
the conformation of the antigen, an oligomerization of the antigen,
etc. In further embodiments, the binding is exemplified by the
specificity of a "key-lock-principle". Thus in some instances,
specific motifs in the amino acid sequence of the
antigen-interaction-site and the antigen bind to each other as a
result of their primary, secondary or tertiary structure as well as
the result of secondary modifications of said structure. In such
cases, the specific interaction of the antigen-interaction-site
with its specific antigen results as well in a simple binding of
the site to the antigen.
[0359] In some instances, specific interaction further refers to a
reduced cross-reactivity of the antibody or its binding fragment or
a reduced off-target effect. For example, the antibody or its
binding fragment that bind to the polypeptide/protein of interest
but do not or do not essentially bind to any of the other
polypeptides are considered as specific for the polypeptide/protein
of interest. Examples for the specific interaction of an
antigen-interaction-site with a specific antigen comprise the
specificity of a ligand for its receptor, for example, the
interaction of an antigenic determinant (epitope) with the
antigenic binding site of an antibody.
Additional Binding Moieties
[0360] In some embodiments, the binding moiety is a plasma protein.
In some instances, the plasma protein comprises albumin. In some
instances, the binding moiety A is albumin. In some instances,
albumin is conjugated by one or more of a conjugation chemistry
described herein to a polynucleic acid molecule. In some instances,
albumin is conjugated by native ligation chemistry to a polynucleic
acid molecule. In some instances, albumin is conjugated by lysine
conjugation to a polynucleic acid molecule.
[0361] In some instances, the binding moiety is a steroid.
Exemplary steroids include cholesterol, phospholipids, di- and
triacylglycerols, fatty acids, hydrocarbons that are saturated,
unsaturated, comprise substitutions, or combinations thereof. In
some instances, the steroid is cholesterol. In some instances, the
binding moiety is cholesterol. In some instances, cholesterol is
conjugated by one or more of a conjugation chemistry described
herein to a polynucleic acid molecule. In some instances,
cholesterol is conjugated by native ligation chemistry to a
polynucleic acid molecule. In some instances, cholesterol is
conjugated by lysine conjugation to a polynucleic acid
molecule.
[0362] In some instances, the binding moiety is a polymer,
including but not limited to poly nucleic acid molecule aptamers
that bind to specific surface markers on cells. In this instance
the binding moiety is a polynucleic acid that does not hybridize to
a target gene or mRNA, but instead is capable of selectively
binding to a cell surface marker similarly to an antibody binding
to its specific epitope of a cell surface marker.
[0363] In some cases, the binding moiety is a peptide. In some
cases, the peptide comprises between about 1 and about 3 kDa. In
some cases, the peptide comprises between about 1.2 and about 2.8
kDa, about 1.5 and about 2.5 kDa, or about 1.5 and about 2 kDa. In
some instances, the peptide is a bicyclic peptide. In some cases,
the bicyclic peptide is a constrained bicyclic peptide. In some
instances, the binding moiety is a bicyclic peptide (e.g., bicycles
from Bicycle Therapeutics).
[0364] In additional cases, the binding moiety is a small molecule.
In some instances, the small molecule is an antibody-recruiting
small molecule. In some cases, the antibody-recruiting small
molecule comprises a target-binding terminus and an
antibody-binding terminus, in which the target-binding terminus is
capable of recognizing and interacting with a cell surface
receptor. For example, in some instances, the target-binding
terminus comprising a glutamate urea compound enables interaction
with PSMA, thereby, enhances an antibody interaction with a cell
(e.g., a cancerous cell) that expresses PSMA. In some instances, a
binding moiety is a small molecule described in Zhang et al., "A
remote arene-binding site on prostate specific membrane antigen
revealed by antibody-recruiting small molecules," J Am Chem Soc.
132(36): 12711-12716 (2010); or McEnaney, et al.,
"Antibody-recruiting molecules: an emerging paradigm for engaging
immune function in treating human disease," ACS Chem Biol. 7(7):
1139-1151 (2012).
Production of Antibodies or Binding Fragments Thereof
[0365] In some embodiments, polypeptides described herein (e.g.,
antibodies and its binding fragments) are produced using any method
known in the art to be useful for the synthesis of polypeptides
(e.g., antibodies), in particular, by chemical synthesis or by
recombinant expression, and are preferably produced by recombinant
expression techniques.
[0366] In some instances, an antibody or its binding fragment
thereof is expressed recombinantly, and the nucleic acid encoding
the antibody or its binding fragment is assembled from chemically
synthesized oligonucleotides (e.g., as described in Kutmeier et
al., 1994, Bio Techniques 17:242), which involves the synthesis of
overlapping oligonucleotides containing portions of the sequence
encoding the antibody, annealing and ligation of those
oligonucleotides, and then amplification of the ligated
oligonucleotides by PCR.
[0367] Alternatively, a nucleic acid molecule encoding an antibody
is optionally generated from a suitable source (e.g., an antibody
cDNA library, or cDNA library generated from any tissue or cells
expressing the immunoglobulin) by PCR amplification using synthetic
primers hybridizable to the 3' and 5' ends of the sequence or by
cloning using an oligonucleotide probe specific for the particular
gene sequence.
[0368] In some instances, an antibody or its binding is optionally
generated by immunizing an animal, such as a rabbit, to generate
polyclonal antibodies or, more preferably, by generating monoclonal
antibodies, e.g., as described by Kohler and Milstein (1975, Nature
256:495-497) or, as described by Kozbor et al. (1983, Immunology
Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a
clone encoding at least the Fab portion of the antibody is
optionally obtained by screening Fab expression libraries (e.g., as
described in Huse et al., 1989, Science 246:1275-1281) for clones
of Fab fragments that bind the specific antigen or by screening
antibody libraries (See, e.g., Clackson et al., 1991, Nature
352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
[0369] In some embodiments, techniques developed for the production
of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity are used. A chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0370] In some embodiments, techniques described for the production
of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988,
Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted
to produce single chain antibodies. Single chain antibodies are
formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli are also optionally used (Skerra et al., 1988, Science
242:1038-1041).
[0371] In some embodiments, an expression vector comprising the
nucleotide sequence of an antibody or the nucleotide sequence of an
antibody is transferred to a host cell by conventional techniques
(e.g., electroporation, liposomal transfection, and calcium
phosphate precipitation), and the transfected cells are then
cultured by conventional techniques to produce the antibody. In
specific embodiments, the expression of the antibody is regulated
by a constitutive, an inducible or a tissue, specific promoter.
[0372] In some embodiments, a variety of host-expression vector
systems is utilized to express an antibody or its binding fragment
described herein. Such host-expression systems represent vehicles
by which the coding sequences of the antibody is produced and
subsequently purified, but also represent cells that are, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody or its binding fragment in situ.
These include, but are not limited to, microorganisms such as
bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing an antibody or its binding fragment coding
sequences; yeast (e.g., Saccharomyces Pichia) transformed with
recombinant yeast expression vectors containing an antibody or its
binding fragment coding sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing an antibody or its binding fragment coding sequences;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic
virus (TMV)) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing an antibody or its binding
fragment coding sequences; or mammalian cell systems (e.g., COS,
CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K
promoter).
[0373] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. In some instances, cell
lines that stably express an antibody are optionally engineered.
Rather than using expression vectors that contain viral origins of
replication, host cells are transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells are then allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci that in turn are cloned and
expanded into cell lines. This method can advantageously be used to
engineer cell lines which express the antibody or its binding
fragments.
[0374] In some instances, a number of selection systems are used,
including but not limited to the herpes simplex virus thymidine
kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt-
or aprt-cells, respectively. Also, antimetabolite resistance are
used as the basis of selection for the following genes: dhfr, which
confers resistance to methotrexate (Wigler et al., 1980, Proc.
Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad.
Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA
78:2072); neo, which confers resistance to the aminoglycoside G-418
(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,
Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215)
and hygro, which confers resistance to hygromycin (Santerre et al.,
1984, Gene 30:147). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology,
John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY; and in
Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols
in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et
al., 1981, J. Mol. Biol. 150:1).
[0375] In some instances, the expression levels of an antibody are
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing an antibody is amplifiable, an increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the nucleotide sequence of the antibody,
production of the antibody will also increase (Crouse et al., 1983,
Mol. Cell Biol. 3:257).
[0376] In some instances, any method known in the art for
purification of an antibody is used, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins.
Polymer Conjugating Moiety
[0377] In some embodiments, a polymer moiety C is further
conjugated to a polynucleic acid molecule described herein, a
binding moiety described herein, or in combinations thereof. In
some instances, a polymer moiety C is conjugated a polynucleic acid
molecule. In some cases, a polymer moiety C is conjugated to a
binding moiety. In other cases, a polymer moiety C is conjugated to
a polynucleic acid molecule-binding moiety molecule. In additional
cases, a polymer moiety C is conjugated, as illustrated in FIG. 1,
and as discussed under the Therapeutic Molecule Platform
section.
[0378] In some instances, the polymer moiety C is a natural or
synthetic polymer, consisting of long chains of branched or
unbranched monomers, and/or cross-linked network of monomers in two
or three dimensions. In some instances, the polymer moiety C
includes a polysaccharide, lignin, rubber, or polyalkylen oxide
(e.g., polyethylene glycol). In some instances, the at least one
polymer moiety C includes, but is not limited to, alpha-,
omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based
polymer, e.g. polyacrylic acid, polylactide acid (PLA),
poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin,
polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat
(PET, PETG), polyethylene terephthalate (PETE), polytetramethylene
glycol (PTG), or polyurethane as well as mixtures thereof. As used
herein, a mixture refers to the use of different polymers within
the same compound as well as in reference to block copolymers. In
some cases, block copolymers are polymers wherein at least one
section of a polymer is build up from monomers of another polymer.
In some instances, the polymer moiety C comprises polyalkylene
oxide. In some instances, the polymer moiety C comprises PEG. In
some instances, the polymer moiety C comprises polyethylene imide
(PEI) or hydroxy ethyl starch (HES).
[0379] In some instances, C is a PEG moiety. In some instances, the
PEG moiety is conjugated at the 5' terminus of the polynucleic acid
molecule while the binding moiety is conjugated at the 3' terminus
of the polynucleic acid molecule. In some instances, the PEG moiety
is conjugated at the 3' terminus of the polynucleic acid molecule
while the binding moiety is conjugated at the 5' terminus of the
polynucleic acid molecule. In some instances, the PEG moiety is
conjugated to an internal site of the polynucleic acid molecule. In
some instances, the PEG moiety, the binding moiety, or a
combination thereof, are conjugated to an internal site of the
polynucleic acid molecule. In some instances, the conjugation is a
direct conjugation. In some instances, the conjugation is via
native ligation.
[0380] In some embodiments, the polyalkylene oxide (e.g., PEG) is a
polydispers or monodispers compound. In some instances, polydispers
material comprises disperse distribution of different molecular
weight of the material, characterized by mean weight (weight
average) size and dispersity. In some instances, the monodisperse
PEG comprises one size of molecules. In some embodiments, C is
poly- or monodispersed polyalkylene oxide (e.g., PEG) and the
indicated molecular weight represents an average of the molecular
weight of the polyalkylene oxide, e.g., PEG, molecules.
[0381] In some embodiments, the molecular weight of the
polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600,
1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,
2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600,
4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,
20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
[0382] In some embodiments, C is polyalkylene oxide (e.g., PEG) and
has a molecular weight of about 200, 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800,
1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,
3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000,
40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is
PEG and has a molecular weight of about 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600,
1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,
2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600,
4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,
20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some
instances, the molecular weight of C is about 200 Da. In some
instances, the molecular weight of C is about 300 Da. In some
instances, the molecular weight of C is about 400 Da. In some
instances, the molecular weight of C is about 500 Da. In some
instances, the molecular weight of C is about 600 Da. In some
instances, the molecular weight of C is about 700 Da. In some
instances, the molecular weight of C is about 800 Da. In some
instances, the molecular weight of C is about 900 Da. In some
instances, the molecular weight of C is about 1000 Da. In some
instances, the molecular weight of C is about 1100 Da. In some
instances, the molecular weight of C is about 1200 Da. In some
instances, the molecular weight of C is about 1300 Da. In some
instances, the molecular weight of C is about 1400 Da. In some
instances, the molecular weight of C is about 1450 Da. In some
instances, the molecular weight of C is about 1500 Da. In some
instances, the molecular weight of C is about 1600 Da. In some
instances, the molecular weight of C is about 1700 Da. In some
instances, the molecular weight of C is about 1800 Da. In some
instances, the molecular weight of C is about 1900 Da. In some
instances, the molecular weight of C is about 2000 Da. In some
instances, the molecular weight of C is about 2100 Da. In some
instances, the molecular weight of C is about 2200 Da. In some
instances, the molecular weight of C is about 2300 Da. In some
instances, the molecular weight of C is about 2400 Da. In some
instances, the molecular weight of C is about 2500 Da. In some
instances, the molecular weight of C is about 2600 Da. In some
instances, the molecular weight of C is about 2700 Da. In some
instances, the molecular weight of C is about 2800 Da. In some
instances, the molecular weight of C is about 2900 Da. In some
instances, the molecular weight of C is about 3000 Da. In some
instances, the molecular weight of C is about 3250 Da. In some
instances, the molecular weight of C is about 3350 Da. In some
instances, the molecular weight of C is about 3500 Da. In some
instances, the molecular weight of C is about 3750 Da. In some
instances, the molecular weight of C is about 4000 Da. In some
instances, the molecular weight of C is about 4250 Da. In some
instances, the molecular weight of C is about 4500 Da. In some
instances, the molecular weight of C is about 4600 Da. In some
instances, the molecular weight of C is about 4750 Da. In some
instances, the molecular weight of C is about 5000 Da. In some
instances, the molecular weight of C is about 5500 Da. In some
instances, the molecular weight of C is about 6000 Da. In some
instances, the molecular weight of C is about 6500 Da. In some
instances, the molecular weight of C is about 7000 Da. In some
instances, the molecular weight of C is about 7500 Da. In some
instances, the molecular weight of C is about 8000 Da. In some
instances, the molecular weight of C is about 10,000 Da. In some
instances, the molecular weight of C is about 12,000 Da. In some
instances, the molecular weight of C is about 20,000 Da. In some
instances, the molecular weight of C is about 35,000 Da. In some
instances, the molecular weight of C is about 40,000 Da. In some
instances, the molecular weight of C is about 50,000 Da. In some
instances, the molecular weight of C is about 60,000 Da. In some
instances, the molecular weight of C is about 100,000 Da.
[0383] In some embodiments, the polyalkylene oxide (e.g., PEG) is a
discrete PEG, in which the discrete PEG is a polymeric PEG
comprising more than one repeating ethylene oxide units. In some
instances, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to
50, or from 2 to 48 repeating ethylene oxide units. In some
instances, a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42,
48, 50 or more repeating ethylene oxide units. In some instances, a
dPEG comprises about 2 or more repeating ethylene oxide units. In
some instances, a dPEG comprises about 3 or more repeating ethylene
oxide units. In some instances, a dPEG comprises about 4 or more
repeating ethylene oxide units. In some instances, a dPEG comprises
about 5 or more repeating ethylene oxide units. In some instances,
a dPEG comprises about 6 or more repeating ethylene oxide units. In
some instances, a dPEG comprises about 7 or more repeating ethylene
oxide units. In some instances, a dPEG comprises about 8 or more
repeating ethylene oxide units. In some instances, a dPEG comprises
about 9 or more repeating ethylene oxide units. In some instances,
a dPEG comprises about 10 or more repeating ethylene oxide units.
In some instances, a dPEG comprises about 11 or more repeating
ethylene oxide units. In some instances, a dPEG comprises about 12
or more repeating ethylene oxide units. In some instances, a dPEG
comprises about 13 or more repeating ethylene oxide units. In some
instances, a dPEG comprises about 14 or more repeating ethylene
oxide units. In some instances, a dPEG comprises about 15 or more
repeating ethylene oxide units. In some instances, a dPEG comprises
about 16 or more repeating ethylene oxide units. In some instances,
a dPEG comprises about 17 or more repeating ethylene oxide units.
In some instances, a dPEG comprises about 18 or more repeating
ethylene oxide units. In some instances, a dPEG comprises about 19
or more repeating ethylene oxide units. In some instances, a dPEG
comprises about 20 or more repeating ethylene oxide units. In some
instances, a dPEG comprises about 22 or more repeating ethylene
oxide units. In some instances, a dPEG comprises about 24 or more
repeating ethylene oxide units. In some instances, a dPEG comprises
about 26 or more repeating ethylene oxide units. In some instances,
a dPEG comprises about 28 or more repeating ethylene oxide units.
In some instances, a dPEG comprises about 30 or more repeating
ethylene oxide units. In some instances, a dPEG comprises about 35
or more repeating ethylene oxide units. In some instances, a dPEG
comprises about 40 or more repeating ethylene oxide units. In some
instances, a dPEG comprises about 42 or more repeating ethylene
oxide units. In some instances, a dPEG comprises about 48 or more
repeating ethylene oxide units. In some instances, a dPEG comprises
about 50 or more repeating ethylene oxide units. In some cases, a
dPEG is synthesized as a single molecular weight compound from pure
(e.g., about 95%, 98%, 99%, or 99.5%) staring material in a
step-wise fashion. In some cases, a dPEG has a specific molecular
weight, rather than an average molecular weight. In some cases, a
dPEG described herein is a dPEG from Quanta Biodesign, LMD.
[0384] In some embodiments, the polymer moiety C comprises a
cationic mucic acid-based polymer (cMAP). In some instances, cMPA
comprises one or more subunit of at least one repeating subunit,
and the subunit structure is represented as Formula (III):
##STR00010##
[0385] wherein m is independently at each occurrence 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10, preferably 4-6 or 5; and n is independently at
each occurrence 1, 2, 3, 4, or 5. In some embodiments, m and n are,
for example, about 10.
[0386] In some instances, cMAP is further conjugated to a PEG
moiety, generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock
polymer, or a cMAP-PEG-cMAP triblock polymer. In some instances,
the PEG moiety is in a range of from about 500 Da to about 50,000
Da. In some instances, the PEG moiety is in a range of from about
500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da,
greater than 5000 Da to about 10,000 Da, greater than 10,000 to
about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any
combination of two or more of these ranges.
[0387] In some instances, the polymer moiety C is cMAP-PEG
copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP
triblock polymer. In some cases, the polymer moiety C is cMAP-PEG
copolymer. In other cases, the polymer moiety C is an
mPEG-cMAP-PEGm triblock polymer. In additional cases, the polymer
moiety C is a cMAP-PEG-cMAP triblock polymer.
[0388] In some embodiments, the polymer moiety C is conjugated to
the polynucleic acid molecule, the binding moiety, and optionally
to the endosomolytic moiety as illustrated in FIG. 1.
Endosomolytic Moiety
[0389] In some embodiments, a molecule of Formula (I): A-X-B-Y-C,
further comprises an additional conjugating moiety. In some
instances, the additional conjugating moiety is an endosomolytic
moiety. In some cases, the endosomolytic moiety is a cellular
compartmental release component, such as a compound capable of
releasing from any of the cellular compartments known in the art,
such as the endosome, lysosome, endoplasmic reticulum (ER), golgi
apparatus, microtubule, peroxisome, or other vesicular bodies with
the cell. In some cases, the endosomolytic moiety comprises an
endosomolytic polypeptide, an endosomolytic polymer, an
endosomolytic lipid, or an endosomolytic small molecule. In some
cases, the endosomolytic moiety comprises an endosomolytic
polypeptide. In other cases, the endosomolytic moiety comprises an
endosomolytic polymer.
Endosomolytic Polypeptides
[0390] In some embodiments, a molecule of Formula (I): A-X-B-Y-C,
is further conjugated with an endosomolytic polypeptide. In some
cases, the endosomolytic polypeptide is a pH-dependent membrane
active peptide. In some cases, the endosomolytic polypeptide is an
amphipathic polypeptide. In additional cases, the endosomolytic
polypeptide is a peptidomimetic. In some instances, the
endosomolytic polypeptide comprises INF, melittin, meucin, or their
respective derivatives thereof. In some instances, the
endosomolytic polypeptide comprises INF or its derivatives thereof.
In other cases, the endosomolytic polypeptide comprises melittin or
its derivatives thereof. In additional cases, the endosomolytic
polypeptide comprises meucin or its derivatives thereof.
[0391] In some instances, INF7 is a 24 residue polypeptide those
sequence comprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 2055), or
GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2056). In some instances, INF7
or its derivatives comprise a sequence of:
GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 2057), GLFEAIEGFIENGWEGMIDG
WYG-(PEG)6-NH2 (SEQ ID NO: 2058), or
GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 2059).
[0392] In some cases, melittin is a 26 residue polypeptide those
sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 2060),
or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 2061). In some instances,
melittin comprises a polypeptide sequence as described in U.S. Pat.
No. 8,501,930.
[0393] In some instances, meucin is an antimicrobial peptide (AMP)
derived from the venom gland of the scorpion Mesobuthus eupeus. In
some instances, meucin comprises of meucin-13 those sequence
comprises IFGAIAGLLKNIF-NH.sub.2 (SEQ ID NO: 2062) and meucin-18
those sequence comprises FFGHLFKLATKIIPSLFQ (SEQ ID NO: 2063).
[0394] In some instances, the endosomolytic polypeptide comprises a
polypeptide in which its sequence is at least 50%, 60%, 70%, 80%,
90%, 95%, or 99% sequence identity to INF7 or its derivatives
thereof, melittin or its derivatives thereof, or meucin or its
derivatives thereof. In some instances, the endosomolytic moiety
comprises INF7 or its derivatives thereof, melittin or its
derivatives thereof, or meucin or its derivatives thereof.
[0395] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055-2059. In some cases, the endosomolytic
moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NO: 2055. In some cases, the endosomolytic
moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NO: 2056-2059. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2056-2059. In some cases, the
endosomolytic moiety consists of SEQ ID NO: 2055. In some cases,
the endosomolytic moiety consists of SEQ ID NO: 2056-2059.
[0396] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060 or 2061. In some cases, the
endosomolytic moiety comprises a polypeptide having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NO: 2060. In some cases, the
endosomolytic moiety comprises a polypeptide having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NO: 2061. In some cases, the
endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the
endosomolytic moiety comprises SEQ ID NO: 2061. In some cases, the
endosomolytic moiety consists of SEQ ID NO: 2060. In some cases,
the endosomolytic moiety consists of SEQ ID NO: 2061.
[0397] In some instances, the endosomolytic moiety is meucin or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2062 or 2063. In some cases, the
endosomolytic moiety comprises a polypeptide having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NO: 2062. In some cases, the
endosomolytic moiety comprises a polypeptide having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NO: 2063. In some cases, the
endosomolytic moiety comprises SEQ ID NO: 2062. In some cases, the
endosomolytic moiety comprises SEQ ID NO: 2063. In some cases, the
endosomolytic moiety consists of SEQ ID NO: 2062. In some cases,
the endosomolytic moiety consists of SEQ ID NO: 2063.
[0398] In some instances, the endosomolytic moiety comprises a
sequence as illustrated in Table 62.
TABLE-US-00001 TABLE 62 SEQ ID Name Origin Amino Acid Sequence NO:
Type Pep-1 NLS from Simian Virus KETWWETWWTEWSQPKKKRKV 2064 Primary
40 large antigen and amphipathic Reverse transcriptase of HIV pVEC
VE-cadherin LLIILRRRRIRKQAHAHSK 2065 Primary amphipathic VT5
Synthetic peptide DPKGDPKGVTVTVTVTVTGKGDP 2066 .beta.-sheet KPD
amphipathic C105Y 1-antitrypsin CSIPPEVKFNKPFVYLI 2067 -- Trans-
Galanin and mastoparan GWTLNSAGYLLGKINLKALAALA 2068 Primary portan
KKIL amphipathic TP10 Galanin and mastoparan AGYLLGKINLKALAALAKKIL
2069 Primary amphipathic MPG A hydrofobic domain GALFLGFLGAAGSTMGA
2070 .beta.-sheet from the fusion amphipathic sequence of HIV gp4l
and NLS of SV40 T antigen gH625 Glycoprotein gH of
HGLASTLTRWAHYNALIRAF 2071 Secondary HSV type I amphipathic
.alpha.-helical CADY PPTG1 peptide GLWRALWRLLRSLWRLLWRA 2072
Secondary amphipathic .alpha.-helical GALA Synthetic peptide
WEAALAEALAEALAEHLAEALAE 2073 Secondary ALEALAA amphipathic
.alpha.-helical INF Influenza HA2 fusion GLFEAIEGFIENGWEGMIDGWYGC
2074 Secondary peptide amphipathic .alpha.-helical/ PH dependent
membrane active peptide HA2E5- Influenza HA2 subunit
GLFGAIAGFIENGWEGMIDGWYG 2075 Secondary TAT of influenza virus X31
amphipathic strain fusion peptide .alpha.-helical/ PH dependent
membrane active peptide HA2- Influenza HA2 subunit
GLFGAIAGFIENGWEGMIDGRQIKI 2076 pH- penetratin of influenza virus
X31 WFQNRRMKW dependent strain fusion peptide KK-amide membrane
active peptide HA-K4 Influenza HA2 subunit GLFGAIAGFIENGWEGMIDG-
2077 pH- of influenza virus X31 SSKKKK dependent strain fusion
peptide membrane active peptide HA2E4 Influenza HA2 subunit
GLFEAIAGFIENGWEGMIDGGGYC 2078 pH- of influenza virus X31 dependent
strain fusion peptide membrane active peptide H5WYG HA2 analogue
GLFHAIAHFIHGGWH 2079 pH- GLIHGWYG dependent membrane active peptide
GALA- INF3 fusion peptide GLFEAIEGFIENGWEGLAEALAEAL 2080
pH-dependent INF3- EALAA- membrane (PEG)6-NH (PEG)6-NH2 active
peptide CM18- Cecropin-A-Melittin.sub.2-12 KWKLFKKIGAVLKVLTTG- 2081
pH-dependent TAT11 (CM.sub.18) fusion peptide YGRKKRRQRRR membrane
active peptide
[0399] In some cases, the endosomolytic moiety comprises a Bak BH3
polypeptide which induces apoptosis through antagonization of
suppressor targets such as Bcl-2 and/or Bcl-x.sub.L. In some
instances, the endosomolytic moiety comprises a Bak BH3 polypeptide
described in Albarran, et al., "Efficient intracellular delivery of
a pro-apoptotic peptide with a pH-responsive carrier," Reactive
& Functional Polymers 71: 261-265 (2011).
[0400] In some instances, the endosomolytic moiety comprises a
polypeptide (e.g., a cell-penetrating polypeptide) as described in
PCT Publication Nos. WO2013/166155 or WO2015/069587.
Endosomolytic Polymers
[0401] In some embodiments, a molecule of Formula (I): A-X-B-Y-C,
is further conjugated with an endosomolytic polymer. As used
herein, an endosomolytic polymer comprises a linear, a branched
network, a star, a comb, or a ladder type of polymer. In some
instances, an endosomolytic polymer is a homopolymer or a copolymer
comprising two ro more different types of monomers. In some cases,
an endosomolytic polymer is a polycation polymer. In other cases,
an endosomolytic polymer is a polyanion polymer.
[0402] In some instances, a polycation polymer comprises monomer
units that are charge positive, charge neutral, or charge negative,
with a net charge being positive. In other cases, a polycation
polymer comprises a non-polymeric molecule that contains two or
more positive charges. Exemplary cationic polymers include, but are
not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA),
polyethyleneimine (PEI), poly[.alpha.-(4-aminobutyl)-L-glycolic
acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or
N,N-Diethylaminoethyl Methacrylate (DEAEMA).
[0403] In some cases, a polyanion polymer comprises monomer units
that are charge positive, charge neutral, or charge negative, with
a net charge being negative. In other cases, a polyanion polymer
comprises a non-polymeric molecule that contains two or more
negative charges. Exemplary anionic polymers include
p(alkylacrylates) (e.g., poly(propyl acrylic acid) (PPAA)) or
poly(N-isopropylacrylamide) (NIPAM). Additional examples include
PP75, a L-phenylalanine-poly(L-lysine isophthalamide) polymer
described in Khormaee, et al., "Edosomolytic anionic polymer for
the cytoplasmic delivery of siRNAs in localized in vivo
applications," Advanced Functional Materials 23: 565-574
(2013).
[0404] In some embodiments, an endosomolytic polymer described
herein is a pH-responsive endosomolytic polymer. A pH-responsive
polymer comprises a polymer that increases in size (swell) or
collapses depending on the pH of the environment. Polyacrylic acid
and chitosan are examples of pH-responsive polymers.
[0405] In some instances, an endosomolytic moiety described herein
is a membrane-disruptive polymer. In some cases, the
membrane-disruptive polymer comprises a cationic polymer, a neutral
or hydrophobic polymer, or an anionic polymer. In some instances,
the membrane-disruptive polymer is a hydrophilic polymer.
[0406] In some instances, an endosomolytic moiety described herein
is a pH-responsive membrane-disruptive polymer. Exemplary
pH-responsive membrane-disruptive polymers include p(alkylacrylic
acids), poly(N-isopropylacrylamide) (NIPAM) copolymers,
succinylated p(glycidols), and p(.beta.-malic acid) polymers.
[0407] In some instances, p(alkylacrylic acids) include
poly(propylacrylic acid) (polyPAA), poly(methacrylic acid) (PMAA),
poly(ethylacrylic acid) (PEAA), and poly(propyl acrylic acid)
(PPAA). In some instances, a p(alkylacrylic acid) include a
p(alkylacrylic acid) described in Jones, et al., Biochemistry
Journal 372: 65-75 (2003).
[0408] In some embodiments, a pH-responsive membrane-disruptive
polymer comprises p(butyl acrylate-co-methacrylic acid). (see
Bulmus, et al., Journal of Controlled Release 93: 105-120 (2003);
and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38
(2003))
[0409] In some embodiments, a pH-responsive membrane-disruptive
polymer comprises p(styrene-alt-maleic anhydride). (see Henry, et
al., Biomacromolecules 7: 2407-2414 (2006))
[0410] In some embodiments, a pH-responsive membrane-disruptive
polymer comprises pyridyldisulfide acrylate (PDSA) polymers such as
poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA),
poly(MAA-co-BA-co-PDSA), poly(EAA-co-BA-co-PDSA), or
poly(PAA-co-BA-co-PDSA) polymers. (see El-Sayed, et al., "Rational
design of composition and activity correlations for pH-responsive
and glutathione-reactive polymer therapeutics," Journal of
Controlled Release 104: 417-427 (2005); or Flanary et al., "Antigen
delivery with poly(propylacrylic acid) conjugation enhanced MHC-1
presentation and T-cell activation," Bioconjugate Chem. 20: 241-248
(2009))
[0411] In some embodiments, a pH-responsive membrane-disruptive
polymer comprises a lytic polymer comprising the base structure
of:
##STR00011##
[0412] In some instances, an endosomolytic moiety described herein
is further conjugated to an additional conjugate, e.g., a polymer
(e.g., PEG), or a modified polymer (e.g., cholesterol-modified
polymer).
[0413] In some instances, the additional conjugate comprises a
detergent (e.g., Triton X-100). In some instances, an endosomolytic
moiety described herein comprises a polymer (e.g., a
poly(amidoamine)) conjugated with a detergent (e.g., Triton X-100).
In some instances, an endosomolytic moiety described herein
comprises poly(amidoamine)-Triton X-100 conjugate (Duncan, et al.,
"A polymer-Triton X-100 conjugate capable of pH-dependent red blood
cell lysis: a model system illustrating the possibility of drug
delivery within acidic intracellular compartments," Journal of Drug
Targeting 2: 341-347 (1994)).
Endosomolytic Lipids
[0414] In some embodiments, the endosomolytic moiety is a lipid
(e.g., a fusogenic lipid). In some embodiments, a molecule of
Formula (I): A-X-B-Y-C, is further conjugated with an endosomolytic
lipid (e.g., fusogenic lipid). Exemplary fusogenic lipids include
1,2-dileoyl-sn-3-phosphoethanolamine (DOPE),
phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine
(POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol
(Di-Lin),
N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanam-
ine (DLin-k-DMA) and
N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethan-
amine (XTC).
[0415] In some instances, an endosomolytic moiety is a lipid (e.g.,
a fusogenic lipid) described in PCT Publication No.
WO09/126,933.
Endosomolytic Small Molecules
[0416] In some embodiments, the endosomolytic moiety is a small
molecule. In some embodiments, a molecule of Formula (I):
A-X-B-Y-C, is further conjugated with an endosomolytic small
molecule. Exemplary small molecules suitable as endosomolytic
moieties include, but are not limited to, quinine, chloroquine,
hydroxychloroquines, amodiaquins (carnoquines), amopyroquines,
primaquines, mefloquines, nivaquines, halofantrines, quinone
imines, or a combination thereof. In some instances, quinoline
endosomolytic moieties include, but are not limited to,
7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline
(chloroquine);
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline
(hydroxychloroquine);
7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline;
4-(4-diethylamino-1-methylbutylamino) quinoline;
7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline;
7-chloro-4-(4-diethylamino-1-butylamino)quinoline
(desmethylchloroquine);
7-fluoro-4-(4-diethylamino-1-butylamino)quinoline);
4-(4-diethyl-amino-1-butylamino)quinoline;
7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline;
4-(1-carboxy-4-diethylamino-1-butylamino) quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline;
4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;
4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline;
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;
hydroxychloroquine phosphate;
7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline
(desmethylhydroxychloroquine);
7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;
4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)
quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoli-
ne;
7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quin-
oline;
4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinol-
ine;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylami-
no)quinoline;
7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)q-
uinoline;
4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)q-
uinoline;
7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbu-
tylamino)quinoline;
8-[(4-aminopentyl)amino-6-methoxydihydrochloride quinoline;
1-acetyl-1,2,3,4-tetrahydroquinoline;
8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;
1-butyryl-1,2,3,4-tetrahydroquinoline;
3-chloro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylid-
inoquinoline,
4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;
3-fluoro-4-(4-hydroxy-alpha,
alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,
4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;
4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinol-
ine; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;
3,4-dihydro-1-(2H)-quinolinecarboxyaldehyde; 1,1'-pentamethylene
diquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde,
carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl
derivatives or analogs thereof. In some instances, an endosomolytic
moiety is a small molecule described in Naisbitt et al (1997, J
Pharmacol Exp Therapy 280:884-893) and in U.S. Pat. No.
5,736,557.
Formula (I) Molecule-Endosomolytic Moiety Conjugates
[0417] In some embodiments, one or more endosomolytic moieties are
conjugated to a molecule comprising at least one binding moiety, at
least one polynucleotide, at least one polymer, or any combinations
thereof. In some instances, the endosomolytic moiety is conjugated
according to Formula (II):
(A-X-B-Y-C.sub.c)-L-D Formula II
wherein,
[0418] A is a binding moiety;
[0419] B is a polynucleotide;
[0420] C is a polymer;
[0421] X is a bond or first linker;
[0422] Y is a bond or second linker;
[0423] L is a bond or third linker;
[0424] D is an endosomolytic moiety; and
[0425] c is an integer between 0 and 1; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety; and D is conjugated anywhere on
A, B, or C.
[0426] In some embodiments, A and C are not attached to B at the
same terminus.
[0427] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
instances, the at least one 2' modified nucleotide comprises locked
nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases,
the at least one modified internucleotide linkage comprises a
phosphorothioate linkage or a phosphorodithioate linkage. In some
embodiments, the polynucleotide comprises a first polynucleotide
and a second polynucleotide hybridized to the first polynucleotide
to form a double-stranded polynucleic acid molecule. In some
instances, the second polynucleotide comprises at least one
modification. In some cases, the first polynucleotide and the
second polynucleotide are RNA molecules. In some cases, the first
polynucleotide and the second polynucleotide are siRNA molecules.
In some embodiments, X, Y, and L are independently a bond or a
non-polymeric linker group. In some instances, A is an antibody or
binding fragment thereof. In some instances, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some cases, C is
polyethylene glycol.
[0428] In some instances, the endosomolytic moiety comprises a
polypeptide, a polymer, a lipid, or a small molecule. In some
instances, the endosomolytic moiety is an endosomolytic
polypeptide. In some cases, the endosomolytic moiety is an
endosomolytic polymer. In other cases, the endosomolytic moiety is
an endosomolytic lipid. In additional cases, the endosomolytic
moiety is an endosomolytic small molecule.
[0429] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2055.
[0430] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2060.
[0431] In some instances, the endosomolytic moiety is a sequence as
illustrated in Table 62.
[0432] In additional cases, the endosomolytic moiety is an
endosomolytic polymer, such as for example, a pH-responsive
endosomolytic polymer, a membrane-disruptive polymer, a polycation
polymer, a polyanion polymer, a pH-responsive membrane-disruptive
polymer, or a combination thereof. In additional cases, the
endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a
p(butyl acrylate-co-methacrylic acid) polymer, a
p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide
acrylate (PDSA) polymer, a polymer-PEG conjugate, a
polymer-detergent conjugate, or a combination thereof.
[0433] In some embodiments, the endosomolytic moiety conjugate is
according to Formula (IIa):
D-L-A-X-B-Y-C.sub.c Formula IIa
wherein,
[0434] A is a binding moiety;
[0435] B is a polynucleotide;
[0436] C is a polymer;
[0437] X is a bond or first linker;
[0438] Y is a bond or second linker;
[0439] L is a bond or third linker;
[0440] D is an endosomolytic moiety; and
[0441] c is an integer of 1; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety.
[0442] In some embodiments, A and C are not attached to B at the
same terminus.
[0443] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
instances, the at least one 2' modified nucleotide comprises locked
nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases,
the at least one modified internucleotide linkage comprises a
phosphorothioate linkage or a phosphorodithioate linkage. In some
embodiments, the polynucleotide comprises a first polynucleotide
and a second polynucleotide hybridized to the first polynucleotide
to form a double-stranded polynucleic acid molecule. In some
instances, the second polynucleotide comprises at least one
modification. In some cases, the first polynucleotide and the
second polynucleotide are RNA molecules. In some cases, the first
polynucleotide and the second polynucleotide are siRNA molecules.
In some embodiments, X, Y, and L are independently a bond or a
non-polymeric linker group. In some instances, A is an antibody or
binding fragment thereof. In some instances, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some cases, C is
polyethylene glycol.
[0444] In some instances, the endosomolytic moiety comprises a
polypeptide, a polymer, a lipid, or a small molecule. In some
instances, the endosomolytic moiety is an endosomolytic
polypeptide. In some cases, the endosomolytic moiety is an
endosomolytic polymer. In other cases, the endosomolytic moiety is
an endosomolytic lipid. In additional cases, the endosomolytic
moiety is an endosomolytic small molecule.
[0445] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2055.
[0446] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2060.
[0447] In some instances, the endosomolytic moiety is a sequence as
illustrated in Table 62.
[0448] In additional cases, the endosomolytic moiety is an
endosomolytic polymer, such as for example, a pH-responsive
endosomolytic polymer, a membrane-disruptive polymer, a polycation
polymer, a polyanion polymer, a pH-responsive membrane-disruptive
polymer, or a combination thereof. In additional cases, the
endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a
p(butyl acrylate-co-methacrylic acid) polymer, a
p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide
acrylate (PDSA) polymer, a polymer-PEG conjugate, a
polymer-detergent conjugate, or a combination thereof.
[0449] In some instances, the endosomolytic moiety conjugate is
according to Formula (IIb):
A-X-B-L-D Formula IIb
wherein,
[0450] A is a binding moiety;
[0451] B is a polynucleotide;
[0452] X is a bond or first linker;
[0453] L is a bond or third linker; and
[0454] D is an endosomolytic moiety; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety.
[0455] In some embodiments, A and C are not attached to B at the
same terminus.
[0456] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
instances, the at least one 2' modified nucleotide comprises locked
nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases,
the at least one modified internucleotide linkage comprises a
phosphorothioate linkage or a phosphorodithioate linkage. In some
embodiments, the polynucleotide comprises a first polynucleotide
and a second polynucleotide hybridized to the first polynucleotide
to form a double-stranded polynucleic acid molecule. In some
instances, the second polynucleotide comprises at least one
modification. In some cases, the first polynucleotide and the
second polynucleotide are RNA molecules. In some cases, the first
polynucleotide and the second polynucleotide are siRNA molecules.
In some embodiments, X and L are independently a bond or a
non-polymeric linker group. In some instances, A is an antibody or
binding fragment thereof. In some instances, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some cases, C is
polyethylene glycol.
[0457] In some instances, the endosomolytic moiety comprises a
polypeptide, a polymer, a lipid, or a small molecule. In some
instances, the endosomolytic moiety is an endosomolytic
polypeptide. In some cases, the endosomolytic moiety is an
endosomolytic polymer. In other cases, the endosomolytic moiety is
an endosomolytic lipid. In additional cases, the endosomolytic
moiety is an endosomolytic small molecule.
[0458] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2055.
[0459] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2060.
[0460] In some instances, the endosomolytic moiety is a sequence as
illustrated in Table 62.
[0461] In additional cases, the endosomolytic moiety is an
endosomolytic polymer, such as for example, a pH-responsive
endosomolytic polymer, a membrane-disruptive polymer, a polycation
polymer, a polyanion polymer, a pH-responsive membrane-disruptive
polymer, or a combination thereof. In additional cases, the
endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a
p(butyl acrylate-co-methacrylic acid) polymer, a
p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide
acrylate (PDSA) polymer, a polymer-PEG conjugate, a
polymer-detergent conjugate, or a combination thereof.
[0462] In some instances, the endosomolytic moiety conjugate is
according to Formula (IIc):
A-X-B-Y-C.sub.c-L-D Formula IIc
wherein,
[0463] A is a binding moiety;
[0464] B is a polynucleotide;
[0465] C is a polymer;
[0466] X is a bond or first linker;
[0467] Y is a bond or second linker;
[0468] L is a bond or third linker;
[0469] D is an endosomolytic moiety; and
[0470] c is an integer of 1; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety.
[0471] In some embodiments, A and C are not attached to B at the
same terminus.
[0472] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
instances, the at least one 2' modified nucleotide comprises locked
nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases,
the at least one modified internucleotide linkage comprises a
phosphorothioate linkage or a phosphorodithioate linkage. In some
embodiments, the polynucleotide comprises a first polynucleotide
and a second polynucleotide hybridized to the first polynucleotide
to form a double-stranded polynucleic acid molecule. In some
instances, the second polynucleotide comprises at least one
modification. In some cases, the first polynucleotide and the
second polynucleotide are RNA molecules. In some cases, the first
polynucleotide and the second polynucleotide are siRNA molecules.
In some embodiments, X, Y, and L are independently a bond or a
non-polymeric linker group. In some instances, A is an antibody or
binding fragment thereof. In some instances, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some cases, C is
polyethylene glycol.
[0473] In some instances, the endosomolytic moiety comprises a
polypeptide, a polymer, a lipid, or a small molecule. In some
instances, the endosomolytic moiety is an endosomolytic
polypeptide. In some cases, the endosomolytic moiety is an
endosomolytic polymer. In other cases, the endosomolytic moiety is
an endosomolytic lipid. In additional cases, the endosomolytic
moiety is an endosomolytic small molecule.
[0474] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2055.
[0475] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2060.
[0476] In some instances, the endosomolytic moiety is a sequence as
illustrated in Table 62.
[0477] In additional cases, the endosomolytic moiety is an
endosomolytic polymer, such as for example, a pH-responsive
endosomolytic polymer, a membrane-disruptive polymer, a polycation
polymer, a polyanion polymer, a pH-responsive membrane-disruptive
polymer, or a combination thereof. In additional cases, the
endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a
p(butyl acrylate-co-methacrylic acid) polymer, a
p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide
acrylate (PDSA) polymer, a polymer-PEG conjugate, a
polymer-detergent conjugate, or a combination thereof.
[0478] In some instances, the endosomolytic moiety conjugate is
according to Formula (IId):
A-L-D-X-B-Y-C.sub.c Formula IId
wherein,
[0479] A is a binding moiety;
[0480] B is a polynucleotide;
[0481] C is a polymer;
[0482] X is a bond or first linker;
[0483] Y is a bond or second linker;
[0484] L is a bond or third linker;
[0485] D is an endosomolytic moiety; and
[0486] c is an integer of 1; and
wherein the polynucleotide comprises at least one 2' modified
nucleotide, at least one modified internucleotide linkage, or at
least one inverted abasic moiety.
[0487] In some embodiments, A and C are not attached to B at the
same terminus.
[0488] In some embodiments, the at least one 2' modified nucleotide
comprises 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA) modified nucleotide. In some
instances, the at least one 2' modified nucleotide comprises locked
nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases,
the at least one modified internucleotide linkage comprises a
phosphorothioate linkage or a phosphorodithioate linkage. In some
embodiments, the polynucleotide comprises a first polynucleotide
and a second polynucleotide hybridized to the first polynucleotide
to form a double-stranded polynucleic acid molecule. In some
instances, the second polynucleotide comprises at least one
modification. In some cases, the first polynucleotide and the
second polynucleotide are RNA molecules. In some cases, the first
polynucleotide and the second polynucleotide are siRNA molecules.
In some embodiments, X, Y, and L are independently a bond or a
non-polymeric linker group. In some instances, A is an antibody or
binding fragment thereof. In some instances, the antibody or
binding fragment thereof comprises a humanized antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, single-chain variable fragment (scFv), diabody,
minibody, nanobody, single-domain antibody (sdAb), or camelid
antibody or binding fragment thereof. In some cases, C is
polyethylene glycol.
[0489] In some instances, the endosomolytic moiety comprises a
polypeptide, a polymer, a lipid, or a small molecule. In some
instances, the endosomolytic moiety is an endosomolytic
polypeptide. In some cases, the endosomolytic moiety is an
endosomolytic polymer. In other cases, the endosomolytic moiety is
an endosomolytic lipid. In additional cases, the endosomolytic
moiety is an endosomolytic small molecule.
[0490] In some instances, the endosomolytic moiety is INF7 or its
derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2055. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2055.
[0491] In some instances, the endosomolytic moiety is melittin or
its derivatives thereof. In some cases, the endosomolytic moiety
comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to SEQ ID NOs: 2060. In some cases, the endosomolytic
moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic
moiety consists of SEQ ID NO: 2060.
[0492] In some instances, the endosomolytic moiety is a sequence as
illustrated in Table 62.
[0493] In additional cases, the endosomolytic moiety is an
endosomolytic polymer, such as for example, a pH-responsive
endosomolytic polymer, a membrane-disruptive polymer, a polycation
polymer, a polyanion polymer, a pH-responsive membrane-disruptive
polymer, or a combination thereof. In additional cases, the
endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a
p(butyl acrylate-co-methacrylic acid) polymer, a
p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide
acrylate (PDSA) polymer, a polymer-PEG conjugate, a
polymer-detergent conjugate, or a combination thereof.
Linkers
[0494] In some embodiments, a linker described herein is a
cleavable linker or a non-cleavable linker. In some instances, the
linker is a cleavable linker. In some instances, the linker is an
acid cleavable linker. In some instances, the linker is a
non-cleavable linker. In some instances, the linker includes a
C.sub.1-C.sub.6 alkyl group (e.g., a C.sub.5, C.sub.4, C.sub.3,
C.sub.2, or C.sub.1 alkyl group). In some instances, the linker
includes homobifunctional cross linkers, heterobifunctional cross
linkers, and the like. In some instances, the linker is a traceless
linker (or a zero-length linker). In some instances, the linker is
a non-polymeric linker. In some cases, the linker is a non-peptide
linker or a linker that does not contain an amino acid residue.
[0495] In some instances, the linker comprises a homobifuctional
linker. Exemplary homobifuctional linkers include, but are not
limited to, Lomant's reagent dithiobis (succinimidylpropionate)
DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP),
disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS),
disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo
DST), ethylene glycobis(succinimidylsuccinate) (EGS),
disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate
(DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP),
dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate
(DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB),
bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB),
such as e.g. 1,5-difluoro-2,4-dinitrobenzene or
1,3-difluoro-4,6-dinitrobenzene,
4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS),
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide (BASED),
formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether,
adipic acid dihydrazide, carbohydrazide, o-toluidine,
3,3'-dimethylbenzidine, benzidine,
.alpha.,.alpha.'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,
N,N'-ethylene-bis(iodoacetamide), or
N,N'-hexamethylene-bis(iodoacetamide).
[0496] In some embodiments, the linker comprises a
heterobifunctional linker. Exemplary heterobifunctional linker
include, but are not limited to, amine-reactive and sulfhydryl
cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate
(sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate
(LC-sPDP), water-soluble-long-chain N-succinimidyl
3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),
succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluene
(sMPT),
sulfosuccinimidyl-6-[.alpha.-methyl-.alpha.-(2-pyridyldithio)tolu-
amido]hexanoate (sulfo-LC-sMPT),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),
N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),
sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),
succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),
sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),
N-(.gamma.-maleimidobutyryloxy)succinimide ester (GMBs),
N-(.gamma.-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),
succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl
6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX),
succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate
(sIAC), succinimidyl
6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)
hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),
carbonyl-reactive and sulfhydryl-reactive cross-linkers such as
4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),
4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8
(M.sub.2C.sub.2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH),
amine-reactive and photoreactive cross-linkers such as
N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),
N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),
sulfosuccinimidyl-(4-azidosalicylamido)hexanoate
(sulfo-NHs-LC-AsA),
sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate
(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),
N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),
N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH),
sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate
(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),
sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionat-
e (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate
(sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate
(sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate
(sulfo-sAPB), sulfosuccinimidyl
2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate
(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate
(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),
p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),
sulfhydryl-reactive and photoreactive cross-linkers such
as1-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),
N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide
(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide
carbonyl-reactive and photoreactive cross-linkers such as
p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and
photoreactive cross-linkers such as
4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and
photoreactive cross-linkers such as p-azidophenyl glyoxal
(APG).
[0497] In some instances, the linker comprises a reactive
functional group. In some cases, the reactive functional group
comprises a nucleophilic group that is reactive to an electrophilic
group present on a binding moiety. Exemplary electrophilic groups
include carbonyl groups--such as aldehyde, ketone, carboxylic acid,
ester, amide, enone, acyl halide or acid anhydride. In some
embodiments, the reactive functional group is aldehyde. Exemplary
nucleophilic groups include hydrazide, oxime, amino, hydrazine,
thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
[0498] In some embodiments, the linker comprises a maleimide goup.
In some instances, the maleimide group is also referred to as a
maleimide spacer. In some instances, the maleimide group further
encompasses a caproic acid, forming maleimidocaproyl (mc). In some
cases, the linker comprises maleimidocaproyl (mc). In some cases,
the linker is maleimidocaproyl (mc). In other instances, the
maleimide group comprises a maleimidomethyl group, such as
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC)
or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sulfo-sMCC) described above.
[0499] In some embodiments, the maleimide group is a
self-stabilizing maleimide. In some instances, the self-stabilizing
maleimide utilizes diaminopropionic acid (DPR) to incorporate a
basic amino group adjacent to the maleimide to provide
intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby
eliminating maleimide from undergoing an elimination reaction
through a retro-Michael reaction. In some instances, the
self-stabilizing maleimide is a maleimide group described in Lyon,
et al., "Self-hydrolyzing maleimides improve the stability and
pharmacological properties of antibody-drug conjugates," Nat.
Biotechnol. 32(10):1059-1062 (2014). In some instances, the linker
comprises a self-stabilizing maleimide. In some instances, the
linker is a self-stabilizing maleimide.
[0500] In some embodiments, the linker comprises a peptide moiety.
In some instances, the peptide moiety comprises at least 2, 3, 4,
5, 6, 7, 8, or more aminoa cid residues. In some instances, the
peptide moiety is a cleavable peptide moiety (e.g., either
enzymatically or chemically). In some instances, the peptide moiety
is a non-cleavable peptide moiety. In some instances, the peptide
moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ
ID NO: 2111), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys,
Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala,
Ala-Leu-Ala-Leu (SEQ ID NO: 2112), or Gly-Phe-Leu-Gly (SEQ ID NO:
2113). In some instances, the linker comprises a peptide moiety
such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO:
2111), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys,
Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala,
Ala-Leu-Ala-Leu (SEQ ID NO: 2112), or Gly-Phe-Leu-Gly (SEQ ID NO:
2113). In some cases, the linker comprises Val-Cit. In some cases,
the linker is Val-Cit.
[0501] In some embodiments, the linker comprises a benzoic acid
group, or its derivatives thereof. In some instances, the benzoic
acid group or its derivatives thereof comprise paraaminobenzoic
acid (PABA). In some instances, the benzoic acid group or its
derivatives thereof comprise gamma-aminobutyric acid (GABA).
[0502] In some embodiments, the linker comprises one or more of a
maleimide group, a peptide moiety, and/or a benzoic acid group, in
any combination. In some embodiments, the linker comprises a
combination of a maleimide group, a peptide moiety, and/or a
benzoic acid group. In some instances, the maleimide group is
maleimidocaproyl (mc). In some instances, the peptide group is
val-cit. In some instances, the benzoic acid group is PABA. In some
instances, the linker comprises a mc-val-cit group. In some cases,
the linker comprises a val-cit-PABA group. In additional cases, the
linker comprises a mc-val-cit-PABA group.
[0503] In some embodiments, the linker is a self-immolative linker
or a self-elimination linker. In some cases, the linker is a
self-immolative linker. In other cases, the linker is a
self-elimination linker (e.g., a cyclization self-elimination
linker). In some instances, the linker comprises a linker described
in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426.
[0504] In some embodiments, the linker is a dendritic type linker.
In some instances, the dendritic type linker comprises a branching,
multifunctional linker moiety. In some instances, the dendritic
type linker is used to increase the molar ratio of polynucleotide B
to the binding moiety A. In some instances, the dendritic type
linker comprises PAMAM dendrimers.
[0505] In some embodiments, the linker is a traceless linker or a
linker in which after cleavage does not leave behind a linker
moiety (e.g., an atom or a linker group) to a binding moiety A, a
polynucleotide B, a polymer C, or an endosomolytic moiety D.
Exemplary traceless linkers include, but are not limited to,
germanium linkers, silicium linkers, sulfur linkers, selenium
linkers, nitrogen linkers, phosphorus linkers, boron linkers,
chromium linkers, or phenylhydrazide linker. In some cases, the
linker is a traceless aryl-triazene linker as described in Hejesen,
et al., "A traceless aryl-triazene linker for DNA-directed
chemistry," Org Biomol Chem 11(15): 2493-2497 (2013). In some
instances, the linker is a traceless linker described in Blaney, et
al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:
2607-2024 (2002). In some instances, a linker is a traceless linker
as described in U.S. Pat. No. 6,821,783.
[0506] In some instances, the linker comprises a functional group
that exerts steric hinderance at the site of bonding between the
linker and a conjugating moiety (e.g., A, B, C, or D described
herein). In some instances, the steric hinderance is a steric
hindrance around a disulfide bond. Exemplary linkers that exhibit
steric hinderance comprises a heterobifuctional linker, such as a
heterobifuctional linker described above. In some cases, a linker
that exhibits steric hinderance comprises SMCC and SPDB.
[0507] In some instances, the linker is an acid cleavable linker.
In some instances, the acid cleavable linker comprises a hydrazone
linkage, which is susceptible to hydrolytic cleavage. In some
cases, the acid cleavable linker comprises a thiomaleamic acid
linker. In some cases, the acid cleavable linker is a thiomaleamic
acid linker as described in Castaneda, et al, "Acid-cleavable
thiomaleamic acid linker for homogeneous antibody-drug
conjugation," Chem. Commun. 49: 8187-8189 (2013).
[0508] In some instances, the linker is a linker described in U.S.
Pat. Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688;
U.S. Patent Publication Nos. 2014/0127239; 2013/028919;
2014/286970; 2013/0309256; 2015/037360; or 2014/0294851; or PCT
Publication Nos. WO2015057699; WO2014080251; WO2014197854;
WO2014145090; or WO2014177042.
[0509] In some embodiments, X, Y, and L are independently a bond or
a linker. In some instances, X, Y, and L are independently a bond.
In some cases, X, Y, and L are independently a linker.
[0510] In some instances, X is a bond or a linker. In some
instances, X is a bond. In some instances, X is a linker. In some
instances, the linker is a C.sub.1-C.sub.6 alkyl group. In some
cases, X is a C.sub.1-C.sub.6 alkyl group, such as for example, a
C.sub.5, C.sub.4, C.sub.3, C.sub.2, or C.sub.1 alkyl group. In some
cases, the C.sub.1-C.sub.6 alkyl group is an unsubstituted
C.sub.1-C.sub.6 alkyl group. As used in the context of a linker,
and in particular in the context of X, alkyl means a saturated
straight or branched hydrocarbon radical containing up to six
carbon atoms. In some instances, X is a non-polymeric linker. In
some instances, X includes a homobifuctional linker or a
heterobifuctional linker described supra. In some cases, X includes
a heterobifunctional linker. In some cases, X includes sMCC. In
other instances, X includes a heterobifuctional linker optionally
conjugated to a C.sub.1-C.sub.6 alkyl group. In other instances, X
includes sMCC optionally conjugated to a C.sub.1-C.sub.6 alkyl
group. In additional instances, X does not include a
homobifuctional linker or a heterobifunctional linker described
supra.
[0511] In some instances, Y is a bond or a linker. In some
instances, Y is a bond. In other cases, Y is a linker. In some
embodiments, Y is a C.sub.1-C.sub.6 alkyl group. In some instances,
Y is a homobifuctional linker or a heterobifunctional linker
described supra. In some instances, Y is a homobifuctional linker
described supra. In some instances, Y is a heterobifunctional
linker described supra. In some instances, Y comprises a maleimide
group, such as maleimidocaproyl (mc) or a self-stabilizing
maleimide group described above. In some instances, Y comprises a
peptide moiety, such as Val-Cit. In some instances, Y comprises a
benzoic acid group, such as PABA. In additional instances, Y
comprises a combination of a maleimide group, a peptide moiety,
and/or a benzoic acid group. In additional instances, Y comprises a
mc group. In additional instances, Y comprises a mc-val-cit group.
In additional instances, Y comprises a val-cit-PABA group. In
additional instances, Y comprises a mc-val-cit-PABA group.
[0512] In some instances, L is a bond or a linker. In some cases, L
is a bond. In other cases, L is a linker. In some embodiments, L is
a C.sub.1-C.sub.6 alkyl group. In some instances, L is a
homobifuctional linker or a heterobifunctional linker described
supra. In some instances, L is a homobifuctional linker described
supra. In some instances, L is a heterobifunctional linker
described supra. In some instances, L comprises a maleimide group,
such as maleimidocaproyl (mc) or a self-stabilizing maleimide group
described above. In some instances, L comprises a peptide moiety,
such as Val-Cit. In some instances, L comprises a benzoic acid
group, such as PABA. In additional instances, L comprises a
combination of a maleimide group, a peptide moiety, and/or a
benzoic acid group. In additional instances, L comprises a mc
group. In additional instances, L comprises a mc-val-cit group. In
additional instances, L comprises a val-cit-PABA group. In
additional instances, L comprises a mc-val-cit-PABA group.
Methods of Use
[0513] In some embodiments, a composition or a pharmaceutical
formulation described herein comprising a binding moiety conjugated
to a polynucleic acid molecule and a polymer is used for the
treatment of a disease or disorder. In some instances, the disease
or disorder is a cancer. In some embodiments, a composition or a
pharmaceutical formulation described herein is used as an
immunotherapy for the treatment of a disease or disorder. In some
instances, the immunotherapy is an immuno-oncology therapy.
Cancer
[0514] In some embodiments, a composition or a pharmaceutical
formulation described herein is used for the treatment of cancer.
In some instances, the cancer is a solid tumor. In some instances,
the cancer is a hematologic malignancy. In some instances, the
cancer is a relapsed or refractory cancer, or a metastatic cancer.
In some instances, the solid tumor is a relapsed or refractory
solid tumor, or a metastatic solid tumor. In some cases, the
hematologic malignancy is a relapsed or refractory hematologic
malignancy, or a metastatic hematologic malignancy.
[0515] In some embodiments, the cancer is a solid tumor. Exemplary
solid tumor includes, but is not limited to, anal cancer, appendix
cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder
cancer, brain tumor, breast cancer, cervical cancer, colon cancer,
cancer of Unknown Primary (CUP), esophageal cancer, eye cancer,
fallopian tube cancer, gastroenterological cancer, kidney cancer,
liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer,
ovarian cancer, pancreatic cancer, parathyroid disease, penile
cancer, pituitary tumor, prostate cancer, rectal cancer, skin
cancer, stomach cancer, testicular cancer, throat cancer, thyroid
cancer, uterine cancer, vaginal cancer, or vulvar cancer.
[0516] In some instances, a composition or a pharmaceutical
formulation described herein comprising a binding moiety conjugated
to a polynucleic acid molecule and a polymer is used for the
treatment of a solid tumor. In some instances, a composition or a
pharmaceutical formulation described herein comprising a binding
moiety conjugated to a polynucleic acid molecule and a polymer is
used for the treatment of anal cancer, appendix cancer, bile duct
cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor,
breast cancer, cervical cancer, colon cancer, cancer of Unknown
Primary (CUP), esophageal cancer, eye cancer, fallopian tube
cancer, gastroenterological cancer, kidney cancer, liver cancer,
lung cancer, medulloblastoma, melanoma, oral cancer, ovarian
cancer, pancreatic cancer, parathyroid disease, penile cancer,
pituitary tumor, prostate cancer, rectal cancer, skin cancer,
stomach cancer, testicular cancer, throat cancer, thyroid cancer,
uterine cancer, vaginal cancer, or vulvar cancer. In some
instances, the solid tumor is a relapsed or refractory solid tumor,
or a metastatic solid tumor.
[0517] In some instances, the cancer is a hematologic malignancy.
In some instances, the hematologic malignancy is a leukemia, a
lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's
lymphoma. In some instances, the hematologic malignancy comprises
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic
leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell
lymphoma (DLBCL), mantle cell lymphoma (MCL), WaldenstrOm's
macroglobulinemia, multiple myeloma, extranodal marginal zone B
cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's
lymphoma, non-Burkitt high grade B cell lymphoma, primary
mediastinal B-cell lymphoma (PMBL), immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic
leukemia, lymphoplasmacytic lymphoma, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)
large B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, or lymphomatoid granulomatosis.
[0518] In some instances, a composition or a pharmaceutical
formulation described herein comprising a binding moiety conjugated
to a polynucleic acid molecule and a polymer is used for the
treatment of a hematologic malignancy. In some instances, a
composition or a pharmaceutical formulation described herein
comprising a binding moiety conjugated to a polynucleic acid
molecule and a polymer is used for the treatment of a leukemia, a
lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's
lymphoma. In some instances, the hematologic malignancy comprises
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic
leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell
lymphoma (DLBCL), mantle cell lymphoma (MCL), WaldenstrOm's
macroglobulinemia, multiple myeloma, extranodal marginal zone B
cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's
lymphoma, non-Burkitt high grade B cell lymphoma, primary
mediastinal B-cell lymphoma (PMBL), immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic
leukemia, lymphoplasmacytic lymphoma, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)
large B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, or lymphomatoid granulomatosis. In some cases,
the hematologic malignancy is a relapsed or refractory hematologic
malignancy, or a metastatic hematologic malignancy.
[0519] In some instances, the cancer is a KRAS-associated,
EGFR-associated, AR-associated cancer, HPRT1-associated cancer, or
.beta.-catenin associated cancer. In some instances, a composition
or a pharmaceutical formulation described herein comprising a
binding moiety conjugated to a polynucleic acid molecule and a
polymer is used for the treatment of a KRAS-associated,
EGFR-associated, AR-associated cancer, HPRT1-associated cancer, or
.beta.-catenin associated cancer. In some instances, a composition
or a pharmaceutical formulation described herein comprising a
binding moiety conjugated to a polynucleic acid molecule and a
polymer is used for the treatment of a KRAS-associated cancer. In
some instances, a composition or a pharmaceutical formulation
described herein comprising a binding moiety conjugated to a
polynucleic acid molecule and a polymer is used for the treatment
of an EGFR-associated cancer. In some instances, a composition or a
pharmaceutical formulation described herein comprising a binding
moiety conjugated to a polynucleic acid molecule and a polymer is
used for the treatment of an AR-associated cancer. In some
instances, a composition or a pharmaceutical formulation described
herein comprising a binding moiety conjugated to a polynucleic acid
molecule and a polymer is used for the treatment of an
HPRT1-associated cancer. In some instances, a composition or a
pharmaceutical formulation described herein comprising a binding
moiety conjugated to a polynucleic acid molecule and a polymer is
used for the treatment of a .beta.-catenin associated cancer. In
some instances, the cancer is a solid tumor. In some instances, the
cancer is a hematologic malignancy. In some instances, the solid
tumor is a relapsed or refractory solid tumor, or a metastatic
solid tumor. In some cases, the hematologic malignancy is a
relapsed or refractory hematologic malignancy, or a metastatic
hematologic malignancy. In some instances, the cancer comprises
bladder cancer, breast cancer, colorectal cancer, endometrial
cancer, esophageal cancer, glioblastoma multiforme, head and neck
cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic
cancer, prostate cancer, thyroid cancer, acute myeloid leukemia,
CLL, DLBCL, or multiple myeloma. In some instances, the
.beta.-catenin associated cancer further comprises PIK3C-associated
cancer and/or MYC-associated cancer.
Immunotherapy
[0520] In some embodiments, a composition or a pharmaceutical
formulation described herein is used as an immunotherapy for the
treatment of a disease or disorder. In some instances, the
immunotherapy is an immuno-oncology therapy. In some instances,
immuno-oncology therapy is categorized into active, passive, or
combinatory (active and passive) methods. In active immuno-oncology
therapy method, for example, tumor-associated antigens (TAAs) are
presented to the immune system to trigger an attack on cancer cells
presenting these TAAs. In some instances, the active
immune-oncology therapy method includes tumor-targeting and/or
immune-targeting agents (e.g., checkpoint inhibitor agents such as
monoclonal antibodies), and/or vaccines, such as in situ
vaccination and/or cell-based or non-cell based (e.g., dendritic
cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or
vector-based) vaccines. In some instances, the cell-based vaccines
are vaccines which are generated using activated immune cells
obtained from a patient's own immune system which are then
activated by the patient's own cancer. In some instances, the
active immune-oncology therapy is further subdivided into
non-specific active immunotherapy and specific active
immunotherapy. In some instances, non-specific active immunotherapy
utilizes cytokines and/or other cell signaling components to induce
a general immune system response. In some cases, specific active
immunotherapy utilizes specific TAAs to elicite an immune
response.
[0521] In some embodiments, a composition or a pharmaceutical
formulation described herein is used as an active immuno-oncology
therapy method for the treatment of a disease or disorder (e.g.,
cancer). In some embodiments, the composition or a pharmaceutical
formulation described herein comprises a tumor-targeting agent. In
some instances, the tumor-targeting agent is encompassed by a
binding moiety A. In other instances, the tumor-targeting agent is
an additional agent used in combination with a molecule of Formula
(I). In some instances, the tumor-targeting agent is a
tumor-directed polypeptide (e.g., a tumor-directed antibody). In
some instances, the tumor-targeting agent is a tumor-directed
antibody, which exerts its antitumor activity through mechanisms
such as direct killing (e.g., signaling-induced apoptosis),
complement-dependent cytotoxicity (CDC), and/or antibody-dependent
cell-mediated cytotoxicity (ADCC). In additional instances, the
tumor-targeting agent elicits an adaptive immune response, with the
induction of antitumor T cells.
[0522] In some embodiments, the binding moiety A is a
tumor-directed polypeptide (e.g., a tumor-directed antibody). In
some instances, the binding moiety A is a tumor-directed antibody,
which exerts its antitumor activity through mechanisms such as
direct killing (e.g., signaling-induced apoptosis),
complement-dependent cytotoxicity (CDC), and/or antibody-dependent
cell-mediated cytotoxicity (ADCC). In additional instances, the
binding moiety A elicits an adaptive immune response, with the
induction of antitumor T cells.
[0523] In some embodiments, the composition or a pharmaceutical
formulation described herein comprises an immune-targeting agent.
In some instances, the immune-targeting agent is encompassed by a
binding moiety A. In other instances, the immune-targeting agent is
an additional agent used in combination with a molecule of Formula
(I). In some instances, the immune-targeting agent comprises
cytokines, checkpoint inhibitors, or a combination thereof.
[0524] In some embodiments, the immune-targeting agent is a
checkpoint inhibitor. In some cases, an immune checkpoint molecule
is a molecule presented on the cell surface of CD4 and/or CD8 T
cells. Exemplary immune checkpoint molecules include, but are not
limited to, Programmed Death-Ligand 1 (PD-L1, also known as B7-H1,
CD274), Programmed Death 1 (PD-1), CTLA-4, B7H1, B7H4, OX-40,
CD137, CD40, 2B4, IDO1, IDO2, VISTA, CD27, CD28, PD-L2 (B7-DC,
CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR, GAL9, TIM3,
A2aR, MARCO (macrophage receptor with collageneous structure), PS
(phosphatidylserine), ICOS (inducible T cell costimulator), HAVCR2,
CD276, VTCN1, CD70, and CD160.
[0525] In some instances, an immune checkpoint inhibitor refers to
any molecule that modulates or inhibits the activity of an immune
checkpoint molecule. In some instances, immune checkpoint
inhibitors include antibodies, antibody-derivatives (e.g., Fab
fragments, scFvs, minobodies, diabodies), antisense
oligonucleotides, siRNA, aptamers, or peptides. In some
embodiments, an immune checkpoint inhibitor is an inhibitor of
Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274),
Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3,
TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30,
CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9,
GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell
costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with
collageneous structure), PS (phosphatidylserine), OX-40, SLAM,
TIGHT, VISTA, VTCN1, or any combinations thereof.
[0526] In some embodiments, exemplary checkpoint inhibitors
include:
[0527] PD-L1 inhibitors such as Genentech's MPDL3280A (RG7446),
Anti-mouse PD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from
BioXcell, anti-PD-L1 monoclonal antibody MDX-1105 (BMS-936559) and
BMS-935559 from Bristol-Meyer's Squibb, MSB0010718C, mouse
anti-PD-L1 Clone 29E.2A3, and AstraZeneca's MEDI4736;
[0528] PD-L2 inhibitors such as GlaxoSmithKline's AMP-224
(Amplimmune), and rHIgM12B7;
[0529] PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43
(Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone
RMP1-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody
Clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda,
pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody
known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers
Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo.RTM.,
BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and
Pidilizumab (CT-011) from CureTech Ltd;
[0530] CTLA-4 inhibitors such as Bristol Meyers Squibb's
anti-CTLA-4 antibody ipilimumab (also known as Yervoy.RTM.,
MDX-010, BMS-734016 and MDX-101), anti-CTLA4 Antibody, clone 9H10
from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab),
and anti-CTLA4 antibody clone BN13 from Abcam;
[0531] LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W
(C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan
Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody
BMS-986016, and the LAG-3 chimeric antibody A9H12;
[0532] B7-H3 inhibitors such as MGA271;
[0533] KIR inhibitors such as Lirilumab (IPH2101);
[0534] CD137 (41BB) inhibitors such as urelumab (BMS-663513,
Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer),
or XmAb-5592 (Xencor);
[0535] PS inhibitors such as Bavituximab;
[0536] and inhibitors such as an antibody or fragments (e.g., a
monoclonal antibody, a human, humanized, or chimeric antibody)
thereof, RNAi molecules, or small molecules to TIM3, CD52, CD30,
CD20, CD33, CD27, OX40 (CD134), GITR, ICOS, BTLA (CD272), CD160,
2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.
[0537] In some embodiments, a binding moiety A comprising an immune
checkpoint inhibitor is used for the treatment of a disease or
disorder (e.g., cancer). In some instances, the binding moiety A is
a bispecific antibody or a binding fragment thereof that comprises
an immune checkpoint inhibitor. In some cases, a binding moiety A
comprising an inhibitor of Programmed Death-Ligand 1 (PD-L1, also
known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2
(B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2,
CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137,CD160, CD226,
CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T
cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor
with collageneous structure), PS (phosphatidylserine), OX-40, SLAM,
TIGHT, VISTA, VTCN1, or any combinations thereof, is used for the
treatment of a disease or disorder (e.g., cancer).
[0538] In some embodiments, a molecule of Formula (I) in
combination with an immune checkpoint inhibitor is used for the
treatment of a disease or disorder (e.g., cancer). In some
instances, the immune checkpoint inhibitor comprises an inhibitor
of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274),
Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3,
TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30,
CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9,
GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell
costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with
collageneous structure), PS (phosphatidylserine), OX-40, SLAM,
TIGHT, VISTA, VTCN1, or any combinations thereof. In some cases, a
molecule of Formula (I) is used in combination with ipilimumab,
tremelimumab, nivolumab, pemrolizumab, pidilizumab, MPDL3280A,
MEDI4736, MSB0010718C, MK-3475, or BMS-936559, for the treatment of
a disease or disorder (e.g., cancer).
[0539] In some embodiments, the immune-targeting agent is a
cytokine. In some cases, cytokine is further subgrouped into
chemokine, interferon, interleukin, and tumor necrosis factor. In
some embodiments, chemokine plays a role as a chemoattractant to
guide the migration of cells, and is classified into four
subfamilies CXC, CC, CX3C, and XC. Exemplary chemokines include
chemokines from the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4,
CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12,
CCL13, CCL14, CCL1.5, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21,
CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC
subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,
CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and
CXCL17; the XC subfamily: XCL1 and XCL2; and the CX3C subfamily
CX3CL1.
[0540] Interferon (IFNs) comprises interferon type I (e.g.
IFN-.alpha., IFN-.beta., IFN-.epsilon., IFN-.kappa., and
IFN-.omega.), interferon type II (e.g. IFN-.gamma.), and interferon
type III. In some embodiments, IFN-.alpha. is further classified
into about 13 subtypes which include IFNA1, IFNA2, IFNA4, IFNA5,
IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and
IFNA21.
[0541] Interleukin is expressed by leukocyte or white blood cell
and promote the development and differentiation of T and B
lymphocytes and hematopoietic cells. Exemplary interleukins include
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36.
[0542] Tumor necrosis factors (TNFs) are a group of cytokines that
modulate apoptosis. In some instances, there are about 19 members
within the TNF family, including, not limited to, TNF.alpha.,
lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell
antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and
TNF-related apoptosis inducing ligand (TRAIL),
[0543] In some embodiments, a molecule of Formula (I) in
combination with a cytokine is used for the treatment of a disease
or disorder (e.g., cancer). In some cases, a molecule of Formula
(I) in combination with a chemokine is used for the treatment of a
disease or disorder (e.g., cancer). In some cases, a molecule of
Formula (I) in combination with an interferon is used for the
treatment of a disease or disorder (e.g., cancer). In some cases, a
molecule of Formula (I) in combination with an interleukin is used
for the treatment of a disease or disorder (e.g., cancer). In some
cases, a molecule of Formula (I) in combination with a tumor
necrosis factor is used for the treatment of a disease or disorder
(e.g., cancer). In some instances, a molecule of Formula (I) in
combination with IL-1.beta., IL-2, IL-7, IL-8, IL-15, MCP-1 (CCL2),
MIP-1.alpha., RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9,
CXCL10, or CXCL11 is used for the treatment of a disease or
disorder (e.g., cancer).
[0544] In some embodiments, the composition or a pharmaceutical
formulation described herein comprises a vaccine. In some
instances, the vaccine is an in situ vaccination. In some
instances, the vaccine is a cell-based vaccine. In some instances,
the vaccine is a non-cell based vaccine. In some instances, a
molecule of Formula (I) in combination with dendritic cell-based
vaccine is used for the treatment of a disease or disorder (e.g.,
cancer). In some instances, a molecule of Formula (I) in
combination with tumor cell-based vaccine is used for the treatment
of a disease or disorder (e.g., cancer). In some instances, a
molecule of Formula (I) in combination with antigen vaccine is used
for the treatment of a disease or disorder (e.g., cancer). In some
instances, a molecule of Formula (I) in combination with
anti-idiotype vaccine is used for the treatment of a disease or
disorder (e.g., cancer). In some instances, a molecule of Formula
(I) in combination with DNA vaccine is used for the treatment of a
disease or disorder (e.g., cancer). In some instances, a molecule
of Formula (I) in combination with vector-based vaccine is used for
the treatment of a disease or disorder (e.g., cancer).
[0545] In some embodiments, a composition or a pharmaceutical
formulation described herein is used as a passive immuno-oncology
therapy method for the treatment of a disease or disorder (e.g.,
cancer). The passive method, in some instances, utilizes adoptive
immune system components such as T cells, natural killer (NK) T
cells, and/or chimeric antigen receptor (CAR) T cells generated
exogenously to attack cancer cells.
[0546] In some embodiments, a molecule of Formula (I) in
combination with a T-cell based therapeutic agent is used for the
treatment of a disease or disorder (e.g., cancer). In some cases,
the T-cell based therapeutic agent is an activated T-cell agent
that recognizes one or more of a CD cell surface marker described
above. In some instances, the T-cell based therapeutic agent
comprises an activated T-cell agent that recognizes one or more of
CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152,
CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272,
CD274, CD278, CD279, or CD357. In some instances, a molecule of
Formula (I) in combination with an activated T-cell agent
recognizing one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28,
CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226,
CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357 is used
for the treatment of a disease or disorder (e.g., cancer).
[0547] In some embodiments, a molecule of Formula (I) in
combination with natural killer (NK) T cell-based therapeutic agent
is used for the treatment of a disease or disorder (e.g., cancer).
In some instances, the NK-based therapeutic agent is an activated
NK agent that recognizes one or more of a CD cell surface marker
described above. In some cases, the NK-based therapeutic agent is
an activated NK agent that recognizes one or more of CD2, CD11a,
CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c,
CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314, CD335, CD337,
CD244, or CD319. In some instances, a molecule of Formula (I) in
combination with an activated NK agent recognizing one or more of
CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b,
CD158c, CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314, CD335,
CD337, CD244, or CD319 is used for the treatment of a disease or
disorder (e.g., cancer).
[0548] In some embodiments, a molecule of Formula (I) in
combination with CAR-T cell-based therapeutic agent is used for the
treatment of a disease or disorder (e.g., cancer).
[0549] In some embodiments, a molecule of Formula (I) in
combination with an additional agent that destabilizes the
endosomal membrane (or disrupts the endosomal-lysosomal membrane
trafficking) is used for the treatment of a disease or disorder
(e.g., cancer). In some instances, the additional agent comprises
an antimitotic agent. Exemplary antimitotic agents include, but are
not limited to, taxanes such as paclitaxel and docetaxel; vinca
alkaloids such as vinblastine, vincristine, vindesine, and
vinorelbine; cabazitaxel; colchicine; eribulin; estramustine;
etoposide; ixabepilone; podophyllotoxin; teniposide; or
griseofulvin. In some instances, the additional agent comprises
paclitaxel, docetaxel, vinblastine, vincristine, vindesine,
vinorelbine, cabazitaxel, colchicine, eribulin, estramustine,
etoposide, ixabepilone, podophyllotoxin, teniposide, or
griseofulvin. In some instances, the additional agent comprises
taxol. In some instances, the additional agent comprises
paclitaxel. In some instances, the additional agent comprises
etoposide. In other instances, the additional agent comprises
vitamin K3.
[0550] In some embodiments, a composition or a pharmaceutical
formulation described herein is used as a combinatory method
(including for both active and passive methods) in the treatment of
a disease or disorder (e.g., cancer).
Pharmaceutical Formulation
[0551] In some embodiments, the pharmaceutical formulations
described herein are administered to a subject by multiple
administration routes, including but not limited to, parenteral
(e.g., intravenous, subcutaneous, intramuscular), oral, intranasal,
buccal, rectal, or transdermal administration routes. In some
instances, the pharmaceutical composition describe herein is
formulated for parenteral (e.g., intravenous, subcutaneous,
intramuscular) administration. In other instances, the
pharmaceutical composition describe herein is formulated for oral
administration. In still other instances, the pharmaceutical
composition describe herein is formulated for intranasal
administration.
[0552] In some embodiments, the pharmaceutical formulations
include, but are not limited to, aqueous liquid dispersions,
self-emulsifying dispersions, solid solutions, liposomal
dispersions, aerosols, solid dosage forms, powders,
immediate-release formulations, controlled-release formulations,
fast melt formulations, tablets, capsules, pills, delayed release
formulations, extended release formulations, pulsatile release
formulations, multiparticulate formulations (e.g., nanoparticle
formulations), and mixed immediate and controlled release
formulations.
[0553] In some instances, the pharmaceutical formulation includes
multiparticulate formulations. In some instances, the
pharmaceutical formulation includes nanoparticle formulations. In
some instances, nanoparticles comprise cMAP, cyclodextrin, or
lipids. In some cases, nanoparticles comprise solid lipid
nanoparticles, polymeric nanoparticles, self-emulsifying
nanoparticles, liposomes, microemulsions, or micellar solutions.
Additional exemplary nanoparticles include, but are not limited to,
paramagnetic nanoparticles, superparamagnetic nanoparticles, metal
nanoparticles, fullerene-like materials, inorganic nanotubes,
dendrimers (such as with covalently attached metal chelates),
nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantum
dots. In some instances, a nanoparticle is a metal nanoparticle,
e.g., a nanoparticle of scandium, titanium, vanadium, chromium,
manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium,
niobium, molybdenum, ruthenium, rhodium, palladium, silver,
cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,
platinum, gold, gadolinium, aluminum, gallium, indium, tin,
thallium, lead, bismuth, magnesium, calcium, strontium, barium,
lithium, sodium, potassium, boron, silicon, phosphorus, germanium,
arsenic, antimony, and combinations, alloys or oxides thereof.
[0554] In some instances, a nanoparticle includes a core or a core
and a shell, as in a core-shell nanoparticle.
[0555] In some instances, a nanoparticle is further coated with
molecules for attachment of functional elements (e.g., with one or
more of a polynucleic acid molecule or binding moiety described
herein). In some instances, a coating comprises chondroitin
sulfate, dextran sulfate, carboxymethyl dextran, alginic acid,
pectin, carragheenan, fucoidan, agaropectin, porphyran, karaya gum,
gellan gum, xanthan gum, hyaluronic acids, glucosamine,
galactosamine, chitin (or chitosan), polyglutamic acid,
polyaspartic acid, lysozyme, cytochrome C, ribonuclease,
trypsinogen, chymotrypsinogen, .alpha.-chymotrypsin, polylysine,
polyarginine, histone, protamine, ovalbumin, dextrin, or
cyclodextrin. In some instances, a nanoparticle comprises a
graphene-coated nanoparticle.
[0556] In some cases, a nanoparticle has at least one dimension of
less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.
[0557] In some instances, the nanoparticle formulation comprises
paramagnetic nanoparticles, superparamagnetic nanoparticles, metal
nanoparticles, fullerene-like materials, inorganic nanotubes,
dendrimers (such as with covalently attached metal chelates),
nanofibers, nanohorns, nano-onions, nanorods, nanoropes or quantum
dots. In some instances, a polynucleic acid molecule or a binding
moiety described herein is conjugated either directly or indirectly
to the nanoparticle. In some instances, at least 1, 5, 10, 15, 20,
30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules
or binding moieties described herein are conjugated either directly
or indirectly to a nanoparticle.
[0558] In some embodiments, the pharmaceutical formulations include
a carrier or carrier materials selected on the basis of
compatibility with the composition disclosed herein, and the
release profile properties of the desired dosage form. Exemplary
carrier materials include, e.g., binders, suspending agents,
disintegration agents, filling agents, surfactants, solubilizers,
stabilizers, lubricants, wetting agents, diluents, and the like.
Pharmaceutically compatible carrier materials include, but are not
limited to, acacia, gelatin, colloidal silicon dioxide, calcium
glycerophosphate, calcium lactate, maltodextrin, glycerine,
magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol,
cholesterol esters, sodium caseinate, soy lecithin, taurocholic
acid, phosphotidylcholine, sodium chloride, tricalcium phosphate,
dipotassium phosphate, cellulose and cellulose conjugates, sugars
sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride,
pregelatinized starch, and the like. See, e.g., Remington: The
Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack
Publishing Company, 1995); Hoover, John E., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;
Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,
Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage
Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams
& Wilkins1999).
[0559] In some instances, the pharmaceutical formulations further
include pH-adjusting agents or buffering agents which include acids
such as acetic, boric, citric, lactic, phosphoric and hydrochloric
acids; bases such as sodium hydroxide, sodium phosphate, sodium
borate, sodium citrate, sodium acetate, sodium lactate and
tris-hydroxymethylaminomethane; and buffers such as
citrate/dextrose, sodium bicarbonate and ammonium chloride. Such
acids, bases and buffers are included in an amount required to
maintain pH of the composition in an acceptable range.
[0560] In some instances, the pharmaceutical formulation includes
one or more salts in an amount required to bring osmolality of the
composition into an acceptable range. Such salts include those
having sodium, potassium or ammonium cations and chloride, citrate,
ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or
bisulfite anions; suitable salts include sodium chloride, potassium
chloride, sodium thiosulfate, sodium bisulfite and ammonium
sulfate.
[0561] In some instances, the pharmaceutical formulations further
include diluent which are used to stabilize compounds because they
can provide a more stable environment. Salts dissolved in buffered
solutions (which also can provide pH control or maintenance) are
utilized as diluents in the art, including, but not limited to a
phosphate buffered saline solution. In certain instances, diluents
increase bulk of the composition to facilitate compression or
create sufficient bulk for homogenous blend for capsule filling.
Such compounds can include e.g., lactose, starch, mannitol,
sorbitol, dextrose, microcrystalline cellulose such as Avicel.RTM.;
dibasic calcium phosphate, dicalcium phosphate dihydrate;
tricalcium phosphate, calcium phosphate; anhydrous lactose,
spray-dried lactose; pregelatinized starch, compressible sugar,
such as Di-Pac.RTM. (Amstar); mannitol,
hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate
stearate, sucrose-based diluents, confectioner's sugar; monobasic
calcium sulfate monohydrate, calcium sulfate dihydrate; calcium
lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose;
powdered cellulose, calcium carbonate; glycine, kaolin; mannitol,
sodium chloride; inositol, bentonite, and the like.
[0562] In some cases, the pharmaceutical formulations include
disintegration agents or disintegrants to facilitate the breakup or
disintegration of a substance. The term "disintegrate" include both
the dissolution and dispersion of the dosage form when contacted
with gastrointestinal fluid. Examples of disintegration agents
include a starch, e.g., a natural starch such as corn starch or
potato starch, a pregelatinized starch such as National 1551 or
Amijel.RTM., or sodium starch glycolate such as Promogel.RTM. or
Explotab.RTM., a cellulose such as a wood product,
methylcrystalline cellulose, e.g., Avicel.RTM., Avicel.RTM. PH101,
Avicel.RTM. PH102, Avicel.RTM. PH105, Elcema.RTM. P100,
Emcocel.RTM., Vivacel.RTM., Ming Tia.RTM., and Solka-Floc.RTM.,
methylcellulose, croscarmellose, or a cross-linked cellulose, such
as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol.RTM.),
cross-linked carboxymethylcellulose, or cross-linked
croscarmellose, a cross-linked starch such as sodium starch
glycolate, a cross-linked polymer such as crospovidone, a
cross-linked polyvinylpyrrolidone, alginate such as alginic acid or
a salt of alginic acid such as sodium alginate, a clay such as
Veegum.RTM. HV (magnesium aluminum silicate), a gum such as agar,
guar, locust bean, Karaya, pectin, or tragacanth, sodium starch
glycolate, bentonite, a natural sponge, a surfactant, a resin such
as a cation-exchange resin, citrus pulp, sodium lauryl sulfate,
sodium lauryl sulfate in combination starch, and the like.
[0563] In some instances, the pharmaceutical formulations include
filling agents such as lactose, calcium carbonate, calcium
phosphate, dibasic calcium phosphate, calcium sulfate,
microcrystalline cellulose, cellulose powder, dextrose, dextrates,
dextran, starches, pregelatinized starch, sucrose, xylitol,
lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol,
and the like.
[0564] Lubricants and glidants are also optionally included in the
pharmaceutical formulations described herein for preventing,
reducing or inhibiting adhesion or friction of materials. Exemplary
lubricants include, e.g., stearic acid, calcium hydroxide, talc,
sodium stearyl fumerate, a hydrocarbon such as mineral oil, or
hydrogenated vegetable oil such as hydrogenated soybean oil
(Sterotex.RTM.), higher fatty acids and their alkali-metal and
alkaline earth metal salts, such as aluminum, calcium, magnesium,
zinc, stearic acid, sodium stearates, glycerol, talc, waxes,
Stearowet.RTM., boric acid, sodium benzoate, sodium acetate, sodium
chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a
methoxypolyethylene glycol such as Carbowax.TM., sodium oleate,
sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium
or sodium lauryl sulfate, colloidal silica such as Syloid.TM.,
Cab-O-Sil.RTM., a starch such as corn starch, silicone oil, a
surfactant, and the like.
[0565] Plasticizers include compounds used to soften the
microencapsulation material or film coatings to make them less
brittle. Suitable plasticizers include, e.g., polyethylene glycols
such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,
stearic acid, propylene glycol, oleic acid, triethyl cellulose and
triacetin. Plasticizers can also function as dispersing agents or
wetting agents.
[0566] Solubilizers include compounds such as triacetin,
triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl
sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide,
N-methylpyrrolidone, N-hydroxyethylpyrrolidone,
polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl
cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol,
bile salts, polyethylene glycol 200-600, glycofurol, transcutol,
propylene glycol, dimethyl isosorbide, and the like.
[0567] Stabilizers include compounds such as any antioxidation
agents, buffers, acids, preservatives and the like.
[0568] Suspending agents include compounds such as
polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,
polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or
polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer
(S630), polyethylene glycol, e.g., the polyethylene glycol can have
a molecular weight of about 300 to about 6000, or about 3350 to
about 4000, or about 7000 to about 5400, sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, hydroxymethylcellulose acetate
stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate,
gums, such as, e.g., gum tragacanth and gum acacia, guar gum,
xanthans, including xanthan gum, sugars, cellulosics, such as,
e.g., sodium carboxymethylcellulose, methylcellulose, sodium
carboxymethylcellulose, hydroxypropylmethylcellulose,
hydroxyethylcellulose, polysorbate-80, sodium alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan
monolaurate, povidone and the like.
[0569] Surfactants include compounds such as sodium lauryl sulfate,
sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS,
sorbitan monooleate, polyoxyethylene sorbitan monooleate,
polysorbates, polaxomers, bile salts, glyceryl monostearate,
copolymers of ethylene oxide and propylene oxide, e.g.,
Pluronic.RTM. (BASF), and the like. Additional surfactants include
polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,
polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene
alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol
40. Sometimes, surfactants is included to enhance physical
stability or for other purposes.
[0570] Viscosity enhancing agents include, e.g., methyl cellulose,
xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose
acetate stearate, hydroxypropylmethyl cellulose phthalate,
carbomer, polyvinyl alcohol, alginates, acacia, chitosans and
combinations thereof.
[0571] Wetting agents include compounds such as oleic acid,
glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate,
triethanolamine oleate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monolaurate, sodium docusate, sodium
oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween
80, vitamin E TPGS, ammonium salts and the like.
Therapeutic Regimens
[0572] In some embodiments, the pharmaceutical compositions
described herein are administered for therapeutic applications. In
some embodiments, the pharmaceutical composition is administered
once per day, twice per day, three times per day or more. The
pharmaceutical composition is administered daily, every day, every
alternate day, five days a week, once a week, every other week, two
weeks per month, three weeks per month, once a month, twice a
month, three times per month, or more. The pharmaceutical
composition is administered for at least 1 month, 2 months, 3
months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,
10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or
more.
[0573] In some embodiments, one or more pharmaceutical compositions
are administered simutaneously, sequentially, or at an interval
period of time. In some embodiments, one or more pharmaceutical
compositions are administered simutaneously. In some cases, one or
more pharmaceutical compositions are administered sequentially. In
additional cases, one or more pharmaceutical compositions are
administered at an interval period of time (e.g., the first
administration of a first pharmaceutical composition is on day one
followed by an interval of at least 1, 2, 3, 4, 5, or more days
prior to the administration of at least a second pharmaceutical
composition).
[0574] In some embodiments, two or more different pharmaceutical
compositions are coadministered. In some instances, the two or more
different pharmaceutical compositions are coadministered
simutaneously. In some cases, the two or more different
pharmaceutical compositions are coadministered sequentially without
a gap of time between administrations. In other cases, the two or
more different pharmaceutical compositions are coadministered
sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour,
12 hours, 1 day, 2 days, or more between administrations.
[0575] In the case wherein the patient's status does improve, upon
the doctor's discretion the administration of the composition is
given continuously; alternatively, the dose of the composition
being administered is temporarily reduced or temporarily suspended
for a certain length of time (i.e., a "drug holiday"). In some
instances, the length of the drug holiday varies between 2 days and
1 year, including by way of example only, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days,
35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days,
200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365
days. The dose reduction during a drug holiday is from 10%-100%,
including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
[0576] Once improvement of the patient's conditions has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, are optionally
reduced, as a function of the symptoms, to a level at which the
improved disease, disorder or condition is retained.
[0577] In some embodiments, the amount of a given agent that
correspond to such an amount varies depending upon factors such as
the particular compound, the severity of the disease, the identity
(e.g., weight) of the subject or host in need of treatment, but
nevertheless is routinely determined in a manner known in the art
according to the particular circumstances surrounding the case,
including, e.g., the specific agent being administered, the route
of administration, and the subject or host being treated. In some
instances, the desired dose is conveniently presented in a single
dose or as divided doses administered simultaneously (or over a
short period of time) or at appropriate intervals, for example as
two, three, four or more sub-doses per day.
[0578] The foregoing ranges are merely suggestive, as the number of
variables in regard to an individual treatment regime is large, and
considerable excursions from these recommended values are not
uncommon. Such dosages are altered depending on a number of
variables, not limited to the activity of the compound used, the
disease or condition to be treated, the mode of administration, the
requirements of the individual subject, the severity of the disease
or condition being treated, and the judgment of the
practitioner.
[0579] In some embodiments, toxicity and therapeutic efficacy of
such therapeutic regimens are determined by standard pharmaceutical
procedures in cell cultures or experimental animals, including, but
not limited to, the determination of the LD50 (the dose lethal to
50% of the population) and the ED50 (the dose therapeutically
effective in 50% of the population). The dose ratio between the
toxic and therapeutic effects is the therapeutic index and it is
expressed as the ratio between LD50 and ED50. Compounds exhibiting
high therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies are used in formulating a range
of dosage for use in human. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED50 with minimal toxicity. The dosage varies within
this range depending upon the dosage form employed and the route of
administration utilized.
Kits/Article of Manufacture
[0580] Disclosed herein, in certain embodiments, are kits and
articles of manufacture for use with one or more of the
compositions and methods described herein. Such kits include a
carrier, package, or container that is compartmentalized to receive
one or more containers such as vials, tubes, and the like, each of
the container(s) comprising one of the separate elements to be used
in a method described herein. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. In one
embodiment, the containers are formed from a variety of materials
such as glass or plastic.
[0581] The articles of manufacture provided herein contain
packaging materials. Examples of pharmaceutical packaging materials
include, but are not limited to, blister packs, bottles, tubes,
bags, containers, bottles, and any packaging material suitable for
a selected formulation and intended mode of administration and
treatment.
[0582] For example, the container(s) include a molecule of Formula
(I): A-X-B-Y-C, optionally conjugated to an endosomolytic moiety D
as disclosed herein. Such kits optionally include an identifying
description or label or instructions relating to its use in the
methods described herein.
[0583] A kit typically includes labels listing contents and/or
instructions for use and package inserts with instructions for use.
A set of instructions will also typically be included.
[0584] In one embodiment, a label is on or associated with the
container. In one embodiment, a label is on a container when
letters, numbers, or other characters forming the label are
attached, molded or etched into the container itself; a label is
associated with a container when it is present within a receptacle
or carrier that also holds the container, e.g., as a package
insert. In one embodiment, a label is used to indicate that the
contents are to be used for a specific therapeutic application. The
label also indicates directions for use of the contents, such as in
the methods described herein.
[0585] In certain embodiments, the pharmaceutical compositions are
presented in a pack or dispenser device which contains one or more
unit dosage forms containing a compound provided herein. The pack,
for example, contains metal or plastic foil, such as a blister
pack. In one embodiment, the pack or dispenser device is
accompanied by instructions for administration. In one embodiment,
the pack or dispenser is also accompanied with a notice associated
with the container in form prescribed by a governmental agency
regulating the manufacture, use, or sale of pharmaceuticals, which
notice is reflective of approval by the agency of the form of the
drug for human or veterinary administration. Such notice, for
example, is the labeling approved by the U.S. Food and Drug
Administration for prescription drugs, or the approved product
insert. In one embodiment, compositions containing a compound
provided herein formulated in a compatible pharmaceutical carrier
are also prepared, placed in an appropriate container, and labeled
for treatment of an indicated condition.
Certain Terminology
[0586] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. It
is to be understood that the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of any subject matter claimed. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. It must be noted that, as used in
the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly
dictates otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes,"
and "included," is not limiting.
[0587] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5
.mu.L." Generally, the term "about" includes an amount that is
expected to be within experimental error.
[0588] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0589] As used herein, the terms "individual(s)", "subject(s)" and
"patient(s)" mean any mammal. In some embodiments, the mammal is a
human. In some embodiments, the mammal is a non-human. None of the
terms require or are limited to situations characterized by the
supervision (e.g. constant or intermittent) of a health care worker
(e.g. a doctor, a registered nurse, a nurse practitioner, a
physician's assistant, an orderly or a hospice worker).
EXAMPLES
[0590] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1
Sequences
[0591] Tables 1, 4, 7, 8, and 10 illustrate target sequences
described herein. Tables 2, 3, 5, 6, 9, 11, and 12 illustrate
polynucleic acid molecule sequences described herein.
TABLE-US-00002 TABLE 1 KRAS Target Sequences sequence position in
target SEQ ID Id # NM_033360.2 site in NM_033360.2 NO: 182 182-200
AAAUGACUGAAUAUAAACUUGUG 1 183 183-201 AAUGACUGAAUAUAAACUUGUGG 2 197
197-215 AACUUGUGGUAGUUGGAGCUGGU 3 224 224-242
UAGGCAAGAGUGCCUUGACGAUA 4 226 226-244 GGCAAGAGUGCCUUGACGAUACA 5 227
227-245 GCAAGAGUGCCUUGACGAUACAG 6 228 228-246
CAAGAGUGCCUUGACGAUACAGC 7 232 232-250 AGUGCCUUGACGAUACAGCUAAU 8 233
233-251 GUGCCUUGACGAUACAGCUAAUU 9 236 236-254
CCUUGACGAUACAGCUAAUUCAG 10 237 237-255 CUUGACGAUACAGCUAAUUCAGA 11
245 245-263 UACAGCUAAUUCAGAAUCAUUUU 12 266 266-284
UUGUGGACGAAUAUGAUCCAACA 13 269 269-287 UGGACGAAUAUGAUCCAACAAUA 14
270 270-288 GGACGAAUAUGAUCCAACAAUAG 15
TABLE-US-00003 TABLE 2 KRAS siRNA sequences Sequence SEQ SEQ
position in sense strand ID antisense strand ID ID # NM_033360.2
sequence (5'-3') NO: sequence (5'-3') NO: 182 182-200
AUGACUGAAUAUAAACU 16 CAAGUUUAUAUUCAG 17 UGTT UCAUTT 183 183-201
UGACUGAAUAUAAACUU 18 ACAAGUUUAUAUUCA 19 GUTT GUCATT 197 197-215
CUUGUGGUAGUUGGAGC 20 CAGCUCCAACUACCAC 21 UGTT AAGTT 224 224-242
GGCAAGAGUGCCUUGAC 22 UCGUCAAGGCACUCU 23 GATT UGCCTT 226 226-244
CAAGAGUGCCUUGACGA 24 UAUCGUCAAGGCACU 25 UATT CUUGTT 227 227-245
AAGAGUGCCUUGACGAU 26 GUAUCGUCAAGGCAC 27 ACTT UCUUTT 228 228-246
AGAGUGCCUUGACGAUA 28 UGUAUCGUCAAGGCA 29 CATT CUCUTT 232 232-250
UGCCUUGACGAUACAGC 30 UAGCUGUAUCGUCAA 31 UATT GGCATT 233 233-251
GCCUUGACGAUACAGCU 32 UUAGCUGUAUCGUCA 33 AATT AGGCTT 236 236-254
UUGACGAUACAGCUAAU 34 GAAUUAGCUGUAUCG 35 UCTT UCAATT 237 237-255
UGACGAUACAGCUAAUU 36 UGAAUUAGCUGUAUC 37 CATT GUCATT 245 245-263
CAGCUAAUUCAGAAUCA 38 AAUGAUUCUGAAUUA 39 UUTT GCUGTT 266 266-284
GUGGACGAAUAUGAUCC 40 UUGGAUCAUAUUCGU 41 AATT CCACTT 269 269-287
GACGAAUAUGAUCCAAC 42 UUGUUGGAUCAUAUU 43 AATT CGUCTT 270 270-288
ACGAAUAUGAUCCAACA 44 AUUGUUGGAUCAUAU 45 AUTT UCGUTT
TABLE-US-00004 TABLE 3 KRAS siRNA Sequences with Chemical
Modification siRNA sequence with siRNA sequence with sequence
chemical modification SEQ chemical modification SEQ position in
sense strand ID antisense strand ID ID # NM_033360.2 sequence
(5'-3') NO: sequence (5'-3') NO: 182 182-200 auGfaCfuGfaAfuAfuAf 46
CfAfaGfuUfuAfuAfuUfcAfgU aAfcUfuGfdTsdT fcAfudTsdT 47 183 183-201
ugAfcUfgAfaUfaUfaAf 48 AfCfaAfgUfuUfaUfaUfuCfaGf 49 aCfuUfgUfdTsdT
uCfadTsdT 197 197-215 cuUfgUfgGfuAfgUfuGf 50
CfAfgCfuCfcAfaCfuAfcCfaCf 51 gAfgCfuGfdTsdT aAfgdTsdT 224 224-242
ggCfaAfgAfgUfgCfcUf 52 UfCfgUfcAfaGfgCfaCfuCfuUf 53 uGfaCfgAfdTsdT
gCfcdTsdT 226 226-244 caAfgAfgUfgCfcUfuGf 54
UfAfuCfgUfcAfaGfgCfaCfuCf 55 aCfgAfuAfdTsdT uUfgdTsdT 227 227-245
aaGfaGfuGfcCfuUfgAf 56 GfUfaUfcGfuCfaAfgGfcAfcUf 57 cGfaUfaCfdTsdT
cUfudTsdT 228 228-246 agAfgUfgCfcUfuGfaCf 58
UfGfuAfuCfgUfcAfaGfgCfaCf 59 gAfuAfcAfdTsdT uCfudTsdT 232 232-250
ugCfcUfuGfaCfgAfuAf 60 UfAfgCfuGfuAfuCfgUfcAfaGf 61 cAfgCfuAfdTsdT
gCfadTsdT 233 233-251 gcCfuUfgAfcGfaUfaCf 62
UfUfaGfcUfgUfaUfcGfuCfaAf 63 aGfcUfaAfdTsdT gGfcdTsdT 236 236-254
uuGfaCfgAfuAfcAfgCf 64 GfAfaUfuAfgCfuGfuAfuCfgU 65 uAfaUfuCfdTsdT
fcAfadTsdT 237 237-255 ugAfcGfaUfaCfaGfcUf 66
UfGfaAfuUfaGfcUfgUfaUfcGf 67 aAfuUfcAfdTsdT uCfadTsdT 245 245-263
caGfcUfaAfuUfcAfgAf 68 AfAfuGfaUfuCfuGfaAfuUfaGf 69 aUfcAfuUfdTsdT
cUfgdTsdT 266 266-284 guGfgAfcGfaAfuAfuGf 70
UfUfgGfaUfcAfuAfuUfcGfuCf 71 aUfcCfaAfdTsdT cAfcdTsdT 269 269-287
gaCfgAfaUfaUfgAfuCf 72 UfUfgUfuGfgAfuCfaUfaUfuCf 73 cAfaCfaAfdTsdT
gUfcdTsdT 270 270-288 acGfaAfuAfuGfaUfcCf 74
AfUfuGfuUfgGfaUfcAfuAfuU aAfcAfaUfdTsdT fcGfudTsdT 75 siRNA
Sequence with Chemical Modification Info lower case (n) = 2'-O-Me;
Nf = 2'-F; dT = deoxy-T residue; s = phosphorothioate backbone
modification; iB = inverted abasic
TABLE-US-00005 TABLE 4 EGFR Target Sequences sequence of total SEQ
hs 19mer pos. in 23mer target site ID Id # NM_005228.3 in
NM_005228.3 NO: 68 68-86 GGCGGCCGGAGUCCCGAGCUAGC 76 71 71-89
GGCCGGAGUCCCGAGCUAGCCCC 77 72 72-90 GCCGGAGUCCCGAGCUAGCCCCG 78 73
73-91 CCGGAGUCCCGAGCUAGCCCCGG 79 74 74-92 CGGAGUCCCGAGCUAGCCCCGGC
80 75 75-93 GGAGUCCCGAGCUAGCCCCGGCG 81 76 76-94
GAGUCCCGAGCUAGCCCCGGCGG 82 78 78-96 GUCCCGAGCUAGCCCCGGCGGCC 83 114
114-132 CCGGACGACAGGCCACCUCGUCG 84 115 115-133
CGGACGACAGGCCACCUCGUCGG 85 116 116-134 GGACGACAGGCCACCUCGUCGGC 86
117 117-135 GACGACAGGCCACCUCGUCGGCG 87 118 118-136
ACGACAGGCCACCUCGUCGGCGU 88 120 120-138 GACAGGCCACCUCGUCGGCGUCC 89
121 121-139 ACAGGCCACCUCGUCGGCGUCCG 90 122 122-140
CAGGCCACCUCGUCGGCGUCCGC 91 123 123-141 AGGCCACCUCGUCGGCGUCCGCC 92
124 124-142 GGCCACCUCGUCGGCGUCCGCCC 93 125 125-143
GCCACCUCGUCGGCGUCCGCCCG 94 126 126-144 CCACCUCGUCGGCGUCCGCCCGA 95
127 127-145 CACCUCGUCGGCGUCCGCCCGAG 96 128 128-146
ACCUCGUCGGCGUCCGCCCGAGU 97 129 129-147 CCUCGUCGGCGUCCGCCCGAGUC 98
130 130-148 CUCGUCGGCGUCCGCCCGAGUCC 99 131 131-149
UCGUCGGCGUCCGCCCGAGUCCC 100 132 132-150 CGUCGGCGUCCGCCCGAGUCCCC 101
135 135-153 CGGCGUCCGCCCGAGUCCCCGCC 102 136 136-154
GGCGUCCGCCCGAGUCCCCGCCU 103 141 141-159 CCGCCCGAGUCCCCGCCUCGCCG 104
164 164-182 CCAACGCCACAACCACCGCGCAC 105 165 165-183
CAACGCCACAACCACCGCGCACG 106 166 166-184 AACGCCACAACCACCGCGCACGG 107
168 168-186 CGCCACAACCACCGCGCACGGCC 108 169 169-187
GCCACAACCACCGCGCACGGCCC 109 170 170-188 CCACAACCACCGCGCACGGCCCC 110
247 247-265 CGAUGCGACCCUCCGGGACGGCC 111 248 248-266
GAUGCGACCCUCCGGGACGGCCG 112 249 249-267 AUGCGACCCUCCGGGACGGCCGG 113
251 251-269 GCGACCCUCCGGGACGGCCGGGG 114 252 252-270
CGACCCUCCGGGACGGCCGGGGC 115 254 254-272 ACCCUCCGGGACGGCCGGGGCAG 116
329 329-347 AAAGAAAGUUUGCCAAGGCACGA 117 330 330-348
AAGAAAGUUUGCCAAGGCACGAG 118 332 332-350 GAAAGUUUGCCAAGGCACGAGUA 119
333 333-351 AAAGUUUGCCAAGGCACGAGUAA 120 334 334-352
AAGUUUGCCAAGGCACGAGUAAC 121 335 335-353 AGUUUGCCAAGGCACGAGUAACA 122
336 336-354 GUUUGCCAAGGCACGAGUAACAA 123 337 337-355
UUUGCCAAGGCACGAGUAACAAG 124 338 338-356 UUGCCAAGGCACGAGUAACAAGC 125
361 361-379 UCACGCAGUUGGGCACUUUUGAA 126 362 362-380
CACGCAGUUGGGCACUUUUGAAG 127 363 363-381 ACGCAGUUGGGCACUUUUGAAGA 128
364 364-382 CGCAGUUGGGCACUUUUGAAGAU 129 365 365-383
GCAGUUGGGCACUUUUGAAGAUC 130 366 366-384 CAGUUGGGCACUUUUGAAGAUCA 131
367 367-385 AGUUGGGCACUUUUGAAGAUCAU 132 368 368-386
GUUGGGCACUUUUGAAGAUCAUU 133 369 369-387 UUGGGCACUUUUGAAGAUCAUUU 134
377 377-395 UUUUGAAGAUCAUUUUCUCAGCC 135 379 379-397
UUGAAGAUCAUUUUCUCAGCCUC 136 380 380-398 UGAAGAUCAUUUUCUCAGCCUCC 137
385 385-403 AUCAUUUUCUCAGCCUCCAGAGG 138 394 394-412
UCAGCCUCCAGAGGAUGUUCAAU 139 396 396-414 AGCCUCCAGAGGAUGUUCAAUAA 140
397 397-415 GCCUCCAGAGGAUGUUCAAUAAC 141 401 401-419
CCAGAGGAUGUUCAAUAACUGUG 142 403 403-421 AGAGGAUGUUCAAUAACUGUGAG 143
407 407-425 GAUGUUCAAUAACUGUGAGGUGG 144 409 409-427
UGUUCAAUAACUGUGAGGUGGUC 145 410 410-428 GUUCAAUAACUGUGAGGUGGUCC 146
411 411-429 UUCAAUAACUGUGAGGUGGUCCU 147 412 412-430
UCAAUAACUGUGAGGUGGUCCUU 148 413 413-431 CAAUAACUGUGAGGUGGUCCUUG 149
414 414-432 AAUAACUGUGAGGUGGUCCUUGG 150 416 416-434
UAACUGUGAGGUGGUCCUUGGGA 151 418 418-436 ACUGUGAGGUGGUCCUUGGGAAU 152
419 419-437 CUGUGAGGUGGUCCUUGGGAAUU 153 425 425-443
GGUGGUCCUUGGGAAUUUGGAAA 154 431 431-449 CCUUGGGAAUUUGGAAAUUACCU 155
432 432-450 CUUGGGAAUUUGGAAAUUACCUA 156 433 433-451
UUGGGAAUUUGGAAAUUACCUAU 157 434 434-452 UGGGAAUUUGGAAAUUACCUAUG 158
458 458-476 GCAGAGGAAUUAUGAUCUUUCCU 159 459 459-477
CAGAGGAAUUAUGAUCUUUCCUU 160 463 463-481 GGAAUUAUGAUCUUUCCUUCUUA 161
464 464-482 GAAUUAUGAUCUUUCCUUCUUAA 162 466 466-484
AUUAUGAUCUUUCCUUCUUAAAG 163 468 468-486 UAUGAUCUUUCCUUCUUAAAGAC 164
471 471-489 GAUCUUUCCUUCUUAAAGACCAU 165 476 476-494
UUCCUUCUUAAAGACCAUCCAGG 166 477 477-495 UCCUUCUUAAAGACCAUCCAGGA 167
479 479-497 CUUCUUAAAGACCAUCCAGGAGG 168 481 481-499
UCUUAAAGACCAUCCAGGAGGUG 169 482 482-500 CUUAAAGACCAUCCAGGAGGUGG 170
492 492-510 AUCCAGGAGGUGGCUGGUUAUGU 171 493 493-511
UCCAGGAGGUGGCUGGUUAUGUC 172 494 494-512 CCAGGAGGUGGCUGGUUAUGUCC 173
495 495-513 CAGGAGGUGGCUGGUUAUGUCCU 174 496 496-514
AGGAGGUGGCUGGUUAUGUCCUC 175 497 497-515 GGAGGUGGCUGGUUAUGUCCUCA 176
499 499-517 AGGUGGCUGGUUAUGUCCUCAUU 177 520 520-538
UUGCCCUCAACACAGUGGAGCGA 178 542 542-560 AAUUCCUUUGGAAAACCUGCAGA 179
543 543-561 AUUCCUUUGGAAAACCUGCAGAU 180 550 550-568
UGGAAAACCUGCAGAUCAUCAGA 181 551 551-569 GGAAAACCUGCAGAUCAUCAGAG 182
553 553-571 AAAACCUGCAGAUCAUCAGAGGA 183 556 556-574
ACCUGCAGAUCAUCAGAGGAAAU 184 586 586-604 ACGAAAAUUCCUAUGCCUUAGCA 185
587 587-605 CGAAAAUUCCUAUGCCUUAGCAG 186 589 589-607
AAAAUUCCUAUGCCUUAGCAGUC 187 592 592-610 AUUCCUAUGCCUUAGCAGUCUUA 188
593 593-611 UUCCUAUGCCUUAGCAGUCUUAU 189 594 594-612
UCCUAUGCCUUAGCAGUCUUAUC 190 596 596-614 CUAUGCCUUAGCAGUCUUAUCUA 191
597 597-615 UAUGCCUUAGCAGUCUUAUCUAA 192 598 598-616
AUGCCUUAGCAGUCUUAUCUAAC 193 599 599-617 UGCCUUAGCAGUCUUAUCUAACU 194
600 600-618 GCCUUAGCAGUCUUAUCUAACUA 195 601 601-619
CCUUAGCAGUCUUAUCUAACUAU 196 602 602-620 CUUAGCAGUCUUAUCUAACUAUG
197
603 603-621 UUAGCAGUCUUAUCUAACUAUGA 198 604 604-622
UAGCAGUCUUAUCUAACUAUGAU 199 605 605-623 AGCAGUCUUAUCUAACUAUGAUG 200
608 608-626 AGUCUUAUCUAACUAUGAUGCAA 201 609 609-627
GUCUUAUCUAACUAUGAUGCAAA 202 610 610-628 UCUUAUCUAACUAUGAUGCAAAU 203
611 611-629 CUUAUCUAACUAUGAUGCAAAUA 204 612 612-630
UUAUCUAACUAUGAUGCAAAUAA 205 613 613-631 UAUCUAACUAUGAUGCAAAUAAA 206
614 614-632 AUCUAACUAUGAUGCAAAUAAAA 207 616 616-634
CUAACUAUGAUGCAAAUAAAACC 208 622 622-640 AUGAUGCAAAUAAAACCGGACUG 209
623 623-641 UGAUGCAAAUAAAACCGGACUGA 210 624 624-642
GAUGCAAAUAAAACCGGACUGAA 211 626 626-644 UGCAAAUAAAACCGGACUGAAGG 212
627 627-645 GCAAAUAAAACCGGACUGAAGGA 213 628 628-646
CAAAUAAAACCGGACUGAAGGAG 214 630 630-648 AAUAAAACCGGACUGAAGGAGCU 215
631 631-649 AUAAAACCGGACUGAAGGAGCUG 216 632 632-650
UAAAACCGGACUGAAGGAGCUGC 217 633 633-651 AAAACCGGACUGAAGGAGCUGCC 218
644 644-662 GAAGGAGCUGCCCAUGAGAAAUU 219 665 665-683
UUUACAGGAAAUCCUGCAUGGCG 220 668 668-686 ACAGGAAAUCCUGCAUGGCGCCG 221
669 669-687 CAGGAAAUCCUGCAUGGCGCCGU 222 670 670-688
AGGAAAUCCUGCAUGGCGCCGUG 223 671 671-689 GGAAAUCCUGCAUGGCGCCGUGC 224
672 672-690 GAAAUCCUGCAUGGCGCCGUGCG 225 674 674-692
AAUCCUGCAUGGCGCCGUGCGGU 226 676 676-694 UCCUGCAUGGCGCCGUGCGGUUC 227
677 677-695 CCUGCAUGGCGCCGUGCGGUUCA 228 678 678-696
CUGCAUGGCGCCGUGCGGUUCAG 229 680 680-698 GCAUGGCGCCGUGCGGUUCAGCA 230
681 681-699 CAUGGCGCCGUGCGGUUCAGCAA 231 682 682-700
AUGGCGCCGUGCGGUUCAGCAAC 232 683 683-701 UGGCGCCGUGCGGUUCAGCAACA 233
684 684-702 GGCGCCGUGCGGUUCAGCAACAA 234 685 685-703
GCGCCGUGCGGUUCAGCAACAAC 235 686 686-704 CGCCGUGCGGUUCAGCAACAACC 236
688 688-706 CCGUGCGGUUCAGCAACAACCCU 237 690 690-708
GUGCGGUUCAGCAACAACCCUGC 238 692 692-710 GCGGUUCAGCAACAACCCUGCCC 239
698 698-716 CAGCAACAACCCUGCCCUGUGCA 240 700 700-718
GCAACAACCCUGCCCUGUGCAAC 241 719 719-737 CAACGUGGAGAGCAUCCAGUGGC 242
720 720-738 AACGUGGAGAGCAUCCAGUGGCG 243 721 721-739
ACGUGGAGAGCAUCCAGUGGCGG 244 724 724-742 UGGAGAGCAUCCAGUGGCGGGAC 245
725 725-743 GGAGAGCAUCCAGUGGCGGGACA 246 726 726-744
GAGAGCAUCCAGUGGCGGGACAU 247 733 733-751 UCCAGUGGCGGGACAUAGUCAGC 248
734 734-752 CCAGUGGCGGGACAUAGUCAGCA 249 736 736-754
AGUGGCGGGACAUAGUCAGCAGU 250 737 737-755 GUGGCGGGACAUAGUCAGCAGUG 251
763 763-781 UUCUCAGCAACAUGUCGAUGGAC 252 765 765-783
CUCAGCAACAUGUCGAUGGACUU 253 766 766-784 UCAGCAACAUGUCGAUGGACUUC 254
767 767-785 CAGCAACAUGUCGAUGGACUUCC 255 769 769-787
GCAACAUGUCGAUGGACUUCCAG 256 770 770-788 CAACAUGUCGAUGGACUUCCAGA 257
771 771-789 AACAUGUCGAUGGACUUCCAGAA 258 772 772-790
ACAUGUCGAUGGACUUCCAGAAC 259 775 775-793 UGUCGAUGGACUUCCAGAACCAC 260
789 789-807 CAGAACCACCUGGGCAGCUGCCA 261 798 798-816
CUGGGCAGCUGCCAAAAGUGUGA 262 800 800-818 GGGCAGCUGCCAAAAGUGUGAUC 263
805 805-823 GCUGCCAAAAGUGUGAUCCAAGC 264 806 806-824
CUGCCAAAAGUGUGAUCCAAGCU 265 807 807-825 UGCCAAAAGUGUGAUCCAAGCUG 266
810 810-828 CAAAAGUGUGAUCCAAGCUGUCC 267 814 814-832
AGUGUGAUCCAAGCUGUCCCAAU 268 815 815-833 GUGUGAUCCAAGCUGUCCCAAUG 269
817 817-835 GUGAUCCAAGCUGUCCCAAUGGG 270 818 818-836
UGAUCCAAGCUGUCCCAAUGGGA 271 819 819-837 GAUCCAAGCUGUCCCAAUGGGAG 272
820 820-838 AUCCAAGCUGUCCCAAUGGGAGC 273 821 821-839
UCCAAGCUGUCCCAAUGGGAGCU 274 823 823-841 CAAGCUGUCCCAAUGGGAGCUGC 275
826 826-844 GCUGUCCCAAUGGGAGCUGCUGG 276 847 847-865
GGGGUGCAGGAGAGGAGAACUGC 277 871 871-889 AGAAACUGACCAAAAUCAUCUGU 278
872 872-890 GAAACUGACCAAAAUCAUCUGUG 279 873 873-891
AAACUGACCAAAAUCAUCUGUGC 280 877 877-895 UGACCAAAAUCAUCUGUGCCCAG 281
878 878-896 GACCAAAAUCAUCUGUGCCCAGC 282 881 881-899
CAAAAUCAUCUGUGCCCAGCAGU 283 890 890-908 CUGUGCCCAGCAGUGCUCCGGGC 284
892 892-910 GUGCCCAGCAGUGCUCCGGGCGC 285 929 929-947
CCCCAGUGACUGCUGCCACAACC 286 930 930-948 CCCAGUGACUGCUGCCACAACCA 287
979 979-997 GGGAGAGCGACUGCCUGGUCUGC 288 980 980-998
GGAGAGCGACUGCCUGGUCUGCC 289 981 981-999 GAGAGCGACUGCCUGGUCUGCCG 290
982 982-1000 AGAGCGACUGCCUGGUCUGCCGC 291 983 983-1001
GAGCGACUGCCUGGUCUGCCGCA 292 984 984-1002 AGCGACUGCCUGGUCUGCCGCAA
293 989 989-1007 CUGCCUGGUCUGCCGCAAAUUCC 294 990 990-1008
UGCCUGGUCUGCCGCAAAUUCCG 295 991 991-1009 GCCUGGUCUGCCGCAAAUUCCGA
296 992 992-1010 CCUGGUCUGCCGCAAAUUCCGAG 297 994 994-1012
UGGUCUGCCGCAAAUUCCGAGAC 298 995 995-1013 GGUCUGCCGCAAAUUCCGAGACG
299 996 996-1014 GUCUGCCGCAAAUUCCGAGACGA 300 997 997-1015
UCUGCCGCAAAUUCCGAGACGAA 301 999 999-1017 UGCCGCAAAUUCCGAGACGAAGC
302 1004 1004-1022 CAAAUUCCGAGACGAAGCCACGU 303 1005 1005-1023
AAAUUCCGAGACGAAGCCACGUG 304 1006 1006-1024 AAUUCCGAGACGAAGCCACGUGC
305 1007 1007-1025 AUUCCGAGACGAAGCCACGUGCA 306 1008 1008-1026
UUCCGAGACGAAGCCACGUGCAA 307 1010 1010-1028 CCGAGACGAAGCCACGUGCAAGG
308 1013 1013-1031 AGACGAAGCCACGUGCAAGGACA 309 1014 1014-1032
GACGAAGCCACGUGCAAGGACAC 310 1015 1015-1033 ACGAAGCCACGUGCAAGGACACC
311 1016 1016-1034 CGAAGCCACGUGCAAGGACACCU 312 1040 1040-1058
CCCCCCACUCAUGCUCUACAACC 313 1042 1042-1060 CCCCACUCAUGCUCUACAACCCC
314 1044 1044-1062 CCACUCAUGCUCUACAACCCCAC 315 1047 1047-1065
CUCAUGCUCUACAACCCCACCAC 316 1071 1071-1089 UACCAGAUGGAUGUGAACCCCGA
317 1073 1073-1091 CCAGAUGGAUGUGAACCCCGAGG 318 1074 1074-1092
CAGAUGGAUGUGAACCCCGAGGG 319 1075 1075-1093 AGAUGGAUGUGAACCCCGAGGGC
320 1077 1077-1095 AUGGAUGUGAACCCCGAGGGCAA 321 1078 1078-1096
UGGAUGUGAACCCCGAGGGCAAA 322
1080 1080-1098 GAUGUGAACCCCGAGGGCAAAUA 323 1084 1084-1102
UGAACCCCGAGGGCAAAUACAGC 324 1085 1085-1103 GAACCCCGAGGGCAAAUACAGCU
325 1087 1087-1105 ACCCCGAGGGCAAAUACAGCUUU 326 1088 1088-1106
CCCCGAGGGCAAAUACAGCUUUG 327 1089 1089-1107 CCCGAGGGCAAAUACAGCUUUGG
328 1096 1096-1114 GCAAAUACAGCUUUGGUGCCACC 329 1097 1097-1115
CAAAUACAGCUUUGGUGCCACCU 330 1098 1098-1116 AAAUACAGCUUUGGUGCCACCUG
331 1104 1104-1122 AGCUUUGGUGCCACCUGCGUGAA 332 1106 1106-1124
CUUUGGUGCCACCUGCGUGAAGA 333 1112 1112-1130 UGCCACCUGCGUGAAGAAGUGUC
334 1116 1116-1134 ACCUGCGUGAAGAAGUGUCCCCG 335 1117 1117-1135
CCUGCGUGAAGAAGUGUCCCCGU 336 1118 1118-1136 CUGCGUGAAGAAGUGUCCCCGUA
337 1119 1119-1137 UGCGUGAAGAAGUGUCCCCGUAA 338 1120 1120-1138
GCGUGAAGAAGUGUCCCCGUAAU 339 1121 1121-1139 CGUGAAGAAGUGUCCCCGUAAUU
340 1122 1122-1140 GUGAAGAAGUGUCCCCGUAAUUA 341 1123 1123-1141
UGAAGAAGUGUCCCCGUAAUUAU 342 1124 1124-1142 GAAGAAGUGUCCCCGUAAUUAUG
343 1125 1125-1143 AAGAAGUGUCCCCGUAAUUAUGU 344 1126 1126-1144
AGAAGUGUCCCCGUAAUUAUGUG 345 1127 1127-1145 GAAGUGUCCCCGUAAUUAUGUGG
346 1128 1128-1146 AAGUGUCCCCGUAAUUAUGUGGU 347 1129 1129-1147
AGUGUCCCCGUAAUUAUGUGGUG 348 1130 1130-1148 GUGUCCCCGUAAUUAUGUGGUGA
349 1132 1132-1150 GUCCCCGUAAUUAUGUGGUGACA 350 1134 1134-1152
CCCCGUAAUUAUGUGGUGACAGA 351 1136 1136-1154 CCGUAAUUAUGUGGUGACAGAUC
352 1137 1137-1155 CGUAAUUAUGUGGUGACAGAUCA 353 1138 1138-1156
GUAAUUAUGUGGUGACAGAUCAC 354 1139 1139-1157 UAAUUAUGUGGUGACAGAUCACG
355 1140 1140-1158 AAUUAUGUGGUGACAGAUCACGG 356 1142 1142-1160
UUAUGUGGUGACAGAUCACGGCU 357 1145 1145-1163 UGUGGUGACAGAUCACGGCUCGU
358 1147 1147-1165 UGGUGACAGAUCACGGCUCGUGC 359 1148 1148-1166
GGUGACAGAUCACGGCUCGUGCG 360 1149 1149-1167 GUGACAGAUCACGGCUCGUGCGU
361 1150 1150-1168 UGACAGAUCACGGCUCGUGCGUC 362 1151 1151-1169
GACAGAUCACGGCUCGUGCGUCC 363 1152 1152-1170 ACAGAUCACGGCUCGUGCGUCCG
364 1153 1153-1171 CAGAUCACGGCUCGUGCGUCCGA 365 1154 1154-1172
AGAUCACGGCUCGUGCGUCCGAG 366 1155 1155-1173 GAUCACGGCUCGUGCGUCCGAGC
367 1156 1156-1174 AUCACGGCUCGUGCGUCCGAGCC 368 1157 1157-1175
UCACGGCUCGUGCGUCCGAGCCU 369 1160 1160-1178 CGGCUCGUGCGUCCGAGCCUGUG
370 1200 1200-1218 AUGGAGGAAGACGGCGUCCGCAA 371 1201 1201-1219
UGGAGGAAGACGGCGUCCGCAAG 372 1203 1203-1221 GAGGAAGACGGCGUCCGCAAGUG
373 1204 1204-1222 AGGAAGACGGCGUCCGCAAGUGU 374 1205 1205-1223
GGAAGACGGCGUCCGCAAGUGUA 375 1207 1207-1225 AAGACGGCGUCCGCAAGUGUAAG
376 1208 1208-1226 AGACGGCGUCCGCAAGUGUAAGA 377 1211 1211-1229
CGGCGUCCGCAAGUGUAAGAAGU 378 1212 1212-1230 GGCGUCCGCAAGUGUAAGAAGUG
379 1213 1213-1231 GCGUCCGCAAGUGUAAGAAGUGC 380 1214 1214-1232
CGUCCGCAAGUGUAAGAAGUGCG 381 1215 1215-1233 GUCCGCAAGUGUAAGAAGUGCGA
382 1216 1216-1234 UCCGCAAGUGUAAGAAGUGCGAA 383 1217 1217-1235
CCGCAAGUGUAAGAAGUGCGAAG 384 1219 1219-1237 GCAAGUGUAAGAAGUGCGAAGGG
385 1220 1220-1238 CAAGUGUAAGAAGUGCGAAGGGC 386 1221 1221-1239
AAGUGUAAGAAGUGCGAAGGGCC 387 1222 1222-1240 AGUGUAAGAAGUGCGAAGGGCCU
388 1223 1223-1241 GUGUAAGAAGUGCGAAGGGCCUU 389 1224 1224-1242
UGUAAGAAGUGCGAAGGGCCUUG 390 1225 1225-1243 GUAAGAAGUGCGAAGGGCCUUGC
391 1226 1226-1244 UAAGAAGUGCGAAGGGCCUUGCC 392 1229 1229-1247
GAAGUGCGAAGGGCCUUGCCGCA 393 1230 1230-1248 AAGUGCGAAGGGCCUUGCCGCAA
394 1231 1231-1249 AGUGCGAAGGGCCUUGCCGCAAA 395 1232 1232-1250
GUGCGAAGGGCCUUGCCGCAAAG 396 1233 1233-1251 UGCGAAGGGCCUUGCCGCAAAGU
397 1235 1235-1253 CGAAGGGCCUUGCCGCAAAGUGU 398 1236 1236-1254
GAAGGGCCUUGCCGCAAAGUGUG 399 1237 1237-1255 AAGGGCCUUGCCGCAAAGUGUGU
400 1238 1238-1256 AGGGCCUUGCCGCAAAGUGUGUA 401 1239 1239-1257
GGGCCUUGCCGCAAAGUGUGUAA 402 1241 1241-1259 GCCUUGCCGCAAAGUGUGUAACG
403 1261 1261-1279 ACGGAAUAGGUAUUGGUGAAUUU 404 1262 1262-1280
CGGAAUAGGUAUUGGUGAAUUUA 405 1263 1263-1281 GGAAUAGGUAUUGGUGAAUUUAA
406 1264 1264-1282 GAAUAGGUAUUGGUGAAUUUAAA 407 1266 1266-1284
AUAGGUAUUGGUGAAUUUAAAGA 408 1267 1267-1285 UAGGUAUUGGUGAAUUUAAAGAC
409 1289 1289-1307 CUCACUCUCCAUAAAUGCUACGA 410 1313 1313-1331
UAUUAAACACUUCAAAAACUGCA 411 1320 1320-1338 CACUUCAAAAACUGCACCUCCAU
412 1321 1321-1339 ACUUCAAAAACUGCACCUCCAUC 413 1322 1322-1340
CUUCAAAAACUGCACCUCCAUCA 414 1323 1323-1341 UUCAAAAACUGCACCUCCAUCAG
415 1324 1324-1342 UCAAAAACUGCACCUCCAUCAGU 416 1328 1328-1346
AAACUGCACCUCCAUCAGUGGCG 417 1332 1332-1350 UGCACCUCCAUCAGUGGCGAUCU
418 1333 1333-1351 GCACCUCCAUCAGUGGCGAUCUC 419 1335 1335-1353
ACCUCCAUCAGUGGCGAUCUCCA 420 1338 1338-1356 UCCAUCAGUGGCGAUCUCCACAU
421 1344 1344-1362 AGUGGCGAUCUCCACAUCCUGCC 422 1345 1345-1363
GUGGCGAUCUCCACAUCCUGCCG 423 1346 1346-1364 UGGCGAUCUCCACAUCCUGCCGG
424 1347 1347-1365 GGCGAUCUCCACAUCCUGCCGGU 425 1348 1348-1366
GCGAUCUCCACAUCCUGCCGGUG 426 1353 1353-1371 CUCCACAUCCUGCCGGUGGCAUU
427 1354 1354-1372 UCCACAUCCUGCCGGUGGCAUUU 428 1355 1355-1373
CCACAUCCUGCCGGUGGCAUUUA 429 1357 1357-1375 ACAUCCUGCCGGUGGCAUUUAGG
430 1360 1360-1378 UCCUGCCGGUGGCAUUUAGGGGU 431 1361 1361-1379
CCUGCCGGUGGCAUUUAGGGGUG 432 1362 1362-1380 CUGCCGGUGGCAUUUAGGGGUGA
433 1363 1363-1381 UGCCGGUGGCAUUUAGGGGUGAC 434 1366 1366-1384
CGGUGGCAUUUAGGGGUGACUCC 435 1369 1369-1387 UGGCAUUUAGGGGUGACUCCUUC
436 1370 1370-1388 GGCAUUUAGGGGUGACUCCUUCA 437 1371 1371-1389
GCAUUUAGGGGUGACUCCUUCAC 438 1372 1372-1390 CAUUUAGGGGUGACUCCUUCACA
439 1373 1373-1391 AUUUAGGGGUGACUCCUUCACAC 440 1374 1374-1392
UUUAGGGGUGACUCCUUCACACA 441 1404 1404-1422 CCUCUGGAUCCACAGGAACUGGA
442 1408 1408-1426 UGGAUCCACAGGAACUGGAUAUU 443 1409 1409-1427
GGAUCCACAGGAACUGGAUAUUC 444 1411 1411-1429 AUCCACAGGAACUGGAUAUUCUG
445 1412 1412-1430 UCCACAGGAACUGGAUAUUCUGA 446 1419 1419-1437
GAACUGGAUAUUCUGAAAACCGU 447 1426 1426-1444 AUAUUCUGAAAACCGUAAAGGAA
448
1427 1427-1445 UAUUCUGAAAACCGUAAAGGAAA 449 1430 1430-1448
UCUGAAAACCGUAAAGGAAAUCA 450 1431 1431-1449 CUGAAAACCGUAAAGGAAAUCAC
451
TABLE-US-00006 TABLE 5 EGFR siRNA Sequences Sequence SEQ SEQ hs Id
position in sense strand ID antisense strand ID # NM_005228.3
sequence (5'-3') NO: sequence (5'-3') NO: 68 68-86 CGGCCGGAGUCCCGAG
452 UAGCUCGGGACUCCGGC 453 CUATT CGTT 71 71-89 CCGGAGUCCCGAGCUA 454
GGCUAGCUCGGGACUCC 455 GCCTT GGTT 72 72-90 CGGAGUCCCGAGCUAG 456
GGGCUAGCUCGGGACUC 457 CCCTT CGTT 73 73-91 GGAGUCCCGAGCUAGC 458
GGGGCUAGCUCGGGACU 459 CCCTT CCTT 74 74-92 GAGUCCCGAGCUAGCC 460
CGGGGCUAGCUCGGGAC 461 CCGTT UCTT 75 75-93 AGUCCCGAGCUAGCCC 462
CCGGGGCUAGCUCGGGA 463 CGGTT CUTT 76 76-94 GUCCCGAGCUAGCCCC 464
GCCGGGGCUAGCUCGGG 465 GGCTT ACTT 78 78-96 CCCGAGCUAGCCCCGG 466
CCGCCGGGGCUAGCUCG 467 CGGTT GGTT 114 114-132 GGACGACAGGCCACCU 468
ACGAGGUGGCCUGUCGU 469 CGUTT CCTT 115 115-133 GACGACAGGCCACCUC 470
GACGAGGUGGCCUGUCG 471 GUCTT UCTT 116 116-134 ACGACAGGCCACCUCG 472
CGACGAGGUGGCCUGUC 473 UCGTT GUTT 117 117-135 CGACAGGCCACCUCGU 474
CCGACGAGGUGGCCUGU 475 CGGTT CGTT 118 118-136 GACAGGCCACCUCGUC 476
GCCGACGAGGUGGCCUG 477 GGCTT UCTT 120 120-138 CAGGCCACCUCGUCGG 478
ACGCCGACGAGGUGGCC 479 CGUTT UGTT 121 121-139 AGGCCACCUCGUCGGC 480
GACGCCGACGAGGUGGC 481 GUCTT CUTT 122 122-140 GGCCACCUCGUCGGCG 482
GGACGCCGACGAGGUGG 483 UCCTT CCTT 123 123-141 GCCACCUCGUCGGCGU 484
CGGACGCCGACGAGGUG 485 CCGTT GCTT 124 124-142 CCACCUCGUCGGCGUC 486
GCGGACGCCGACGAGGU 487 CGCTT GGTT 125 125-143 CACCUCGUCGGCGUCC 488
GGCGGACGCCGACGAGG 489 GCCTT UGTT 126 126-144 ACCUCGUCGGCGUCCG 490
GGGCGGACGCCGACGAG 491 CCCTT GUTT 127 127-145 CCUCGUCGGCGUCCGC 492
CGGGCGGACGCCGACGA 493 CCGTT GGTT 128 128-146 CUCGUCGGCGUCCGCC 494
UCGGGCGGACGCCGACG 495 CGATT AGTT 129 129-147 UCGUCGGCGUCCGCCC 496
CUCGGGCGGACGCCGAC 497 GAGTT GATT 130 130-148 CGUCGGCGUCCGCCCG 498
ACUCGGGCGGACGCCGA 499 AGUTT CGTT 131 131-149 GUCGGCGUCCGCCCGA 500
GACUCGGGCGGACGCCG 501 GUCTT ACTT 132 132-150 UCGGCGUCCGCCCGAG 502
GGACUCGGGCGGACGCC 503 UCCTT GATT 135 135-153 GCGUCCGCCCGAGUCC 504
CGGGGACUCGGGCGGAC 505 CCGTT GCTT 136 136-154 CGUCCGCCCGAGUCCC 506
GCGGGGACUCGGGCGGA 507 CGCTT CGTT 141 141-159 GCCCGAGUCCCCGCCU 508
GCGAGGCGGGGACUCGG 509 CGCTT GCTT 164 164-182 AACGCCACAACCACCG 510
GCGCGGUGGUUGUGGCG 511 CGCTT UUTT 165 165-183 ACGCCACAACCACCGC 512
UGCGCGGUGGUUGUGGC 513 GCATT GUTT 166 166-184 CGCCACAACCACCGCG 514
GUGCGCGGUGGUUGUGG 515 CACTT CGTT 168 168-186 CCACAACCACCGCGCA 516
CCGUGCGCGGUGGUUGU 517 CGGTT GGTT 169 169-187 CACAACCACCGCGCAC 518
GCCGUGCGCGGUGGUUG 519 GGCTT UGTT 170 170-188 ACAACCACCGCGCACG 520
GGCCGUGCGCGGUGGUU 521 GCCTT GUTT 247 247-265 AUGCGACCCUCCGGGA 522
CCGUCCCGGAGGGUCGC 523 CGGTT AUTT 248 248-266 UGCGACCCUCCGGGAC 524
GCCGUCCCGGAGGGUCG 525 GGCTT CATT 249 249-267 GCGACCCUCCGGGACG 526
GGCCGUCCCGGAGGGUC 527 GCCTT GCTT 251 251-269 GACCCUCCGGGACGGC 528
CCGGCCGUCCCGGAGGG 529 CGGTT UCTT 252 252-270 ACCCUCCGGGACGGCC 530
CCCGGCCGUCCCGGAGG 531 GGGTT GUTT 254 254-272 CCUCCGGGACGGCCGG 532
GCCCCGGCCGUCCCGGA 533 GGCTT GGTT 329 329-347 AGAAAGUUUGCCAAGG 534
GUGCCUUGGCAAACUUU 535 CACTT CUTT 330 330-348 GAAAGUUUGCCAAGGC 536
CGUGCCUUGGCAAACUU 537 ACGTT UCTT 332 332-350 AAGUUUGCCAAGGCAC 538
CUCGUGCCUUGGCAAAC 539 GAGTT UUTT 333 333-351 AGUUUGCCAAGGCACG 540
ACUCGUGCCUUGGCAAA 541 AGUTT CUTT 334 334-352 GUUUGCCAAGGCACGA 542
UACUCGUGCCUUGGCAA 543 GUATT ACTT 335 335-353 UUUGCCAAGGCACGAG 544
UUACUCGUGCCUUGGCA 545 UAATT AATT 336 336-354 UUGCCAAGGCACGAGU 546
GUUACUCGUGCCUUGGC 547 AACTT AATT 337 337-355 UGCCAAGGCACGAGUA 548
UGUUACUCGUGCCUUGG 549 ACATT CATT 338 338-356 GCCAAGGCACGAGUAA 550
UUGUUACUCGUGCCUUG 551 CAATT GCTT 361 361-379 ACGCAGUUGGGCACUU 552
CAAAAGUGCCCAACUGC 553 UUGTT GUTT 362 362-380 CGCAGUUGGGCACUUU 554
UCAAAAGUGCCCAACUG 555 UGATT CGTT 363 363-381 GCAGUUGGGCACUUUU 556
UUCAAAAGUGCCCAACU 557 GAATT GCTT 364 364-382 CAGUUGGGCACUUUUG 558
CUUCAAAAGUGCCCAAC 559 AAGTT UGTT 365 365-383 AGUUGGGCACUUUUGA 560
UCUUCAAAAGUGCCCAA 561 AGATT CUTT 366 366-384 GUUGGGCACUUUUGAA 562
AUCUUCAAAAGUGCCCA 563 GAUTT ACTT 367 367-385 UUGGGCACUUUUGAAG 564
GAUCUUCAAAAGUGCCC 565 AUCTT AATT 368 368-386 UGGGCACUUUUGAAGA 566
UGAUCUUCAAAAGUGCC 567 UCATT CATT 369 369-387 GGGCACUUUUGAAGAU 568
AUGAUCUUCAAAAGUGC 569 CAUTT CCTT 377 377-395 UUGAAGAUCAUUUUCU 570
CUGAGAAAAUGAUCUUC 571 CAGTT AATT 379 379-397 GAAGAUCAUUUUCUCA 572
GGCUGAGAAAAUGAUCU 573 GCCTT UCTT 380 380-398 AAGAUCAUUUUCUCAG 574
AGGCUGAGAAAAUGAUC 575 CCUTT UUTT 385 385-403 CAUUUUCUCAGCCUCC 576
UCUGGAGGCUGAGAAAA 577 AGATT UGTT 394 394-412 AGCCUCCAGAGGAUGU 578
UGAACAUCCUCUGGAGG 579 UCATT CUTT 396 396-414 CCUCCAGAGGAUGUUC 580
AUUGAACAUCCUCUGGA 581 AAUTT GGTT 397 397-415 CUCCAGAGGAUGUUCA 582
UAUUGAACAUCCUCUGG 583 AUATT AGTT 401 401-419 AGAGGAUGUUCAAUAA 584
CAGUUAUUGAACAUCCU 585 CUGTT CUTT 403 403-421 AGGAUGUUCAAUAACU 586
CACAGUUAUUGAACAUC 587 GUGTT CUTT 407 407-425 UGUUCAAUAACUGUGA 588
ACCUCACAGUUAUUGAA 589 GGUTT CATT 409 409-427 UUCAAUAACUGUGAGG 590
CCACCUCACAGUUAUUG 591 UGGTT AATT 410 410-428 UCAAUAACUGUGAGGU 592
ACCACCUCACAGUUAUU 593 GGUTT GATT 411 411-429 CAAUAACUGUGAGGUG 594
GACCACCUCACAGUUAU 595 GUCTT UGTT 412 412-430 AAUAACUGUGAGGUGG 596
GGACCACCUCACAGUUA 597 UCCTT UUTT 413 413-431 AUAACUGUGAGGUGGU 598
AGGACCACCUCACAGUU 599 CCUTT AUTT 414 414-432 UAACUGUGAGGUGGUC 600
AAGGACCACCUCACAGU 601 CUUTT UATT 416 416-434 ACUGUGAGGUGGUCCU 602
CCAAGGACCACCUCACA 603 UGGTT GUTT 418 418-436 UGUGAGGUGGUCCUUG 604
UCCCAAGGACCACCUCA 605 GGATT CATT 419 419-437 GUGAGGUGGUCCUUGG 606
UUCCCAAGGACCACCUC 607 GAATT ACTT 425 425-443 UGGUCCUUGGGAAUUU 608
UCCAAAUUCCCAAGGAC 609 GGATT CATT 431 431-449 UUGGGAAUUUGGAAAU 610
GUAAUUUCCAAAUUCCC 611 UACTT AATT 432 432-450 UGGGAAUUUGGAAAUU 612
GGUAAUUUCCAAAUUCC 613 ACCTT CATT
433 433-451 GGGAAUUUGGAAAUUA 614 AGGUAAUUUCCAAAUUC 615 CCUTT CCTT
434 434-452 GGAAUUUGGAAAUUAC 616 UAGGUAAUUUCCAAAUU 617 CUATT CCTT
458 458-476 AGAGGAAUUAUGAUCU 618 GAAAGAUCAUAAUUCCU 619 UUCTT CUTT
459 459-477 GAGGAAUUAUGAUCUU 620 GGAAAGAUCAUAAUUCC 621 UCCTT UCTT
463 463-481 AAUUAUGAUCUUUCCU 622 AGAAGGAAAGAUCAUAA 623 UCUTT UUTT
464 464-482 AUUAUGAUCUUUCCUU 624 AAGAAGGAAAGAUCAUA 625 CUUTT AUTT
466 466-484 UAUGAUCUUUCCUUCU 626 UUAAGAAGGAAAGAUCA 627 UAATT UATT
468 468-486 UGAUCUUUCCUUCUUA 628 CUUUAAGAAGGAAAGAU 629 AAGTT CATT
471 471-489 UCUUUCCUUCUUAAAG 630 GGUCUUUAAGAAGGAAA 631 ACCTT GATT
476 476-494 CCUUCUUAAAGACCAU 632 UGGAUGGUCUUUAAGAA 633 CCATT GGTT
477 477-495 CUUCUUAAAGACCAUC 634 CUGGAUGGUCUUUAAGA 635 CAGTT AGTT
479 479-497 UCUUAAAGACCAUCCA 636 UCCUGGAUGGUCUUUAA 637 GGATT GATT
481 481-499 UUAAAGACCAUCCAGG 638 CCUCCUGGAUGGUCUUU 639 AGGTT AATT
482 482-500 UAAAGACCAUCCAGGA 640 ACCUCCUGGAUGGUCUU 641 GGUTT UATT
492 492-510 CCAGGAGGUGGCUGGU 642 AUAACCAGCCACCUCCU 643 UAUTT GGTT
493 493-511 CAGGAGGUGGCUGGUU 644 CAUAACCAGCCACCUCC 645 AUGTT UGTT
494 494-512 AGGAGGUGGCUGGUUA 646 ACAUAACCAGCCACCUC 647 UGUTT CUTT
495 495-513 GGAGGUGGCUGGUUAU 648 GACAUAACCAGCCACCU 649 GUCTT CCTT
496 496-514 GAGGUGGCUGGUUAUG 650 GGACAUAACCAGCCACC 651 UCCTT UCTT
497 497-515 AGGUGGCUGGUUAUGU 652 AGGACAUAACCAGCCAC 653 CCUTT CUTT
499 499-517 GUGGCUGGUUAUGUCC 654 UGAGGACAUAACCAGCC 655 UCATT ACTT
520 520-538 GCCCUCAACACAGUGG 656 GCUCCACUGUGUUGAGG 657 AGCTT GCTT
542 542-560 UUCCUUUGGAAAACCU 658 UGCAGGUUUUCCAAAGG 659 GCATT AATT
543 543-561 UCCUUUGGAAAACCUG 660 CUGCAGGUUUUCCAAAG 661 CAGTT GATT
550 550-568 GAAAACCUGCAGAUCA 662 UGAUGAUCUGCAGGUUU 663 UCATT UCTT
551 551-569 AAAACCUGCAGAUCAU 664 CUGAUGAUCUGCAGGUU 665 CAGTT UUTT
553 553-571 AACCUGCAGAUCAUCA 666 CUCUGAUGAUCUGCAGG 667 GAGTT UUTT
556 556-574 CUGCAGAUCAUCAGAG 668 UUCCUCUGAUGAUCUGC 669 GAATT AGTT
586 586-604 GAAAAUUCCUAUGCCU 670 CUAAGGCAUAGGAAUUU 671 UAGTT UCTT
587 587-605 AAAAUUCCUAUGCCUU 672 GCUAAGGCAUAGGAAUU 673 AGCTT UUTT
589 589-607 AAUUCCUAUGCCUUAG 674 CUGCUAAGGCAUAGGAA 675 CAGTT UUTT
592 592-610 UCCUAUGCCUUAGCAG 676 AGACUGCUAAGGCAUAG 677 UCUTT GATT
593 593-611 CCUAUGCCUUAGCAGU 678 AAGACUGCUAAGGCAUA 679 CUUTT GGTT
594 594-612 CUAUGCCUUAGCAGUC 680 UAAGACUGCUAAGGCAU 681 UUATT AGTT
596 596-614 AUGCCUUAGCAGUCUU 682 GAUAAGACUGCUAAGGC 683 AUCTT AUTT
597 597-615 UGCCUUAGCAGUCUUA 684 AGAUAAGACUGCUAAGG 685 UCUTT CATT
598 598-616 GCCUUAGCAGUCUUAU 686 UAGAUAAGACUGCUAAG 687 CUATT GCTT
599 599-617 CCUUAGCAGUCUUAUC 688 UUAGAUAAGACUGCUAA 689 UAATT GGTT
600 600-618 CUUAGCAGUCUUAUCU 690 GUUAGAUAAGACUGCUA 691 AACTT AGTT
601 601-619 UUAGCAGUCUUAUCUA 692 AGUUAGAUAAGACUGCU 693 ACUTT AATT
602 602-620 UAGCAGUCUUAUCUAA 694 UAGUUAGAUAAGACUGC 695 CUATT UATT
603 603-621 AGCAGUCUUAUCUAAC 696 AUAGUUAGAUAAGACUG 697 UAUTT CUTT
604 604-622 GCAGUCUUAUCUAACU 698 CAUAGUUAGAUAAGACU 699 AUGTT GCTT
605 605-623 CAGUCUUAUCUAACUA 700 UCAUAGUUAGAUAAGAC 701 UGATT UGTT
608 608-626 UCUUAUCUAACUAUGA 702 GCAUCAUAGUUAGAUAA 703 UGCTT GATT
609 609-627 CUUAUCUAACUAUGAU 704 UGCAUCAUAGUUAGAUA 705 GCATT AGTT
610 610-628 UUAUCUAACUAUGAUG 706 UUGCAUCAUAGUUAGAU 707 CAATT AATT
611 611-629 UAUCUAACUAUGAUGC 708 UUUGCAUCAUAGUUAGA 709 AAATT UATT
612 612-630 AUCUAACUAUGAUGCA 710 AUUUGCAUCAUAGUUAG 711 AAUTT AUTT
613 613-631 UCUAACUAUGAUGCAA 712 UAUUUGCAUCAUAGUUA 713 AUATT GATT
614 614-632 CUAACUAUGAUGCAAA 714 UUAUUUGCAUCAUAGUU 715 UAATT AGTT
616 616-634 AACUAUGAUGCAAAUA 716 UUUUAUUUGCAUCAUAG 717 AAATT UUTT
622 622-640 GAUGCAAAUAAAACCG 718 GUCCGGUUUUAUUUGCA 719 GACTT UCTT
623 623-641 AUGCAAAUAAAACCGG 720 AGUCCGGUUUUAUUUGC 721 ACUTT AUTT
624 624-642 UGCAAAUAAAACCGGA 722 CAGUCCGGUUUUAUUUG 723 CUGTT CATT
626 626-644 CAAAUAAAACCGGACU 724 UUCAGUCCGGUUUUAUU 725 GAATT UGTT
627 627-645 AAAUAAAACCGGACUG 726 CUUCAGUCCGGUUUUAU 727 AAGTT UUTT
628 628-646 AAUAAAACCGGACUGA 728 CCUUCAGUCCGGUUUUA 729 AGGTT UUTT
630 630-648 UAAAACCGGACUGAAG 730 CUCCUUCAGUCCGGUUU 731 GAGTT UATT
631 631-649 AAAACCGGACUGAAGG 732 GCUCCUUCAGUCCGGUU 733 AGCTT UUTT
632 632-650 AAACCGGACUGAAGGA 734 AGCUCCUUCAGUCCGGU 735 GCUTT UUTT
633 633-651 AACCGGACUGAAGGAG 736 CAGCUCCUUCAGUCCGG 737 CUGTT UUTT
644 644-662 AGGAGCUGCCCAUGAG 738 UUUCUCAUGGGCAGCUC 739 AAATT CUTT
665 665-683 UACAGGAAAUCCUGCA 740 CCAUGCAGGAUUUCCUG 741 UGGTT UATT
668 668-686 AGGAAAUCCUGCAUGG 742 GCGCCAUGCAGGAUUUC 743 CGCTT CUTT
669 669-687 GGAAAUCCUGCAUGGC 744 GGCGCCAUGCAGGAUUU 745 GCCTT CCTT
670 670-688 GAAAUCCUGCAUGGCG 746 CGGCGCCAUGCAGGAUU 747 CCGTT UCTT
671 671-689 AAAUCCUGCAUGGCGC 748 ACGGCGCCAUGCAGGAU 749 CGUTT UUTT
672 672-690 AAUCCUGCAUGGCGCC 750 CACGGCGCCAUGCAGGA 751 GUGTT UUTT
674 674-692 UCCUGCAUGGCGCCGU 752 CGCACGGCGCCAUGCAG 753 GCGTT GATT
676 676-694 CUGCAUGGCGCCGUGC 754 ACCGCACGGCGCCAUGC 755 GGUTT AGTT
677 677-695 UGCAUGGCGCCGUGCG 756 AACCGCACGGCGCCAUG 757 GUUTT CATT
678 678-696 GCAUGGCGCCGUGCGG 758 GAACCGCACGGCGCCAU 759 UUCTT GCTT
680 680-698 AUGGCGCCGUGCGGUU 760 CUGAACCGCACGGCGCC 761 CAGTT AUTT
681 681-699 UGGCGCCGUGCGGUUC 762 GCUGAACCGCACGGCGC 763 AGCTT CATT
682 682-700 GGCGCCGUGCGGUUCA 764 UGCUGAACCGCACGGCG 765 GCATT CCTT
683 683-701 GCGCCGUGCGGUUCAG 766 UUGCUGAACCGCACGGC 767 CAATT GCTT
684 684-702 CGCCGUGCGGUUCAGC 768 GUUGCUGAACCGCACGG 769 AACTT CGTT
685 685-703 GCCGUGCGGUUCAGCA 770 UGUUGCUGAACCGCACG 771 ACATT GCTT
686 686-704 CCGUGCGGUUCAGCAA 772 UUGUUGCUGAACCGCAC 773 CAATT GGTT
688 688-706 GUGCGGUUCAGCAACA 774 GGUUGUUGCUGAACCGC 775 ACCTT ACTT
690 690-708 GCGGUUCAGCAACAAC 776 AGGGUUGUUGCUGAACC 777 CCUTT GCTT
692 692-710 GGUUCAGCAACAACCC 778 GCAGGGUUGUUGCUGAA 779 UGCTT CCTT
698 698-716 GCAACAACCCUGCCCU 780 CACAGGGCAGGGUUGUU 781 GUGTT
GCTT
700 700-718 AACAACCCUGCCCUGU 782 UGCACAGGGCAGGGUUG 783 GCATT UUTT
719 719-737 ACGUGGAGAGCAUCCA 784 CACUGGAUGCUCUCCAC 785 GUGTT GUTT
720 720-738 CGUGGAGAGCAUCCAG 786 CCACUGGAUGCUCUCCA 787 UGGTT CGTT
721 721-739 GUGGAGAGCAUCCAGU 788 GCCACUGGAUGCUCUCC 789 GGCTT ACTT
724 724-742 GAGAGCAUCCAGUGGC 790 CCCGCCACUGGAUGCUC 791 GGGTT UCTT
725 725-743 AGAGCAUCCAGUGGCG 792 UCCCGCCACUGGAUGCU 793 GGATT CUTT
726 726-744 GAGCAUCCAGUGGCGG 794 GUCCCGCCACUGGAUGC 795 GACTT UCTT
733 733-751 CAGUGGCGGGACAUAG 796 UGACUAUGUCCCGCCAC 797 UCATT UGTT
734 734-752 AGUGGCGGGACAUAGU 798 CUGACUAUGUCCCGCCA 799 CAGTT CUTT
736 736-754 UGGCGGGACAUAGUCA 800 UGCUGACUAUGUCCCGC 801 GCATT CATT
737 737-755 GGCGGGACAUAGUCAG 802 CUGCUGACUAUGUCCCG 803 CAGTT CCTT
763 763-781 CUCAGCAACAUGUCGA 804 CCAUCGACAUGUUGCUG 805 UGGTT AGTT
765 765-783 CAGCAACAUGUCGAUG 806 GUCCAUCGACAUGUUGC 807 GACTT UGTT
766 766-784 AGCAACAUGUCGAUGG 808 AGUCCAUCGACAUGUUG 809 ACUTT CUTT
767 767-785 GCAACAUGUCGAUGGA 810 AAGUCCAUCGACAUGUU 811 CUUTT GCTT
769 769-787 AACAUGUCGAUGGACU 812 GGAAGUCCAUCGACAUG 813 UCCTT UUTT
770 770-788 ACAUGUCGAUGGACUU 814 UGGAAGUCCAUCGACAU 815 CCATT GUTT
771 771-789 CAUGUCGAUGGACUUC 816 CUGGAAGUCCAUCGACA 817 CAGTT UGTT
772 772-790 AUGUCGAUGGACUUCC 818 UCUGGAAGUCCAUCGAC 819 AGATT AUTT
775 775-793 UCGAUGGACUUCCAGA 820 GGUUCUGGAAGUCCAUC 821 ACCTT GATT
789 789-807 GAACCACCUGGGCAGC 822 GCAGCUGCCCAGGUGGU 823 UGCTT UCTT
798 798-816 GGGCAGCUGCCAAAAG 824 ACACUUUUGGCAGCUGC 825 UGUTT CCTT
800 800-818 GCAGCUGCCAAAAGUG 826 UCACACUUUUGGCAGCU 827 UGATT GCTT
805 805-823 UGCCAAAAGUGUGAUC 828 UUGGAUCACACUUUUGG 829 CAATT CATT
806 806-824 GCCAAAAGUGUGAUCC 830 CUUGGAUCACACUUUUG 831 AAGTT GCTT
807 807-825 CCAAAAGUGUGAUCCA 832 GCUUGGAUCACACUUUU 833 AGCTT GGTT
810 810-828 AAAGUGUGAUCCAAGC 834 ACAGCUUGGAUCACACU 835 UGUTT UUTT
814 814-832 UGUGAUCCAAGCUGUC 836 UGGGACAGCUUGGAUCA 837 CCATT CATT
815 815-833 GUGAUCCAAGCUGUCC 838 UUGGGACAGCUUGGAUC 839 CAATT ACTT
817 817-835 GAUCCAAGCUGUCCCA 840 CAUUGGGACAGCUUGGA 841 AUGTT UCTT
818 818-836 AUCCAAGCUGUCCCAA 842 CCAUUGGGACAGCUUGG 843 UGGTT AUTT
819 819-837 UCCAAGCUGUCCCAAU 844 CCCAUUGGGACAGCUUG 845 GGGTT GATT
820 820-838 CCAAGCUGUCCCAAUG 846 UCCCAUUGGGACAGCUU 847 GGATT GGTT
821 821-839 CAAGCUGUCCCAAUGG 848 CUCCCAUUGGGACAGCU 849 GAGTT UGTT
823 823-841 AGCUGUCCCAAUGGGA 850 AGCUCCCAUUGGGACAG 851 GCUTT CUTT
826 826-844 UGUCCCAAUGGGAGCU 852 AGCAGCUCCCAUUGGGA 853 GCUTT CATT
847 847-865 GGUGCAGGAGAGGAGA 854 AGUUCUCCUCUCCUGCA 855 ACUTT CCTT
871 871-889 AAACUGACCAAAAUCA 856 AGAUGAUUUUGGUCAGU 857 UCUTT UUTT
872 872-890 AACUGACCAAAAUCAU 858 CAGAUGAUUUUGGUCAG 859 CUGTT UUTT
873 873-891 ACUGACCAAAAUCAUC 860 ACAGAUGAUUUUGGUCA 861 UGUTT GUTT
877 877-895 ACCAAAAUCAUCUGUG 862 GGGCACAGAUGAUUUUG 863 CCCTT GUTT
878 878-896 CCAAAAUCAUCUGUGC 864 UGGGCACAGAUGAUUUU 865 CCATT GGTT
881 881-899 AAAUCAUCUGUGCCCA 866 UGCUGGGCACAGAUGAU 867 GCATT UUTT
890 890-908 GUGCCCAGCAGUGCUC 868 CCGGAGCACUGCUGGGC 869 CGGTT ACTT
892 892-910 GCCCAGCAGUGCUCCG 870 GCCCGGAGCACUGCUGG 871 GGCTT GCTT
929 929-947 CCAGUGACUGCUGCCA 872 UUGUGGCAGCAGUCACU 873 CAATT GGTT
930 930-948 CAGUGACUGCUGCCAC 874 GUUGUGGCAGCAGUCAC 875 AACTT UGTT
979 979-997 GAGAGCGACUGCCUGG 876 AGACCAGGCAGUCGCUC 877 UCUTT UCTT
980 980-998 AGAGCGACUGCCUGGU 878 CAGACCAGGCAGUCGCU 879 CUGTT CUTT
981 981-999 GAGCGACUGCCUGGUC 880 GCAGACCAGGCAGUCGC 881 UGCTT UCTT
982 982-1000 AGCGACUGCCUGGUCU 882 GGCAGACCAGGCAGUCG 883 GCCTT CUTT
983 983-1001 GCGACUGCCUGGUCUG 884 CGGCAGACCAGGCAGUC 885 CCGTT GCTT
984 984-1002 CGACUGCCUGGUCUGC 886 GCGGCAGACCAGGCAGU 887 CGCTT CGTT
989 989-1007 GCCUGGUCUGCCGCAA 888 AAUUUGCGGCAGACCAG 889 AUUTT GCTT
990 990-1008 CCUGGUCUGCCGCAAA 890 GAAUUUGCGGCAGACCA 891 UUCTT GGTT
991 991-1009 CUGGUCUGCCGCAAAU 892 GGAAUUUGCGGCAGACC 893 UCCTT AGTT
992 992-1010 UGGUCUGCCGCAAAUU 894 CGGAAUUUGCGGCAGAC 895 CCGTT CATT
994 994-1012 GUCUGCCGCAAAUUCC 896 CUCGGAAUUUGCGGCAG 897 GAGTT ACTT
995 995-1013 UCUGCCGCAAAUUCCG 898 UCUCGGAAUUUGCGGCA 899 AGATT GATT
996 996-1014 CUGCCGCAAAUUCCGA 900 GUCUCGGAAUUUGCGGC 901 GACTT AGTT
997 997-1015 UGCCGCAAAUUCCGAG 902 CGUCUCGGAAUUUGCGG 903 ACGTT CATT
999 999-1017 CCGCAAAUUCCGAGAC 904 UUCGUCUCGGAAUUUGC 905 GAATT GGTT
1004 1004-1022 AAUUCCGAGACGAAGC 906 GUGGCUUCGUCUCGGAA 907 CACTT
UUTT 1005 1005-1023 AUUCCGAGACGAAGCC 908 CGUGGCUUCGUCUCGGA 909
ACGTT AUTT 1006 1006-1024 UUCCGAGACGAAGCCA 910 ACGUGGCUUCGUCUCGG
911 CGUTT AATT 1007 1007-1025 UCCGAGACGAAGCCAC 912
CACGUGGCUUCGUCUCG 913 GUGTT GATT 1008 1008-1026 CCGAGACGAAGCCACG
914 GCACGUGGCUUCGUCUC 915 UGCTT GGTT 1010 1010-1028
GAGACGAAGCCACGUG 916 UUGCACGUGGCUUCGUC 917 CAATT UCTT 1013
1013-1031 ACGAAGCCACGUGCAA 918 UCCUUGCACGUGGCUUC 919 GGATT GUTT
1014 1014-1032 CGAAGCCACGUGCAAG 920 GUCCUUGCACGUGGCUU 921 ACTT CGTT
1015 1015-1033 GAAGCCACGUGCAAGG 922 UGUCCUUGCACGUGGCU 923 ACATT
UCTT 1016 1016-1034 AAGCCACGUGCAAGGA 924 GUGUCCUUGCACGUGGC 925
CACTT UUTT 1040 1040-1058 CCCCACUCAUGCUCUA 926 UUGUAGAGCAUGAGUGG
927 CAATT GGTT 1042 1042-1060 CCACUCAUGCUCUACA 928
GGUUGUAGAGCAUGAGU 929 ACCTT GGTT 1044 1044-1062 ACUCAUGCUCUACAAC
930 GGGGUUGUAGAGCAUGA 931 CCCTT GUTT 1047 1047-1065
CAUGCUCUACAACCCC 932 GGUGGGGUUGUAGAGCA 933 ACCTT UGTT 1071
1071-1089 CCAGAUGGAUGUGAAC 934 GGGGUUCACAUCCAUCU 935 CCCTT GGTT
1073 1073-1091 AGAUGGAUGUGAACCC 936 UCGGGGUUCACAUCCAU 937 CGATT
CUTT 1074 1074-1092 GAUGGAUGUGAACCCC 938 CUCGGGGUUCACAUCCA 939
GAGTT UCTT 1075 1075-1093 AUGGAUGUGAACCCCG 940 CCUCGGGGUUCACAUCC
941 AGGTT AUTT 1077 1077-1095 GGAUGUGAACCCCGAG 942
GCCCUCGGGGUUCACAU 943 GGCTT CCTT 1078 1078-1096 GAUGUGAACCCCGAGG
944 UGCCCUCGGGGUUCACA 945 GCATT UCTT 1080 1080-1098
UGUGAACCCCGAGGGC 946 UUUGCCCUCGGGGUUCA 947 AAATT CATT 1084
1084-1102 AACCCCGAGGGCAAAU 948 UGUAUUUGCCCUCGGGG 949
ACATT UUTT 1085 1085-1103 ACCCCGAGGGCAAAUA 950 CUGUAUUUGCCCUCGGG
951 CAGTT GUTT 1087 1087-1105 CCCGAGGGCAAAUACA 952
AGCUGUAUUUGCCCUCG 953 GCUTT GGTT 1088 1088-1106 CCGAGGGCAAAUACAG
954 AAGCUGUAUUUGCCCUC 955 CUUTT GGTT 1089 1089-1107
CGAGGGCAAAUACAGC 956 AAAGCUGUAUUUGCCCU 957 UUUTT CGTT 1096
1096-1114 AAAUACAGCUUUGGUG 958 UGGCACCAAAGCUGUAU 959 CCATT UUTT
1097 1097-1115 AAUACAGCUUUGGUGC 960 GUGGCACCAAAGCUGUA 961 CACTT
UUTT 1098 1098-1116 AUACAGCUUUGGUGCC 962 GGUGGCACCAAAGCUGU 963
ACCTT AUTT 1104 1104-1122 CUUUGGUGCCACCUGC 964 CACGCAGGUGGCACCAA
965 GUGTT AGTT 1106 1106-1124 UUGGUGCCACCUGCGU 966
UUCACGCAGGUGGCACC 967 GAATT AATT 1112 1112-1130 CCACCUGCGUGAAGAA
968 CACUUCUUCACGCAGGU 969 GUGTT GGTT 1116 1116-1134
CUGCGUGAAGAAGUGU 970 GGGACACUUCUUCACGC 971 CCCTT AGTT 1117
1117-1135 UGCGUGAAGAAGUGUC 972 GGGGACACUUCUUCACG 973 CCCTT CATT
1118 1118-1136 GCGUGAAGAAGUGUCC 974 CGGGGACACUUCUUCAC 975 CCGTT
GCTT 1119 1119-1137 CGUGAAGAAGUGUCCC 976 ACGGGGACACUUCUUCA 977
CGUTT CGTT 1120 1120-1138 GUGAAGAAGUGUCCCC 978 UACGGGGACACUUCUUC
979 GUATT ACTT 1121 1121-1139 UGAAGAAGUGUCCCCG 980
UUACGGGGACACUUCUU 981 UAATT CATT 1122 1122-1140 GAAGAAGUGUCCCCGU
982 AUUACGGGGACACUUCU 983 AAUTT UCTT 1123 1123-1141
AAGAAGUGUCCCCGUA 984 AAUUACGGGGACACUUC 985 AUUTT UUTT 1124
1124-1142 AGAAGUGUCCCCGUAA 986 UAAUUACGGGGACACUU 987 UUATT CUTT
1125 1125-1143 GAAGUGUCCCCGUAAU 988 AUAAUUACGGGGACACU 989 UAUTT
UCTT 1126 1126-1144 AAGUGUCCCCGUAAUU 990 CAUAAUUACGGGGACAC 991
AUGTT UUTT 1127 1127-1145 AGUGUCCCCGUAAUUA 992 ACAUAAUUACGGGGACA
993 UGUTT CUTT 1128 1128-1146 GUGUCCCCGUAAUUAU 994
CACAUAAUUACGGGGAC 995 GUGTT ACTT 1129 1129-1147 UGUCCCCGUAAUUAUG
996 CCACAUAAUUACGGGGA 997 UGGTT CATT 1130 1130-1148
GUCCCCGUAAUUAUGU 998 ACCACAUAAUUACGGGG 999 GGUTT ACTT 1132
1132-1150 CCCCGUAAUUAUGUGG 1000 UCACCACAUAAUUACGG 1001 UGATT GGTT
1134 1134-1152 CCGUAAUUAUGUGGUG 1002 UGUCACCACAUAAUUAC 1003 ACATT
GGTT 1136 1136-1154 GUAAUUAUGUGGUGAC 1004 UCUGUCACCACAUAAUU 1005
AGATT ACTT 1137 1137-1155 UAAUUAUGUGGUGACA 1006 AUCUGUCACCACAUAAU
1007 GAUTT UATT 1138 1138-1156 AAUUAUGUGGUGACAG 1008
GAUCUGUCACCACAUAA 1009 AUCTT UUTT 1139 1139-1157 AUUAUGUGGUGACAGA
1010 UGAUCUGUCACCACAUA 1011 UCATT AUTT 1140 1140-1158
UUAUGUGGUGACAGAU 1012 GUGAUCUGUCACCACAU 1013 ACTT AATT 1142
1142-1160 AUGUGGUGACAGAUCA 1014 CCGUGAUCUGUCACCAC 1015 CGGTT AUTT
1145 1145-1163 UGGUGACAGAUCACGG 1016 GAGCCGUGAUCUGUCAC 1017 CUCTT
CATT 1147 1147-1165 GUGACAGAUCACGGCU 1018 ACGAGCCGUGAUCUGUC 1019
CGUTT ACTT 1148 1148-1166 UGACAGAUCACGGCUC 1020 CACGAGCCGUGAUCUGU
1021 GUGTT CATT 1149 1149-1167 GACAGAUCACGGCUCG 1022
GCACGAGCCGUGAUCUG 1023 UGCTT UCTT 1150 1150-1168 ACAGAUCACGGCUCGU
1024 CGCACGAGCCGUGAUCU 1025 GCGTT GUTT 1151 1151-1169
CAGAUCACGGCUCGUG 1026 ACGCACGAGCCGUGAUC 1027 CGUTT UGTT 1152
1152-1170 AGAUCACGGCUCGUGC 1028 GACGCACGAGCCGUGAU 1029 GUCTT CUTT
1153 1153-1171 GAUCACGGCUCGUGCG 1030 GGACGCACGAGCCGUGA 1031 UCCTT
UCTT 1154 1154-1172 AUCACGGCUCGUGCGU 1032 CGGACGCACGAGCCGUG 1033
CCGTT AUTT 1155 1155-1173 UCACGGCUCGUGCGUC 1034 UCGGACGCACGAGCCGU
1035 CGATT GATT 1156 1156-1174 CACGGCUCGUGCGUCC 1036
CUCGGACGCACGAGCCG 1037 GAGTT UGTT 1157 1157-1175 ACGGCUCGUGCGUCCG
1038 GCUCGGACGCACGAGCC 1039 AGCTT GUTT 1160 1160-1178
GCUCGUGCGUCCGAGC 1040 CAGGCUCGGACGCACGA 1041 CUGTT GCTT 1200
1200-1218 GGAGGAAGACGGCGUC 1042 GCGGACGCCGUCUUCCU 1043 CGCTT CCTT
1201 1201-1219 GAGGAAGACGGCGUCC 1044 UGCGGACGCCGUCUUCC 1045 GCATT
UCTT 1203 1203-1221 GGAAGACGGCGUCCGC 1046 CUUGCGGACGCCGUCUU 1047
AAGTT CCTT 1204 1204-1222 GAAGACGGCGUCCGCA 1048 ACUUGCGGACGCCGUCU
1049 AGUTT UCTT 1205 1205-1223 AAGACGGCGUCCGCAA 1050
CACUUGCGGACGCCGUC 1051 GUGTT UUTT 1207 1207-1225 GACGGCGUCCGCAAGU
1052 UACACUUGCGGACGCCG 1053 GUATT UCTT 1208 1208-1226
ACGGCGUCCGCAAGUG 1054 UUACACUUGCGGACGCC 1055 UAATT GUTT 1211
1211-1229 GCGUCCGCAAGUGUAA 1056 UUCUUACACUUGCGGAC 1057 GAATT GCTT
1212 1212-1230 CGUCCGCAAGUGUAAG 1058 CUUCUUACACUUGCGGA 1059 AAGTT
CGTT 1213 1213-1231 GUCCGCAAGUGUAAGA 1060 ACUUCUUACACUUGCGG 1061
AGUTT ACTT 1214 1214-1232 UCCGCAAGUGUAAGAA 1062 CACUUCUUACACUUGCG
1063 GUGTT GATT 1215 1215-1233 CCGCAAGUGUAAGAAG 1064
GCACUUCUUACACUUGC 1065 UGCTT GGTT 1216 1216-1234 CGCAAGUGUAAGAAGU
1066 CGCACUUCUUACACUUG 1067 GCGTT CGTT 1217 1217-1235
GCAAGUGUAAGAAGUG 1068 UCGCACUUCUUACACUU 1069 CGATT GCTT 1219
1219-1237 AAGUGUAAGAAGUGCG 1070 CUUCGCACUUCUUACAC 1071 AAGTT UUTT
1220 1220-1238 AGUGUAAGAAGUGCGA 1072 CCUUCGCACUUCUUACA 1073 AGGTT
CUTT 1221 1221-1239 GUGUAAGAAGUGCGAA 1074 CCCUUCGCACUUCUUAC 1075
GGGTT ACTT 1222 1222-1240 UGUAAGAAGUGCGAAG 1076 GCCCUUCGCACUUCUUA
1077 GGCTT CATT 1223 1223-1241 GUAAGAAGUGCGAAGG 1078
GGCCCUUCGCACUUCUU 1079 GCCTT ACTT 1224 1224-1242 UAAGAAGUGCGAAGGG
1080 AGGCCCUUCGCACUUCU 1081 CCUTT UATT 1225 1225-1243
AAGAAGUGCGAAGGGC 1082 AAGGCCCUUCGCACUUC 1083 CUUTT UUTT 1226
1226-1244 AGAAGUGCGAAGGGCC 1084 CAAGGCCCUUCGCACUU 1085 UUGTT CUTT
1229 1229-1247 AGUGCGAAGGGCCUUG 1086 CGGCAAGGCCCUUCGCA 1087 CCGTT
CUTT 1230 1230-1248 GUGCGAAGGGCCUUGC 1088 GCGGCAAGGCCCUUCGC 1089
CGCTT ACTT 1231 1231-1249 UGCGAAGGGCCUUGCC 1090 UGCGGCAAGGCCCUUCG
1091 GCATT CATT 1232 1232-1250 GCGAAGGGCCUUGCCG 1092
UUGCGGCAAGGCCCUUC 1093 CAATT GCTT 1233 1233-1251 CGAAGGGCCUUGCCGC
1094 UUUGCGGCAAGGCCCUU 1095 AAATT CGTT 1235 1235-1253
AAGGGCCUUGCCGCAA 1096 ACUUUGCGGCAAGGCCC 1097 AGUTT UUTT 1236
1236-1254 AGGGCCUUGCCGCAAA 1098 CACUUUGCGGCAAGGCC 1099 GUGTT CUTT
1237 1237-1255 GGGCCUUGCCGCAAAG 1100 ACACUUUGCGGCAAGGC 1101 UGUTT
CCTT 1238 1238-1256 GGCCUUGCCGCAAAGU 1102 CACACUUUGCGGCAAGG 1103
GUGTT CCTT 1239 1239-1257 GCCUUGCCGCAAAGUG 1104 ACACACUUUGCGGCAAG
1105 UGUTT GCTT 1241 1241-1259 CUUGCCGCAAAGUGUG 1106
UUACACACUUUGCGGCA 1107 UAATT AGTT 1261 1261-1279 GGAAUAGGUAUUGGUG
1108 AUUCACCAAUACCUAUU 1109 AAUTT CCTT 1262 1262-1280
GAAUAGGUAUUGGUGA 1110 AAUUCACCAAUACCUAU 1111 AUUTT UCTT 1263
1263-1281 AAUAGGUAUUGGUGAA 1112 AAAUUCACCAAUACCUA 1113 UUUTT UUTT
1264 1264-1282 AUAGGUAUUGGUGAAU 1114 UAAAUUCACCAAUACCU 1115 UUATT
AUTT
1266 1266-1284 AGGUAUUGGUGAAUUU 1116 UUUAAAUUCACCAAUAC 1117 AAATT
CUTT 1267 1267-1285 GGUAUUGGUGAAUUUA 1118 CUUUAAAUUCACCAAUA 1119
AAGTT CCTT 1289 1289-1307 CACUCUCCAUAAAUGC 1120 GUAGCAUUUAUGGAGAG
1121 UACTT UGTT 1313 1313-1331 UUAAACACUUCAAAAA 1122
CAGUUUUUGAAGUGUUU 1123 CUGTT AATT 1320 1320-1338 CUUCAAAAACUGCACC
1124 GGAGGUGCAGUUUUUGA 1125 UCCTT AGTT 1321 1321-1339
UUCAAAAACUGCACCU 1126 UGGAGGUGCAGUUUUUG 1127 CCATT AATT 1322
1322-1340 UCAAAAACUGCACCUC 1128 AUGGAGGUGCAGUUUUU 1129 CAUTT GATT
1323 1323-1341 CAAAAACUGCACCUCC 1130 GAUGGAGGUGCAGUUUU 1131 AUCTT
UGTT 1324 1324-1342 AAAAACUGCACCUCCA 1132 UGAUGGAGGUGCAGUUU 1133
UCATT UUTT 1328 1328-1346 ACUGCACCUCCAUCAG 1134 CCACUGAUGGAGGUGCA
1135 UGGTT GUTT 1332 1332-1350 CACCUCCAUCAGUGGC 1136
AUCGCCACUGAUGGAGG 1137 GAUTT UGTT 1333 1333-1351 ACCUCCAUCAGUGGCG
1138 GAUCGCCACUGAUGGAG 1139 AUCTT GUTT 1335 1335-1353
CUCCAUCAGUGGCGAU 1140 GAGAUCGCCACUGAUGG 1141 CUCTT AGTT 1338
1338-1356 CAUCAGUGGCGAUCUC 1142 GUGGAGAUCGCCACUGA 1143 CACTT UGTT
1344 1344-1362 UGGCGAUCUCCACAUC 1144 CAGGAUGUGGAGAUCGC 1145 CUGTT
CATT 1345 1345-1363 GGCGAUCUCCACAUCC 1146 GCAGGAUGUGGAGAUCG 1147
UGCTT CCTT 1346 1346-1364 GCGAUCUCCACAUCCU 1148 GGCAGGAUGUGGAGAUC
1149 GCCTT GCTT 1347 1347-1365 CGAUCUCCACAUCCUG 1150
CGGCAGGAUGUGGAGAU 1151 CCGTT CGTT 1348 1348-1366 GAUCUCCACAUCCUGC
1152 CCGGCAGGAUGUGGAGA 1153 CGGTT UCTT 1353 1353-1371
CCACAUCCUGCCGGUG 1154 UGCCACCGGCAGGAUGU 1155 GCATT GGTT 1354
1354-1372 CACAUCCUGCCGGUGG 1156 AUGCCACCGGCAGGAUG 1157 CAUTT UGTT
1355 1355-1373 AC AUCCUGCCGGUGGC 1158 AAUGCCACCGGCAGGAU 1159 AUUTT
GUTT 1357 1357-1375 AUCCUGCCGGUGGCAU 1160 UAAAUGCCACCGGCAGG 1161
UUATT AUTT 1360 1360-1378 CUGCCGGUGGCAUUUA 1162 CCCUAAAUGCCACCGGC
1163 GGGTT AGTT 1361 1361-1379 UGCCGGUGGCAUUUAG 1164
CCCCUAAAUGCCACCGG 1165 GGGTT CATT 1362 1362-1380 GCCGGUGGCAUUUAGG
1166 ACCCCUAAAUGCCACCG 1167 GGUTT GCTT 1363 1363-1381
CCGGUGGCAUUUAGGG 1168 CACCCCUAAAUGCCACC 1169 GUGTT GGTT 1366
1366-1384 GUGGCAUUUAGGGGUG 1170 AGUCACCCCUAAAUGCC 1171 ACUTT ACTT
1369 1369-1387 GCAUUUAGGGGUGACU 1172 AGGAGUCACCCCUAAAU 1173 CCUTT
GCTT 1370 1370-1388 CAUUUAGGGGUGACUC 1174 AAGGAGUCACCCCUAAA 1175
CUUTT UGTT 1371 1371-1389 AUUUAGGGGUGACUCC 1176 GAAGGAGUCACCCCUAA
1177 UUCTT AUTT 1372 1372-1390 UUUAGGGGUGACUCCU 1178
UGAAGGAGUCACCCCUA 1179 UCATT AATT 1373 1373-1391 UUAGGGGUGACUCCUU
1180 GUGAAGGAGUCACCCCU 1181 CACTT AATT 1374 1374-1392
UAGGGGUGACUCCUUC 1182 UGUGAAGGAGUCACCCC 1183 ACATT UATT 1404
1404-1422 UCUGGAUCCACAGGAA 1184 CAGUUCCUGUGGAUCCA 1185 CUGTT GATT
1408 1408-1426 GAUCCACAGGAACUGG 1186 UAUCCAGUUCCUGUGGA 1187 AUATT
UCTT 1409 1409-1427 AUCCACAGGAACUGGA 1188 AUAUCCAGUUCCUGUGG 1189
UAUTT AUTT 1411 1411-1429 CCACAGGAACUGGAUA 1190 GAAUAUCCAGUUCCUGU
1191 UUCTT GGTT 1412 1412-1430 CACAGGAACUGGAUAU 1192
AGAAUAUCCAGUUCCUG 1193 UCUTT UGTT 1419 1419-1437 ACUGGAUAUUCUGAAA
1194 GGUUUUCAGAAUAUCCA 1195 ACCTT GUTT 1426 1426-1444
AUUCUGAAAACCGUAA 1196 CCUUUACGGUUUUCAGA 1197 AGGTT AUTT 1427
1427-1445 UUCUGAAAACCGUAAA 1198 UCCUUUACGGUUUUCAG 1199 GGATT AATT
1430 1430-1448 UGAAAACCGUAAAGGA 1200 AUUUCCUUUACGGUUUU 1201 AAUTT
CATT 1431 1431-1449 GAAAACCGUAAAGGAA 1202 GAUUUCCUUUACGGUUU 1203
AUCTT UCTT
TABLE-US-00007 TABLE 6 EGFR siRNA Sequences with Chemical
Modifications Sequence SEQ SEQ hs Id position in sense strand ID
antisense strand ID # NM_005228.3 sequence (5'-3') NO: sequence
(5'-3') NO: 68 68-86 cgGfcCfgGfaGfuCfcCfgAfg 1204
UfAfgCfuCfgGfgAfcUfcCfgGf 1205 CfuAfdTsdT cCfgdTsdT 71 71-89
ccGfgAfgUfcCfcGfaGfcUfa 1206 GfGfcUfaGfcUfcGfgGfaCfuCf 1207
GfcCfdTsdT cGfgdTsdT 72 72-90 cgGfaGfuCfcCfgAfgCfuAfg 1208
GfGfgCfuAfgCfuCfgGfgAfcUf 1209 CfcCfdTsdT cCfgdTsdT 73 73-91
ggAfgUfcCfcGfaGfcUfaGfc 1210 GfGfgGfcUfaGfcUfcGfgGfaCf 1211
CfcCfdTsdT uCfcdTsdT 74 74-92 gaGfuCfcCfgAfgCfuAfgCfc 1212
CfGfgGfgCfuAfgCfuCfgGfgAf 1213 CfcGfdTsdT cUfcdTsdT 75 75-93
agUfcCfcGfaGfcUfaGfcCfcC 1214 CfCfgGfgGfcUfaGfcUfcGfgGf 1215
fgGfdTsdT aCfudTsdT 76 76-94 guCfcCfgAfgCfuAfgCfcCfc 1216
GfCfcGfgGfgCfuAfgCfuCfgGf 1217 GfgCfdTsdT gAfcdTsdT 78 78-96
ccCfgAfgCfuAfgCfcCfcGfg 1218 CfCfgCfcGfgGfgCfuAfgCfuCf 1219
CfgGfdTsdT gGfgdTsdT 114 114-132 ggAfcGfaCfaGfgCfcAfcCfu 1220
AfCfgAfgGfuGfgCfcUfgUfcGf 1221 CfgUfdTsdT uCfcdTsdT 115 115-133
gaCfgAfcAfgGfcCfaCfcUfc 1222 GfAfcGfaGfgUfgGfcCfuGfuCf 1223
GfuCfdTsdT gUfcdTsdT 116 116-134 acGfaCfaGfgCfcAfcCfuCfg 1224
CfGfaCfgAfgGfuGfgCfcUfgUf 1225 UfcGfdTsdT cGfudTsdT 117 117-135
cgAfcAfgGfcCfaCfcUfcGfu 1226 CfCfgAfcGfaGfgUfgGfcCfuGf 1227
CfgGfdTsdT uCfgdTsdT 118 118-136 gaCfaGfgCfcAfcCfuCfgUfc 1228
GfCfcGfaCfgAfgGfuGfgCfcUf 1229 GfgCfdTsdT gUfcdTsdT 120 120-138
caGfgCfcAfcCfuCfgUfcGfg 1230 AfCfgCfcGfaCfgAfgGfuGfgCf 1231
CfgUfdTsdT cUfgdTsdT 121 121-139 agGfcCfaCfcUfcGfuCfgGfc 1232
GfAfcGfcCfgAfcGfaGfgUfgGf 1233 GfuCfdTsdT cCfudTsdT 122 122-140
ggCfcAfcCfuCfgUfcGfgCfg 1234 GfGfaCfgCfcGfaCfgAfgGfuGf 1235
UfcCfdTsdT gCfcdTsdT 123 123-141 gcCfaCfcUfcGfuCfgGfcGfu 1236
CfGfgAfcGfcCfgAfcGfaGfgUf 1237 CfcGfdTsdT gGfcdTsdT 124 124-142
ccAfcCfuCfgUfcGfgCfgUfc 1238 GfCfgGfaCfgCfcGfaCfgAfgGf 1239
CfgCfdTsdT uGfgdTsdT 125 125-143 caCfcUfcGfuCfgGfcGfuCfc 1240
GfGfcGfgAfcGfcCfgAfcGfaGf 1241 GfcCfdTsdT gUfgdTsdT 126 126-144
acCfuCfgUfcGfgCfgUfcCfg 1242 GfGfgCfgGfaCfgCfcGfaCfgAf 1243
CfcCfdTsdT gGfudTsdT 127 127-145 ccUfcGfuCfgGfcGfuCfcGfc 1244
CfGfgGfcGfgAfcGfcCfgAfcGf 1245 CfcGfdTsdT aGfgdTsdT 128 128-146
cuCfgUfcGfgCfgUfcCfgCfc 1246 UfCfgGfgCfgGfaCfgCfcGfaCf 1247
CfgAfdTsdT gAfgdTsdT 129 129-147 ucGfuCfgGfcGfuCfcGfcCfc 1248
CfUfcGfgGfcGfgAfcGfcCfgAf 1249 GfaGfdTsdT cGfadTsdT 130 130-148
cgUfcGfgCfgUfcCfgCfcCfg 1250 AfCfuCfgGfgCfgGfaCfgCfcGf 1251
AfgUfdTsdT aCfgdTsdT 131 131-149 guCfgGfcGfuCfcGfcCfcGfa 1252
GfAfcUfcGfgGfcGfgAfcGfcCf 1253 GfuCfdTsdT gAfcdTsdT 132 132-150
ucGfgCfgUfcCfgCfcCfgAfg 1254 GfGfaCfuCfgGfgCfgGfaCfgCf 1255
UfcCfdTsdT cGfadTsdT 135 135-153 gcGfuCfcGfcCfcGfaGfuCfc 1256
CfGfgGfgAfcUfcGfgGfcGfgAf 1257 CfcGfdTsdT cGfcdTsdT 136 136-154
cgUfcCfgCfcCfgAfgUfcCfc 1258 GfCfgGfgGfaCfuCfgGfgCfgGf 1259
CfgCfdTsdT aCfgdTsdT 141 141-159 gcCfcGfaGfuCfcCfcGfcCfuC 1260
GfCfgAfgGfcGfgGfgAfcUfcGf 1261 fgCfdTsdT gGfcdTsdT 164 164-182
aaCfgCfcAfcAfaCfcAfcCfgC 1262 GfCfgCfgGfuGfgUfuGfuGfgC 1263
fgCfdTsdT fgUfudTsdT 165 165-183 acGfcCfaCfaAfcCfaCfcGfcG 1264
UfGfcGfcGfgUfgGfuUfgUfgG 1265 fcAfdTsdT fcGfudTsdT 166 166-184
cgCfcAfcAfaCfcAfcCfgCfgC 1266 GfUfgCfgCfgGfuGfgUfuGfuG 1267
faCfdTsdT fgCfgdTsdT 168 168-186 ccAfcAfaCfcAfcCfgCfgCfaC 1268
CfCfgUfgCfgCfgGfuGfgUfuGf 1269 fgGfdTsdT uGfgdTsdT 169 169-187
caCfaAfcCfaCfcGfcGfcAfcG 1270 GfCfcGfuGfcGfcGfgUfgGfuUf 1271
fgCfdTsdT gUfgdTsdT 170 170-188 acAfaCfcAfcCfgCfgCfaCfgG 1272
GfGfcCfgUfgCfgCfgGfuGfgUf 1273 fcCfdTsdT uGfudTsdT 247 247-265
auGfcGfaCfcCfuCfcGfgGfa 1274 CfCfgUfcCfcGfgAfgGfgUfcGf 1275
CfgGfdTsdT cAfudTsdT 248 248-266 ugCfgAfcCfcUfcCfgGfgAfc 1276
GfCfcGfuCfcCfgGfaGfgGfuCf 1277 GfgCfdTsdT gCfadTsdT 249 249-267
gcGfaCfcCfuCfcGfgGfaCfg 1278 GfGfcCfgUfcCfcGfgAfgGfgUf 1279
GfcCfdTsdT cGfcdTsdT 251 251-269 gaCfcCfuCfcGfgGfaCfgGfc 1280
CfCfgGfcCfgUfcCfcGfgAfgGf 1281 CfgGfdTsdT gUfcdTsdT 252 252-270
acCfcUfcCfgGfgAfcGfgCfc 1282 CfCfcGfgCfcGfuCfcCfgGfaGf 1283
GfgGfdTsdT gGfudTsdT 254 254-272 ccUfcCfgGfgAfcGfgCfcGfg 1284
GfCfcCfcGfgCfcGfuCfcCfgGf 1285 GfgCfdTsdT aGfgdTsdT 329 329-347
agAfaAfgUfuUfgCfcAfaGfg 1286 GfUfgCfcUfuGfgCfaAfaCfuUf 1287
CfaCfdTsdT uCfudTsdT 330 330-348 gaAfaGfuUfuGfcCfaAfgGfc 1288
CfGfuGfcCfuUfgGfcAfaAfcUf 1289 AfcGfdTsdT uUfcdTsdT 332 332-350
aaGfuUfuGfcCfaAfgGfcAfc 1290 CfUfcGfuGfcCfuUfgGfcAfaAf 1291
GfaGfdTsdT cUfudTsdT 333 333-351 agUfuUfgCfcAfaGfgCfaCfg 1292
AfCfuCfgUfgCfcUfuGfgCfaAf 1293 AfgUfdTsdT aCfudTsdT 334 334-352
guUfuGfcCfaAfgGfcAfcGfa 1294 UfAfcUfcGfuGfcCfuUfgGfcAf 1295
GfuAfdTsdT aAfcdTsdT 335 335-353 uuUfgCfcAfaGfgCfaCfgAfg 1296
UfUfaCfuCfgUfgCfcUfuGfgCf 1297 UfaAfdTsdT aAfadTsdT 336 336-354
uuGfcCfaAfgGfcAfcGfaGfu 1298 GfUfuAfcUfcGfuGfcCfuUfgGf 1299
AfaCfdTsdT cAfadTsdT 337 337-355 ugCfcAfaGfgCfaCfgAfgUfa 1300
UfGfuUfaCfuCfgUfgCfcUfuGf 1301 AfcAfdTsdT gCfadTsdT 338 338-356
gcCfaAfgGfcAfcGfaGfuAfa 1302 UfUfgUfuAfcUfcGfuGfcCfuUf 1303
CfaAfdTsdT gGfcdTsdT 361 361-379 acGfcAfgUfuGfgGfcAfcUfu 1304
CfAfaAfaGfuGfcCfcAfaCfuGf 1305 UfuGfdTsdT cGfudTsdT 362 362-380
cgCfaGfuUfgGfgCfaCfuUfu 1306 UfCfaAfaAfgUfgCfcCfaAfcUf 1307
UfgAfdTsdT gCfgdTsdT 363 363-381 gcAfgUfuGfgGfcAfcUfuUfu 1308
UfUfcAfaAfaGfuGfcCfcAfaCf 1309 GfaAfdTsdT uGfcdTsdT 364 364-382
caGfuUfgGfgCfaCfuUfuUfg 1310 CfUfuCfaAfaAfgUfgCfcCfaAf 1311
AfaGfdTsdT cUfgdTsdT 365 365-383 agUfuGfgGfcAfcUfuUfuGfa 1312
UfCfuUfcAfaAfaGfuGfcCfcAf 1313 AfgAfdTsdT aCfudTsdT 366 366-384
guUfgGfgCfaCfuUfuUfgAfa 1314 AfUfcUfuCfaAfaAfgUfgCfcCf 1315
GfaUfdTsdT aAfcdTsdT 367 367-385 uuGfgGfcAfcUfuUfuGfaAfg 1316
GfAfuCfuUfcAfaAfaGfuGfcCf 1317 AfuCfdTsdT cAfadTsdT 368 368-386
ugGfgCfaCfuUfuUfgAfaGfa 1318 UfGfaUfcUfuCfaAfaAfgUfgCf 1319
UfcAfdTsdT cCfadTsdT 369 369-387 ggGfcAfcUfuUfuGfaAfgAfu 1320
AfUfgAfuCfuUfcAfaAfaGfuGf 1321 CfaUfdTsdT cCfcdTsdT 377 377-395
uuGfaAfgAfuCfaUfuUfuCfu 1322 CfUfgAfgAfaAfaUfgAfuCfuUf 1323
CfaGfdTsdT cAfadTsdT 379 379-397 gaAfgAfuCfaUfuUfuCfuCfa 1324
GfGfcUfgAfgAfaAfaUfgAfuCf 1325 GfcCfdTsdT uUfcdTsdT 380 380-398
aaGfaUfcAfuUfuUfcUfcAfg 1326 AfGfgCfuGfaGfaAfaAfuGfaUf 1327
CfcUfdTsdT cUfudTsdT 385 385-403 caUfuUfuCfuCfaGfcCfuCfc 1328
UfCfuGfgAfgGfcUfgAfgAfaA 1329 AfgAfdTsdT faUfgdTsdT 394 394-412
agCfcUfcCfaGfaGfgAfuGfu 1330 UfGfaAfcAfuCfcUfcUfgGfaGf 1331
UfcAfdTsdT gCfudTsdT 396 396-414 ccUfcCfaGfaGfgAfuGfuUfc 1332
AfUfuGfaAfcAfuCfcUfcUfgGf 1333 AfaUfdTsdT aGfgdTsdT 397 397-415
cuCfcAfgAfgGfaUfgUfuCfa 1334 UfAfuUfgAfaCfaUfcCfuCfuGf 1335
AfuAfdTsdT gAfgdTsdT 401 401-419 agAfgGfaUfgUfuCfaAfuAfa 1336
CfAfgUfuAfuUfgAfaCfaUfcCf 1337 CfuGfdTsdT uCfudTsdT 403 403-421
agGfaUfgUfuCfaAfuAfaCfu 1338 CfAfcAfgUfuAfuUfgAfaCfaUf 1339
GfuGfdTsdT cCfudTsdT 407 407-425 ugUfuCfaAfuAfaCfuGfuGfa 1340
AfCfcUfcAfcAfgUfuAfuUfgAf 1341 GfgUfdTsdT aCfadTsdT 409 409-427
uuCfaAfuAfaCfuGfuGfaGfg 1342 CfCfaCfcUfcAfcAfgUfuAfuUf 1343
UfgGfdTsdT gAfadTsdT 410 410-428 ucAfaUfaAfcUfgUfgAfgGfu 1344
AfCfcAfcCfuCfaCfaGfuUfaUf 1345 GfgUfdTsdT uGfadTsdT 411 411-429
caAfuAfaCfuGfuGfaGfgUfg 1346 GfAfcCfaCfcUfcAfcAfgUfuAf 1347
GfuCfdTsdT uUfgdTsdT 412 412-430 aaUfaAfcUfgUfgAfgGfuGfg 1348
GfGfaCfcAfcCfuCfaCfaGfuUf 1349 UfcCfdTsdT aUfudTsdT 413 413-431
auAfaCfuGfuGfaGfgUfgGfu 1350 AfGfgAfcCfaCfcUfcAfcAfgUf 1351
CfcUfdTsdT uAfudTsdT 414 414-432 uaAfcUfgUfgAfgGfuGfgUfc 1352
AfAfgGfaCfcAfcCfuCfaCfaGf 1353 CfuUfdTsdT uUfadTsdT 416 416-434
acUfgUfgAfgGfuGfgUfcCfu 1354 CfCfaAfgGfaCfcAfcCfuCfaCf 1355
UfgGfdTsdT aGfudTsdT 418 418-436 ugUfgAfgGfuGfgUfcCfuUfg 1356
UfCfcCfaAfgGfaCfcAfcCfuCf 1357 GfgAfdTsdT aCfadTsdT 419 419-437
guGfaGfgUfgGfuCfcUfuGfg 1358 UfUfcCfcAfaGfgAfcCfaCfcUf 1359
GfaAfdTsdT cAfcdTsdT 425 425-443 ugGfuCfcUfuGfgGfaAfuUfu 1360
UfCfcAfaAfuUfcCfcAfaGfgAf 1361 GfgAfdTsdT cCfadTsdT 431 431-449
uuGfgGfaAfuUfuGfgAfaAfu 1362 GfUfaAfuUfuCfcAfaAfuUfcCf 1363
UfaCfdTsdT cAfadTsdT 432 432-450 ugGfgAfaUfuUfgGfaAfaUfu 1364
GfGfuAfaUfuUfcCfaAfaUfuCf 1365 AfcCfdTsdT cCfadTsdT
433 433-451 ggGfaAfuUfuGfgAfaAfuUfa 1366 AfGfgUfaAfuUfuCfcAfaAfuUf
1367 CfcUfdTsdT cCfcdTsdT 434 434-452 ggAfaUfuUfgGfaAfaUfuAfc 1368
UfAfgGfuAfaUfuUfcCfaAfaUf 1369 CfuAfdTsdT uCfcdTsdT 458 458-476
agAfgGfaAfuUfaUfgAfuCfu 1370 GfAfaAfgAfuCfaUfaAfuUfcCf 1371
UfuCfdTsdT uCfudTsdT 459 459-477 gaGfgAfaUfuAfuGfaUfcUfu 1372
GfGfaAfaGfaUfcAfuAfaUfuCf 1373 UfcCfdTsdT cUfcdTsdT 463 463-481
aaUfuAfuGfaUfcUfuUfcCfu 1374 AfGfaAfgGfaAfaGfaUfcAfuAf 1375
UfcUfdTsdT aUfudTsdT 464 464-482 auUfaUfgAfuCfuUfuCfcUfu 1376
AfAfgAfaGfgAfaAfgAfuCfaUf 1377 CfuUfdTsdT aAfudTsdT 466 466-484
uaUfgAfuCfuUfuCfcUfuCfu 1378 UfUfaAfgAfaGfgAfaAfgAfuCf 1379
UfaAfdTsdT aUfadTsdT 468 468-486 ugAfuCfuUfuCfcUfuCfuUfa 1380
CfUfuUfaAfgAfaGfgAfaAfgAf 1381 AfaGfdTsdT uCfadTsdT 471 471-489
ucUfuUfcCfuUfcUfuAfaAfg 1382 GfGfuCfuUfuAfaGfaAfgGfaAf 1383
AfcCfdTsdT aGfadTsdT 476 476-494 ccUfuCfuUfaAfaGfaCfcAfu 1384
UfGfgAfuGfgUfcUfuUfaAfgA 1385 CfcAfdTsdT faGfgdTsdT 477 477-495
cuUfcUfuAfaAfgAfcCfaUfc 1386 CfUfgGfaUfgGfuCfuUfuAfaGf 1387
CfaGfdTsdT aAfgdTsdT 479 479-497 ucUfuAfaAfgAfcCfaUfcCfa 1388
UfCfcUfgGfaUfgGfuCfuUfuAf 1389 GfgAfdTsdT aGfadTsdT 481 481-499
uuAfaAfgAfcCfaUfcCfaGfg 1390 CfCfuCfcUfgGfaUfgGfuCfuUf 1391
AfgGfdTsdT uAfadTsdT 482 482-500 uaAfaGfaCfcAfuCfcAfgGfa 1392
AfCfcUfcCfuGfgAfuGfgUfcUf 1393 GfgUfdTsdT uUfadTsdT 492 492-510
ccAfgGfaGfgUfgGfcUfgGfu 1394 AfUfaAfcCfaGfcCfaCfcUfcCf 1395
UfaUfdTsdT uGfgdTsdT 493 493-511 caGfgAfgGfuGfgCfuGfgUfu 1396
CfAfuAfaCfcAfgCfcAfcCfuCf 1397 AfuGfdTsdT cUfgdTsdT 494 494-512
agGfaGfgUfgGfcUfgGfuUfa 1398 AfCfaUfaAfcCfaGfcCfaCfcUfc 1399
UfgUfdTsdT CfudTsdT 495 495-513 ggAfgGfuGfgCfuGfgUfuAfu 1400
GfAfcAfuAfaCfcAfgCfcAfcCf 1401 GfuCfdTsdT uCfcdTsdT 496 496-514
gaGfgUfgGfcUfgGfuUfaUfg 1402 GfGfaCfaUfaAfcCfaGfcCfaCfc 1403
UfcCfdTsdT UfcdTsdT 497 497-515 agGfuGfgCfuGfgUfuAfuGfu 1404
AfGfgAfcAfuAfaCfcAfgCfcAf 1405 CfcUfdTsdT cCfudTsdT 499 499-517
guGfgCfuGfgUfuAfuGfuCfc 1406 UfGfaGfgAfcAfuAfaCfcAfgCf 1407
UfcAfdTsdT cAfcdTsdT 520 520-538 gcCfcUfcAfaCfaCfaGfuGfg 1408
GfCfuCfcAfcUfgUfgUfuGfaGf 1409 AfgCfdTsdT gGfcdTsdT 542 542-560
uuCfcUfuUfgGfaAfaAfcCfu 1410 UfGfcAfgGfuUfuUfcCfaAfaGf 1411
GfcAfdTsdT gAfadTsdT 543 543-561 ucCfuUfuGfgAfaAfaCfcUfg 1412
CfUfgCfaGfgUfuUfuCfcAfaAf 1413 CfaGfdTsdT gGfadTsdT 550 550-568
gaAfaAfcCfuGfcAfgAfuCfa 1414 UfGfaUfgAfuCfuGfcAfgGfuU 1415
UfcAfdTsdT fuUfcdTsdT 551 551-569 aaAfaCfcUfgCfaGfaUfcAfu 1416
CfUfgAfuGfaUfcUfgCfaGfgUf 1417 CfaGfdTsdT uUfudTsdT 553 553-571
aaCfcUfgCfaGfaUfcAfuCfa 1418 CfUfcUfgAfuGfaUfcUfgCfaGf 1419
GfaGfdTsdT gUfudTsdT 556 556-574 cuGfcAfgAfuCfaUfcAfgAfg 1420
UfUfcCfuCfuGfaUfgAfuCfuGf 1421 GfaAfdTsdT cAfgdTsdT 586 586-604
gaAfaAfuUfcCfuAfuGfcCfu 1422 CfUfaAfgGfcAfuAfgGfaAfuUf 1423
UfaGfdTsdT uUfcdTsdT 587 587-605 aaAfaUfuCfcUfaUfgCfcUfu 1424
GfCfuAfaGfgCfaUfaGfgAfaUf 1425 AfgCfdTsdT uUfudTsdT 589 589-607
aaUfuCfcUfaUfgCfcUfuAfg 1426 CfUfgCfuAfaGfgCfaUfaGfgAf 1427
CfaGfdTsdT aUfudTsdT 592 592-610 ucCfuAfuGfcCfuUfaGfcAfg 1428
AfGfaCfuGfcUfaAfgGfcAfuAf 1429 UfcUfdTsdT gGfadTsdT 593 593-611
ccUfaUfgCfcUfuAfgCfaGfu 1430 AfAfgAfcUfgCfuAfaGfgCfaUf 1431
CfuUfdTsdT aGfgdTsdT 594 594-612 cuAfuGfcCfuUfaGfcAfgUfc 1432
UfAfaGfaCfuGfcUfaAfgGfcAf 1433 UfuAfdTsdT uAfgdTsdT 596 596-614
auGfcCfuUfaGfcAfgUfcUfu 1434 GfAfuAfaGfaCfuGfcUfaAfgGf 1435
AfuCfdTsdT cAfudTsdT 597 597-615 ugCfcUfuAfgCfaGfuCfuUfa 1436
AfGfaUfaAfgAfcUfgCfuAfaGf 1437 UfcUfdTsdT gCfadTsdT 598 598-616
gcCfuUfaGfcAfgUfcUfuAfu 1438 UfAfgAfuAfaGfaCfuGfcUfaAf 1439
CfuAfdTsdT gGfcdTsdT 599 599-617 ccUfuAfgCfaGfuCfuUfaUfc 1440
UfUfaGfaUfaAfgAfcUfgCfuAf 1441 UfaAfdTsdT aGfgdTsdT 600 600-618
cuUfaGfcAfgUfcUfuAfuCfu 1442 GfUfuAfgAfuAfaGfaCfuGfcUf 1443
AfaCfdTsdT aAfgdTsdT 601 601-619 uuAfgCfaGfuCfuUfaUfcUfa 1444
AfGfuUfaGfaUfaAfgAfcUfgCf 1445 AfcUfdTsdT uAfadTsdT 602 602-620
uaGfcAfgUfcUfuAfuCfuAfa 1446 UfAfgUfuAfgAfuAfaGfaCfuG 1447
CfuAfdTsdT fcUfadTsdT 603 603-621 agCfaGfuCfuUfaUfcUfaAfc 1448
AfUfaGfuUfaGfaUfaAfgAfcUf 1449 UfaUfdTsdT gCfudTsdT 604 604-622
gcAfgUfcUfuAfuCfuAfaCfu 1450 CfAfuAfgUfuAfgAfuAfaGfaCf 1451
AfuGfdTsdT uGfcdTsdT 605 605-623 caGfuCfuUfaUfcUfaAfcUfa 1452
UfCfaUfaGfuUfaGfaUfaAfgAf 1453 UfgAfdTsdT cUfgdTsdT 608 608-626
ucUfuAfuCfuAfaCfuAfuGfa 1454 GfCfaUfcAfuAfgUfuAfgAfuA 1455
UfgCfdTsdT faGfadTsdT 609 609-627 cuUfaUfcUfaAfcUfaUfgAfu 1456
UfGfcAfuCfaUfaGfuUfaGfaUf 1457 GfcAfdTsdT aAfgdTsdT 610 610-628
uuAfuCfuAfaCfuAfuGfaUfg 1458 UfUfgCfaUfcAfuAfgUfuAfgA 1459
CfaAfdTsdT fuAfadTsdT 611 611-629 uaUfcUfaAfcUfaUfgAfuGfc 1460
UfUfuGfcAfuCfaUfaGfuUfaGf 1461 AfaAfdTsdT aUfadTsdT 612 612-630
auCfuAfaCfuAfuGfaUfgCfa 1462 AfUfuUfgCfaUfcAfuAfgUfuA 1463
AfaUfdTsdT fgAfudTsdT 613 613-631 ucUfaAfcUfaUfgAfuGfcAfa 1464
UfAfuUfuGfcAfuCfaUfaGfuUf 1465 AfuAfdTsdT aGfadTsdT 614 614-632
cuAfaCfuAfuGfaUfgCfaAfa 1466 UfUfaUfuUfgCfaUfcAfuAfgUf 1467
UfaAfdTsdT uAfgdTsdT 616 616-634 aaCfuAfuGfaUfgCfaAfaUfa 1468
UfUfuUfaUfuUfgCfaUfcAfuAf 1469 AfaAfdTsdT gUfudTsdT 622 622-640
gaUfgCfaAfaUfaAfaAfcCfg 1470 GfUfcCfgGfuUfuUfaUfuUfgCf 1471
GfaCfdTsdT aUfcdTsdT 623 623-641 auGfcAfaAfuAfaAfaCfcGfg 1472
AfGfuCfcGfgUfuUfuAfuUfuG 1473 AfcUfdTsdT fcAfudTsdT 624 624-642
ugCfaAfaUfaAfaAfcCfgGfa 1474 CfAfgUfcCfgGfuUfuUfaUfuUf 1475
CfuGfdTsdT gCfadTsdT 626 626-644 caAfaUfaAfaAfcCfgGfaCfu 1476
UfUfcAfgUfcCfgGfuUfuUfaUf 1477 GfaAfdTsdT uUfgdTsdT 627 627-645
aaAfuAfaAfaCfcGfgAfcUfg 1478 CfUfuCfaGfuCfcGfgUfuUfuAf 1479
AfaGfdTsdT uUfudTsdT 628 628-646 aaUfaAfaAfcCfgGfaCfuGfa 1480
CfCfuUfcAfgUfcCfgGfuUfuUf 1481 AfgGfdTsdT aUfudTsdT 630 630-648
uaAfaAfcCfgGfaCfuGfaAfg 1482 CfUfcCfuUfcAfgUfcCfgGfuUf 1483
GfaGfdTsdT uUfadTsdT 631 631-649 aaAfaCfcGfgAfcUfgAfaGfg 1484
GfCfuCfcUfuCfaGfuCfcGfgUf 1485 AfgCfdTsdT uUfudTsdT 632 632-650
aaAfcCfgGfaCfuGfaAfgGfa 1486 AfGfcUfcCfuUfcAfgUfcCfgGf 1487
GfcUfdTsdT uUfudTsdT 633 633-651 aaCfcGfgAfcUfgAfaGfgAfg 1488
CfAfgCfuCfcUfuCfaGfuCfcGf 1489 CfuGfdTsdT gUfudTsdT 644 644-662
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auUfaUfgUfgGfuGfaCfaGfa 1762 UfGfaUfcUfgUfcAfcCfaCfaUf 1763
UfcAfdTsdT aAfudTsdT 1140 1140-1158 uuAfuGfuGfgUfgAfcAfgAfu 1764
GfUfgAfuCfuGfuCfaCfcAfcAf 1765 CfaCfdTsdT uAfadTsdT 1142 1142-1160
auGfuGfgUfgAfcAfgAfuCfa 1766 CfCfgUfgAfuCfuGfuCfaCfcAf 1767
CfgGfdTsdT cAfudTsdT 1145 1145-1163 ugGfuGfaCfaGfaUfcAfcGfg 1768
GfAfgCfcGfuGfaUfcUfgUfcAf 1769 CfuCfdTsdT cCfadTsdT 1147 1147-1165
guGfaCfaGfaUfcAfcGfgCfu 1770 AfCfgAfgCfcGfuGfaUfcUfgUf 1771
CfgUfdTsdT cAfcdTsdT 1148 1148-1166 ugAfcAfgAfuCfaCfgGfcUfc 1771
CfAfcGfaGfcCfgUfgAfuCfuGf 1773 GfuGfdTsdT uCfadTsdT 1149 1149-1167
gaCfaGfaUfcAfcGfgCfuCfg 1774 GfCfaCfgAfgCfcGfuGfaUfcUf 1775
UfgCfdTsdT gUfcdTsdT 1150 1150-1168 acAfgAfuCfaCfgGfcUfcGfu 177
CfGfcAfcGfaGfcCfgUfgAfuCf 1777 GfcGfdTsdT uGfudTsdT 1151 1151-1169
caGfaUfcAfcGfgCfuCfgUfg 1778 AfCfgCfaCfgAfgCfcGfuGfaUf 1779
CfgUfdTsdT cUfgdTsdT 1152 1152-1170 agAfuCfaCfgGfcUfcGfuGfc 1780
GfAfcGfcAfcGfaGfcCfgUfgAf 1781 GfuCfdTsdT uCfudTsdT 1153 1153-1171
gaUfcAfcGfgCfuCfgUfgCfg 1782 GfGfaCfgCfaCfgAfgCfcGfuGf 1783
UfcCfdTsdT aUfcdTsdT 1154 1154-1172 auCfaCfgGfcUfcGfuGfcGfu 1784
CfGfgAfcGfcAfcGfaGfcCfgUf 1785 CfcGfdTsdT gAfudTsdT 1155 1155-1173
ucAfcGfgCfuCfgUfgCfgUfc 1786 UfCfgGfaCfgCfaCfgAfgCfcGf 1787
CfgAfdTsdT uGfadTsdT 1156 1156-1174 caCfgGfcUfcGfuGfcGfuCfc 1788
CfUfcGfgAfcGfcAfcGfaGfcCf 1789 GfaGfdTsdT gUfgdTsdT 1157 1157-1175
acGfgCfuCfgUfgCfgUfcCfg 1790 GfCfuCfgGfaCfgCfaCfgAfgCf 1791
AfgCfdTsdT cGfudTsdT 1160 1160-1178 gcUfcGfuGfcGfuCfcGfaGfc 1701
CfAfgGfcUfcGfgAfcGfcAfcGf 1793 CfuGfdTsdT aGfcdTsdT 1200 1200-1218
ggAfgGfaAfgAfcGfgCfgUfc 1794 GfCfgGfaCfgCfcGfuCfuUfcCf 1795
CfgCfdTsdT uCfcdTsdT 1201 1201-1219 gaGfgAfaGfaCfgGfcGfuCfc 1796
UfGfcGfgAfcGfcCfgUfcUfuCf 1797 GfcAfdTsdT cUfcdTsdT 1203 1203-1221
ggAfaGfaCfgGfcGfuCfcGfc 1798 CfUfuGfcGfgAfcGfcCfgUfcUf 1799
AfaGfdTsdT uCfcdTsdT 1204 1204-1222 gaAfgAfcGfgCfgUfcCfgCfa 1800
AfCfuUfgCfgGfaCfgCfcGfuCf 1801 AfgUfdTsdT uUfcdTsdT 1205 1205-1223
aaGfaCfgGfcGfuCfcGfcAfa 1802 CfAfcUfuGfcGfgAfcGfcCfgUf 1803
GfuGfdTsdT cUfudTsdT 1207 1207-1225 gaCfgGfcGfuCfcGfcAfaGfu 1804
UfAfcAfcUfuGfcGfgAfcGfcCf 1805 GfuAfdTsdT gUfcdTsdT 1208 1208-1226
acGfgCfgUfcCfgCfaAfgUfg 1806 UfUfaCfaCfuUfgCfgGfaCfgCf 1807
UfaAfdTsdT cGfudTsdT 1211 1211-1229 gcGfuCfcGfcAfaGfuGfuAfa 1808
UfUfcUfuAfcAfcUfuGfcGfgAf 1809 GfaAfdTsdT cGfcdTsdT 1212 1212-1230
cgUfcCfgCfaAfgUfgUfaAfg 1810 CfUfuCfuUfaCfaCfuUfgCfgGf 1811
AfaGfdTsdT aCfgdTsdT 1213 1213-1231 guCfcGfcAfaGfuGfuAfaGfa 1812
AfCfuUfcUfuAfcAfcUfuGfcGf 1813 AfgUfdTsdT gAfcdTsdT 1214 1214-1232
ucCfgCfaAfgUfgUfaAfgAfa 1814 CfAfcUfuCfuUfaCfaCfuUfgCf 1815
GfuGfdTsdT gGfadTsdT 1215 1215-1233 ccGfcAfaGfuGfuAfaGfaAfg 1816
GfCfaCfuUfcUfuAfcAfcUfuGf 1817
UfgCfdTsdT cGfgdTsdT 1216 1216-1234 cgCfaAfgUfgUfaAfgAfaGfu 1818
CfGfcAfcUfuCfuUfaCfaCfuUf 1819 GfcGfdTsdT gCfgdTsdT 1217 1217-1235
gcAfaGfuGfuAfaGfaAfgUfg 1820 UfCfgCfaCfuUfcUfuAfcAfcUf 1821
CfgAfdTsdT uGfcdTsdT 1219 1219-1237 aaGfuGfuAfaGfaAfgUfgCfg 1822
CfUfuCfgCfaCfuUfcUfuAfcAf 1823 AfaGfdTsdT cUfudTsdT 1220 1220-1238
agUfgUfaAfgAfaGfuGfcGfa 1824 CfCfuUfcGfcAfcUfuCfuUfaCf 1825
AfgGfdTsdT aCfudTsdT 1221 1221-1239 guGfuAfaGfaAfgUfgCfgAfa 1826
CfCfcUfuCfgCfaCfuUfcUfuAf 1827 GfgGfdTsdT cAfcdTsdT 1222 1222-1240
ugUfaAfgAfaGfuGfcGfaAfg 1828 GfCfcCfuUfcGfcAfcUfuCfuUf 1829
GfgCfdTsdT aCfadTsdT 1223 1223-1241 guAfaGfaAfgUfgCfgAfaGfg 1830
GfGfcCfcUfuCfgCfaCfuUfcUf 1831 GfcCfdTsdT uAfcdTsdT 1224 1224-1242
uaAfgAfaGfuGfcGfaAfgGfg 1832 AfGfgCfcCfuUfcGfcAfcUfuCf 1833
CfcUfdTsdT uUfadTsdT 1225 1225-1243 aaGfaAfgUfgCfgAfaGfgGfc 1834
AfAfgGfcCfcUfuCfgCfaCfuUf 1835 CfuUfdTsdT cUfudTsdT 1226 1226-1244
agAfaGfuGfcGfaAfgGfgCfc 1836 CfAfaGfgCfcCfuUfcGfcAfcUf 1837
UfuGfdTsdT uCfudTsdT 1229 1229-1247 agUfgCfgAfaGfgGfcCfuUfg 1838
CfGfgCfaAfgGfcCfcUfuCfgCf 1839 CfcGfdTsdT aCfudTsdT 1230 1230-1248
guGfcGfaAfgGfgCfcUfuGfc 1840 GfCfgGfcAfaGfgCfcCfuUfcGf 1841
CfgCfdTsdT cAfcdTsdT 1231 1231-1249 ugCfgAfaGfgGfcCfuUfgCfc 1842
UfGfcGfgCfaAfgGfcCfcUfuCf 1843 GfcAfdTsdT gCfadTsdT 1232 1232-1250
gcGfaAfgGfgCfcUfuGfcCfg 1844 UfUfgCfgGfcAfaGfgCfcCfuUf 1845
CfaAfdTsdT cGfcdTsdT 1233 1233-1251 cgAfaGfgGfcCfuUfgCfcGfc 1846
UfUfuGfcGfgCfaAfgGfcCfcUf 1847 AfaAfdTsdT uCfgdTsdT 1235 1235-1253
aaGfgGfcCfuUfgCfcGfcAfa 1848 AfCfuUfuGfcGfgCfaAfgGfcCf 1849
AfgUfdTsdT cUfudTsdT 1236 1236-1254 agGfgCfcUfuGfcCfgCfaAfa 1850
CfAfcUfuUfgCfgGfcAfaGfgCf 1851 GfuGfdTsdT cCfudTsdT 1237 1237-1255
ggGfcCfuUfgCfcGfcAfaAfg 1852 AfCfaCfuUfuGfcGfgCfaAfgGf 1853
UfgUfdTsdT cCfcdTsdT 1238 1238-1256 ggCfcUfuGfcCfgCfaAfaGfu 1854
CfAfcAfcUfuUfgCfgGfcAfaGf 1855 GfuGfdTsdT gCfcdTsdT 1239 1239-1257
gcCfuUfgCfcGfcAfaAfgUfg 1856 AfCfaCfaCfuUfuGfcGfgCfaAf 1857
UfgUfdTsdT gGfcdTsdT 1241 1241-1259 cuUfgCfcGfcAfaAfgUfgUfg 1858
UfUfaCfaCfaCfuUfuGfcGfgCf 1859 UfaAfdTsdT aAfgdTsdT 1261 1261-1279
ggAfaUfaGfgUfaUfuGfgUfg 1860 AfUfuCfaCfcAfaUfaCfcUfaUf 1861
AfaUfdTsdT uCfcdTsdT 1262 1262-1280 gaAfuAfgGfuAfuUfgGfuGfa 1862
AfAfuUfcAfcCfaAfuAfcCfuAf 1863 AfuUfdTsdT uUfcdTsdT 1263 1263-1281
aaUfaGfgUfaUfuGfgUfgAfa 1864 AfAfaUfuCfaCfcAfaUfaCfcUf 1865
UfuUfdTsdT aUfudTsdT 1264 1264-1282 auAfgGfuAfuUfgGfuGfaAfu 1866
UfAfaAfuUfcAfcCfaAfuAfcCf 1867 UfuAfdTsdT uAfudTsdT 1266 1266-1284
agGfuAfuUfgGfuGfaAfuUfu 1868 UfUfuAfaAfuUfcAfcCfaAfuAf 1869
AfaAfdTsdT cCfudTsdT 1267 1267-1285 ggUfaUfuGfgUfgAfaUfuUfa 1870
CfUfuUfaAfaUfuCfaCfcAfaUf 1871 AfaGfdTsdT aCfcdTsdT 1289 1289-1307
caCfuCfuCfcAfuAfaAfuGfc 1872 GfUfaGfcAfuUfuAfuGfgAfgA 1873
UfaCfdTsdT fgUfgdTsdT 1313 1313-1331 uuAfaAfcAfcUfuCfaAfaAfa 1874
CfAfgUfuUfuUfgAfaGfuGfuU 1875 CfuGfdTsdT fuAfadTsdT 1320 1320-1338
cuUfcAfaAfaAfcUfgCfaCfc 1876 GfGfaGfgUfgCfaGfuUfuUfuG 1877
UfcCfdTsdT faAfgdTsdT 1321 1321-1339 uuCfaAfaAfaCfuGfcAfcCfu 1878
UfGfgAfgGfuGfcAfgUfuUfuU 1879 CfcAfdTsdT fgAfadTsdT 1322 1322-1340
ucAfaAfaAfcUfgCfaCfcUfc 1880 AfUfgGfaGfgUfgCfaGfuUfuU 1881
CfaUfdTsdT fuGfadTsdT 1323 1323-1341 caAfaAfaCfuGfcAfcCfuCfc 1882
GfAfuGfgAfgGfuGfcAfgUfuU 1883 AfuCfdTsdT fuUfgdTsdT 1324 1324-1342
aaAfaAfcUfgCfaCfcUfcCfaU 1884 UfGfaUfgGfaGfgUfgCfaGfuUf 1885
fcAfdTsdT uUfudTsdT 1328 1328-1346 acUfgCfaCfcUfcCfaUfcAfg 1886
CfCfaCfuGfaUfgGfaGfgUfgCf 1887 UfgGfdTsdT aGfudTsdT 1332 1332-1350
caCfcUfcCfaUfcAfgUfgGfc 1888 AfUfcGfcCfaCfuGfaUfgGfaGf 1889
GfaUfdTsdT gUfgdTsdT 1333 1333-1351 acCfuCfcAfuCfaGfuGfgCfg 1890
GfAfuCfgCfcAfcUfgAfuGfgAf 1891 AfuCfdTsdT gGfudTsdT 1335 1335-1353
cuCfcAfuCfaGfuGfgCfgAfu 1892 GfAfgAfuCfgCfcAfcUfgAfuGf 1893
CfuCfdTsdT gAfgdTsdT 1338 1338-1356 caUfcAfgUfgGfcGfaUfcUfc 1894
GfUfgGfaGfaUfcGfcCfaCfuGf 1895 CfaCfdTsdT aUfgdTsdT 1344 1344-1362
ugGfcGfaUfcUfcCfaCfaUfc 1896 CfAfgGfaUfgUfgGfaGfaUfcGf 1897
CfuGfdTsdT cCfadTsdT 1345 1345-1363 ggCfgAfuCfuCfcAfcAfuCfc 1898
GfCfaGfgAfuGfuGfgAfgAfuC 1899 UfgCfdTsdT fgCfcdTsdT 1346 1346-1364
gcGfaUfcUfcCfaCfaUfcCfu 1900 GfGfcAfgGfaUfgUfgGfaGfaUf 1901
GfcCfdTsdT cGfcdTsdT 1347 1347-1365 cgAfuCfuCfcAfcAfuCfcUfg 1902
CfGfgCfaGfgAfuGfuGfgAfgA 1903 CfcGfdTsdT fuCfgdTsdT 1348 1348-1366
gaUfcUfcCfaCfaUfcCfuGfcC 1904 CfCfgGfcAfgGfaUfgUfgGfaGf 1905
fgGfdTsdT aUfcdTsdT 1353 1353-1371 ccAfcAfuCfcUfgCfcGfgUfg 1906
UfGfcCfaCfcGfgCfaGfgAfuGf 1907 GfcAfdTsdT uGfgdTsdT 1354 1354-1372
caCfaUfcCfuGfcCfgGfuGfg 1908 AfUfgCfcAfcCfgGfcAfgGfaUf 1909
CfaUfdTsdT gUfgdTsdT 1355 1355-1373 acAfuCfcUfgCfcGfgUfgGfc 1910
AfAfuGfcCfaCfcGfgCfaGfgAf 1911 AfuUfdTsdT uGfudTsdT 1357 1357-1375
auCfcUfgCfcGfgUfgGfcAfu 1912 UfAfaAfuGfcCfaCfcGfgCfaGf 1913
UfuAfdTsdT gAfudTsdT 1360 1360-1378 cuGfcCfgGfuGfgCfaUfuUfa 1914
CfCfcUfaAfaUfgCfcAfcCfgGf 1915 GfgGfdTsdT cAfgdTsdT 1361 1361-1379
ugCfcGfgUfgGfcAfuUfuAfg 1916 CfCfcCfuAfaAfuGfcCfaCfcGf 1917
GfgGfdTsdT gCfadTsdT 1362 1362-1380 cCfgGfuGfgCfaUfuUfaGfg 1918
AfCfcCfcUfaAfaUfgCfcAfcCf 1919 GfgUfdTsdT gGfcdTsdT 1363 1363-1381
ccGfgUfgGfcAfuUfuAfgGfg 1920 CfAfcCfcCfuAfaAfuGfcCfaCf 1921
GfuGfdTsdT cGfgdTsdT 1366 1366-1384 guGfgCfaUfuUfaGfgGfgUfg 1922
AfGfuCfaCfcCfcUfaAfaUfgCf 1923 AfcUfdTsdT cAfcdTsdT 1369 1369-1387
gcAfuUfuAfgGfgGfuGfaCfu 1924 AfGfgAfgUfcAfcCfcCfuAfaAf 1925
CfcUfdTsdT uGfcdTsdT 1370 1370-1388 caUfuUfaGfgGfgUfgAfcUfc 1926
AfAfgGfaGfuCfaCfcCfcUfaAf 1927 CfuUfdTsdT aUfgdTsdT 1371 1371-1389
auUfuAfgGfgGfuGfaCfuCfc 1928 GfAfaGfgAfgUfcAfcCfcCfuAf 1929
UfuCfdTsdT aAfudTsdT 1372 1372-1390 uuUfaGfgGfgUfgAfcUfcCfu 1930
UfGfaAfgGfaGfuCfaCfcCfcUf 1931 UfcAfdTsdT aAfadTsdT 1373 1373-1391
uuAfgGfgGfuGfaCfuCfcUfu 1932 GfUfgAfaGfgAfgUfcAfcCfcCf 1933
CfaCfdTsdT uAfadTsdT 1374 1374-1392 uaGfgGfgUfgAfcUfcCfuUfc 1934
UfGfuGfaAfgGfaGfuCfaCfcCf 1935 AfcAfdTsdT cUfadTsdT 1404 1404-1422
ucUfgGfaUfcCfaCfaGfgAfa 1936 CfAfgUfuCfcUfgUfgGfaUfcCf 1937
CfuGfdTsdT aGfadTsdT 1408 1408-1426 gaUfcCfaCfaGfgAfaCfuGfg 1938
UfAfuCfcAfgUfuCfcUfgUfgGf 1939 AfuAfdTsdT aUfcdTsdT 1409 1409-1427
auCfcAfcAfgGfaAfcUfgGfa 1940 AfUfaUfcCfaGfuUfcCfuGfuGf 1941
UfaUfdTsdT gAfudTsdT 1411 1411-1429 ccAfcAfgGfaAfcUfgGfaUfa 1942
GfAfaUfaUfcCfaGfuUfcCfuGf 1943 UfuCfdTsdT uGfgdTsdT 1412 1412-1430
caCfaGfgAfaCfuGfgAfuAfu 1944 AfGfaAfuAfuCfcAfgUfuCfcUf 1945
UfcUfdTsdT gUfgdTsdT 1419 1419-1437 acUfgGfaUfaUfuCfuGfaAfa 1946
GfGfuUfuUfcAfgAfaUfaUfcCf 1947
AfcCfdTsdT aGfudTsdT 1426 1426-1444 auUfcUfgAfaAfaCfcGfuAfa 1948
CfCfuUfuAfcGfgUfuUfuCfaGf 1949 AfgGfdTsdT aAfudTsdT 1427 1427-1445
uuCfuGfaAfaAfcCfgUfaAfa 1950 UfCfcUfuUfaCfgGfuUfuUfcAf 1951
GfgAfdTsdT gAfadTsdT 1430 1430-1448 ugAfaAfaCfcGfuAfaAfgGfa 1952
AfUfuUfcCfuUfuAfcGfgUfuU 1953 AfaUfdTsdT fuCfadTsdT 1431 1431-1449
gaAfaAfcCfgUfaAfaGfgAfa 1954 GfAfuUfuCfcUfuUfaCfgGfuUf 1955
AfuCfdTsdT uUfcdTsdT siRNA Sequence with Chemical Modification Info
lower case (n) = 2'-O-Me; Nf = 2'-F; dT = deoxy-T residue; s =
phosphorothioate backbone modification; iB = inverted abasic
TABLE-US-00008 TABLE 7 AR Target Sequences SEQ ID ID Code Target
Sequence NO: NM_000044.3 Exon Species XD- 17 CAAAGGUUCUCUGCUAGAC
1956 1987-2005 1 h 01817K1 GACA XD- 27 UCUGGGUGUCACUAUGGAG 1957
2819-2837 2 h 01827K1 CUCU XD- 28 CUGGGUGUCACUAUGGAGC 1958
2820-2838 2 h 01828K1 UCUC XD- 29 GGGUGUCACUAUGGAGCUC 1959
2822-2840 2 h 01829K1 UCAC XD-01821K1 21 UACUACAACUUUCCACUGGC 1960
2207-2225 1 h UCU XD-01825K1 25 AAGCUUCUGGGUGUCACUA 1961 2814-2832
2 h, m UGGA XD-01826K1 26 CUUCUGGGUGUCACUAUGG 1962 2817-2835 2 h
AGCU
TABLE-US-00009 TABLE 8 .beta.-catenin Target Sequences Generic R #
name Gene Target sequences R- 1797mfm CTNNB1 CUGUUGGAUUGAU SEQ ID
UUUCGAAUCAAUCCA SEQ ID 1146 UCGAAAUU NO: ACAGUU NO: 1963 1964 R-
1870mfm CTNNB1 ACGACUAGUUCAGU SEQ ID AAGCAACUGAACUAG SEQ ID 1147
UGCUUUU NO: UCGUUU NO: 1965 1966
TABLE-US-00010 TABLE 9 .beta.-catenin and .beta.-catenin associated
siRNA Sequences Sense Strand SEQ Antisense Strand SEQ Generic
Sequence (5'-3') ID Sequence (5'-3') ID R # name Gene Passenger
Strand (PS)2 NO: Guide Strand (GS)3 NO: R- 1797mfm CTNNB1
iBcsuGfuUfgGfaUfuGfa 1967 usUfsusCfgAfaUfcAfaUfcC 1968 1146
UfuCfgAfaAfusuiB faAfcAfgusu R- 1870mfm CTNNB1 iBascGfaCfuAfgUfuCfa
1969 asAfsgsCfaAfcUfgAfaCfuA 1970 1147 GfuUfgCfuUfusuiB fgUfcGfuusu
R- PA1746 1746 GCUCAAAGCAAUUU 1971 UGUAGAAAUUGCUUU 1972 1150
CUACAdTsdT GAGCdTsdT R- PA2328 2328 GGAUGAAACACAAA 1973
UACCUUUUGUGUUUCA 1974 1151 AGGUAdTsdT UCCdTsdT R- PA2522 2522
UGUCAGAGUUACUG 1975 UGAAACAGUAACUCUG 1976 1152 UUUCAdTsdT ACAdTsdT
R- PA3484 3484 AGCAAGAACAGAAA 1977 UUUUAUUUCUGUUCU 1978 1153
UAAAAdTsdT UGCUdTsdT R- PA5018 5018 CUAGUUCAUUUCAA 1979
UAAUUUUGAAAUGAA 1980 1154 AAUUAdTsdT CUAGdTsdT R- PB183 183
CAAGUUCACAAUUA 1981 UUGGGUAAUUGUGAA 1982 1155 CCCAAdTsdT CUUGdTsdT
R- PB272 272 GCUUGAAGAUGAAA 1983 UCGUGUUUCAUCUUCA 1984 1156
CACGAdTsdT AGCdTsdT R- PB862 862 AGAUCAAGAAAAUG 1985
UCAUACAUUUUCUUGA 1986 1157 UAUGAdTsdT UCUdTsdT R- PB948 948
CCAAAGAAAACACG 1987 UAAUUCGUGUUUUCU 1988 1158 AAUUAdTsdT UUGGdTsdT
R- PB1520 1520 CUUCGAUAAGAUUA 1989 UUCAAUAAUCUUAUCG 1990 1159
UUGAAdTsdT AAGdTsdT R- Myc953U 953 AGGAACUAUGACCU 1991
AGUCGAGGUCAUAGU 1992 1160 CGACUdTsdT UCCUdTsdT R- Myc622U 622
ACGACGAGACCUUC 1993 UUGAUGAAGGUCUCG 1994 1161 AUCAAdTsdT UCGUdTsdT
R- Myc1370U 1370 AAGAUGAGGAAGAA 1995 UCGAUUUCUUCCUCAU 1996 1162
AUCGAdTsdT CUUdTsdT R- Myc1364U 1364 AGGAAGAAAUCGAU 1997
ACAACAUCGAUUUCUU 1998 1163 GUUGUdTsdT CCUdTsdT R- Myc1711U 1711
AGCUUUUUUGCCCU 1999 CACGCAGGGCAAAAAA 2000 1164 GCGUGdTsdT GCUdTsdT
R- Myc1769U 1769 AGGUAGUUAUCCUU 2001 UUUUUAAGGAUAACU 2002 1165
AAAAAdTsdT ACCUdTsdT siRNA Sequence with Chemical Modification Info
lower case (n) = 2'-O-Me; Nf = 2'-F; dT = deoxy-T residue; s =
phosphorothioate backbone modification; iB = inverted abasic
TABLE-US-00011 TABLE 10 PIK3CA* and PIK3CB* Target Sequences Gene
Gene SEQ ID Symbol ID Name Target Sequences (97-mer) NO: PIK3CA
5290 PIK3CA_ TGCTGTTGACAGTGAGCGCCAGCTCAAAGCAATTT 2003 1746
CTACATAGTGAAGCCACAGATGTATGTAGAAATTG CTTTGAGCTGTTGCCTACTGCCTCGGA
PIK3CA 5290 PIK3CA_ TGCTGTTGACAGTGAGCGAAAGGATGAAACACAAA 2004 2328
AGGTATAGTGAAGCCACAGATGTATACCTTTTGTGT TTCATCCTTCTGCCTACTGCCTCGGA
PIK3CA 5290 PIK3CA_ TGCTGTTGACAGTGAGCGCCATGTCAGAGTTACTG 2005 2522
TTTCATAGTGAAGCCACAGATGTATGAAACAGTAA CTCTGACATGATGCCTACTGCCTCGGA
PIK3CA 5290 PIK3CA_ TGCTGTTGACAGTGAGCGCAACTAGTTCATTTCAA 2006 3555
AATTATAGTGAAGCCACAGATGTATAATTTTGAAA TGAACTAGTTTTGCCTACTGCCTCGGA
PIK3CA 5290 PIK3CA_ TGCTGTTGACAGTGAGCGCACAGCAAGAACAGAAA 2007 3484
TAAAATAGTGAAGCCACAGATGTATTTTATTTCTGT TCTTGCTGTATGCCTACTGCCTCGGA
PIK3CB 5291 PIK3CB_ TGCTGTTGACAGTGAGCGACAAGATCAAGAAAATG 2008 862
TATGATAGTGAAGCCACAGATGTATCATACATTTTC TTGATCTTGCTGCCTACTGCCTCGGA
PIK3CB 5291 PIK3CB_ TGCTGTTGACAGTGAGCGCAGCAAGTTCACAATTA 2009 183
CCCAATAGTGAAGCCACAGATGTATTGGGTAATTG TGAACTTGCTTTGCCTACTGCCTCGGA
PIK3CB 5291 PIK3CB_ TGCTGTTGACAGTGAGCGCCCCTTCGATAAGATTAT 2010 1520
TGAATAGTGAAGCCACAGATGTATTCAATAATCTT ATCGAAGGGATGCCTACTGCCTCGGA
PIK3CB 5291 PIK3CB_ TGCTGTTGACAGTGAGCGAGAGCTTGAAGATGAAA 2011 272
CACGATAGTGAAGCCACAGATGTATCGTGTTTCAT CTTCAAGCTCCTGCCTACTGCCTCGGA
PIK3CB 5291 PIK3CB_ TGCTGTTGACAGTGAGCGACACCAAAGAAAACACG 2012 948
AATTATAGTGAAGCCACAGATGTATAATTCGTGTTT TCTTTGGTGGTGCCTACTGCCTCGGA
*Species is Homo sapiens.
TABLE-US-00012 TABLE 11 PIK3CA and PIK3CB siRNA Sequences SEQ SEQ
Gene Gene ID ID Symbol ID Name siRNA Guide NO: siRNA passenger NO:
PIK3CA 5290 PIK3CA_ UGUAGAAAUUGCUU 2013 AGCUCAAAGCAAUUU 2014 1746
UGAGCUGU CUACAUA PIK3CA 5290 PIK3CA UACCUUUUGUGUUU 2015
AGGAUGAAACACAAA 2016 2328 CAUCCUUC AGGUAUA PIK3CA 5290 PIK3CA_
UGAAACAGUAACUC 2017 AUGUCAGAGUUACUG 2018 2522 UGACAUGA UUUCAUA
PIK3CA 5290 PIK3CA UAAUUUUGAAAUGA 2019 ACUAGUUCAUUUCAA 2020 3555
ACUAGUUU AAUUAUA PIK3CA 5290 PIK3CA_ UUUUAUUUCUGUUC 2021
CAGCAAGAACAGAAA 2022 3484 UUGCUGUA UAAAAUA PIK3CB 5291 PIK3CB_
UCAUACAUUUUCUU 2023 AAGAUCAAGAAAAUG 2024 862 GAUCUUGC UAUGAUA
PIK3CB 5291 PIK3CB_ UUGGGUAAUUGUGA 2025 GCAAGUUCACAAUUA 2026 183
ACUUGCUU CCCAAUA PIK3CB 5291 PIK3CB_ UUCAAUAAUCUUAU 2027
CCUUCGAUAAGAUUA 2028 1520 CGAAGGGA UUGAAUA PIK3CB 5291 PIK3CB_
UCGUGUUUCAUCUU 2029 AGCUUGAAGAUGAAA 2030 272 CAAGCUCC CACGAUA
PIK3CB 5291 PIK3CB_ UAAUUCGUGUUUUC 2031 ACCAAAGAAAACACG 2032 948
UUUGGUGG AAUUAUA
TABLE-US-00013 TABLE 12 Additional polynucleic acid molecule
sequences Base SEQ SEQ start ID ID position Guide strand NO:
Passenger strand NO: EGFR 333 ACUCGUGCCUUGGCAA 2082
AGUUUGCCAAGGCACGAG 2083 R1246 ACUUU UUU EGFR 333 ACUCGUGCCUUGGCAA
2084 AGUUUGCCAAGGCACGAG 2085 R1195 ACUUU UUU EGFR 333
ACUCGUGCCUUGGCAA 2086 AGUUUGCCAAGGCACGAG 2087 R1449 ACUUU UUU KRAS
237 UGAAUUAGCUGUAUCG 2088 TGACGAUACAGCUAAUUC 2089 R1450 UCAUU AUU
KRAS 237 UGAAUUAGCUGUAUCG 2090 UGACGAUACAGCUAAUUC 2091 R1443 UCAUU
AUU KRAS 237 UGAAUUAGCUGUAUCG 2092 UGACGAUACAGCUAAUUC 2093 R1194
UCAUU AUU CTNNB1 1248 UAAGUAUAGGUCCUCA 2094 UAAUGAGGACCUAUACU 2095
R1442 UUAUU UAUU CTNNB1 1797 TUUCGAAUCAAUCCAA 2096
CUGUUGGAUUGAUUCGA 2097 R1404 CAGUU AAUU CTNNB1 1797
UUUCGAAUCAAUCCAA 2098 CUGUUGGAUUGAUUCGA 2099 R1441 CAGUU AAUU
CTNNB1 1797 UUUCGAAUCAAUCCAA 2100 CUGUUGGAUUGAUUCGA 2101 R1523
CAGUU AAUU HPRT 425 AUAAAAUCUACAGUCA 2102 CUAUGACUGUAGAUUUU 2103
R1492 UAGUU AUUU HPRT 425 UUAAAAUCUACAGUCA 2104 CUAUGACUGUAGAUUUU
2105 R1526 UAGUU AAUU HPRT 425 UUAAAAUCUACAGUCA 2106
CUAUGACUGUAGAUUUU 2107 R1527 UAGUU AAUU AR 2822 GAGAGCUCCAUAGUGA
2108 GUGUCACUAUGGAGCUCU 2109 R1245 CACUU CUU
Example 2
General Experimental Protocol
[0592] Stem-Loop qPCR Assay for Quantification of siRNA
[0593] Plasma samples were directly diluted in TE buffer. 50 mg
tissue pieces were homogenized in 1 mL of Trizol using a
FastPrep-24 tissue homogenizer (MP Biomedicals) and then diluted in
TE buffer. Standard curves were generated by spiking siRNA into
plasma or homogenized tissue from untreated animals and then
serially diluting with TE buffer. The antisense strand of the siRNA
was reverse transcribed using a TaqMan MicroRNA reverse
transcription kit (Applied Biosystems) with 25 nM of a
sequence-specific stem-loop RT primer. The cDNA from the RT step
was utilized for real-time PCR using TaqMan Fast Advanced Master
Mix (Applied Biosystems) with 1.5 .mu.M of forward primer, 0.75
.mu.M of reverse primer, and 0.2 .mu.M of probe. The sequences of
KRAS and EGFR siRNA antisense strands and all primers and probes
used to measure them are shown in Table 13. Quantitative PCR
reactions were performed using standard cycling conditions in a
ViiA 7 Real-Time PCR System (Life Technologies). The Ct values were
transformed into plasma or tissue concentrations using the linear
equations derived from the standard curves.
TABLE-US-00014 TABLE 13 Sequences for all siRNA antisense strands,
primers, and probes used in the stem-loop qPCR assay. Target Name
Sequence (5'-3') SEQ ID NO: KRAS Antisense UGAAUUAGCUGUAUCGUCAUU
2033 KRAS RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA 2034
TACGACAATGACG KRAS Forward GGCGGCTGAATTAGCTGTATCGT 2035 KRAS
Reverse AGTGCAGGGTCCGAG 2036 KRAS Probe
(6FAM)-TGGATACGACAATGAC-(NFQ-MGB) 2037 Target Name Sequence (5'-3')
EGFR Antisense ACUCGUGCCUUGGCAAACUUU 2038 EGFR RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA 2039 TACGACAAAGTTTG EGFR
Forward GGCGGCACTCGTGCCTTGGCA 2040 EGFR Reverse AGTGCAGGGTCCGAG
2041 EGFR Probe (6FAM)-TGGATACGACAAAGTT-(NFQ-MGB) 2042
[0594] Comparative qPCR Assay for Determination of mRNA
Knockdown
[0595] Tissue samples were homogenized in Trizol as described
above. Total RNA was isolated using RNeasy RNA isolation 96-well
plates (Qiagen), then 500 ng RNA was reverse transcribed with a
High Capacity RNA to cDNA kit (ThermoFisher). KRAS, EGFR, CTNNB1
and PPIB mRNA was quantified by TaqMan qPCR analysis performed with
a ViiA 7 Real-Time PCR System. The TaqMan primers and probes for
KRAS were designed and validated by Avidity and are shown in Table
14. The TaqMan primers and probes for EGFR and CTNNB1 were
purchased from Applied Biosystems as pre-validated gene expression
assays. PPIB (housekeeping gene) was used as an internal RNA
loading control, with all TaqMan primers and probes for PPIB
purchased from Applied Biosystems as pre-validated gene expression
assays. Results are calculated by the comparative Ct method, where
the difference between the target gene (KRAS, CTNNB1, or EGFR) Ct
value and the PPIB Ct value (.DELTA.Ct) is calculated and then
further normalized relative to the PBS control group by taking a
second difference (.DELTA..DELTA.Ct).
TABLE-US-00015 TABLE 14 Sequences of primers and probes for KRAS
mRNA detection using comparative qPCR assay. Target Species Name
Sequence (5'-3') SEQ ID NO: KRAS Mouse Forward
CGCCTTGACGATACAGCTAAT 2043 KRAS Mouse Reverse
TGTTTCCTGTAGGAGTCCTCTAT 2044 KRAS Mouse Probe (6FAM)- 2045 and 2114
TCACTTTGT(Zen)GGATGAGTATGACCCTACG- (IABkFQ) Target Species Name
Sequence (5-3') KRAS Human Forward GTGCCTTGACGATACAGCTAAT 2046 KRAS
Human Reverse CCAAGAGACAGGTTTCTCCATC 2047 KRAS Human Probe (6FAM)-
2048 and 2115 CCAACAATA(Zen)GAGGATTCCTACAGGAAGCA- (IABkFQ)
[0596] Animals
[0597] All animal studies were conducted following protocols in
accordance with the Institutional Animal Care and Use Committee
(IACUC) at Explora BioLabs, which adhere to the regulations
outlined in the USDA Animal Welfare Act as well as the "Guide for
the Care and Use of Laboratory Animals" (National Research Council
publication, 8th Ed., revised in 2011). All mice were obtained from
either Charles River Laboratories or Harlan Laboratories.
[0598] H358, HCC827, and Hep-3B2 1-7 Subcutaneous Flank Tumor
Model
[0599] For the H358 subcutaneous flank tumor model, tumor cells
were inoculated and tumors were established according to the
following methods. Female NCr nu/nu mice were identified by ear-tag
the day before cell injection. Mice were weighed prior to
inoculation. H358 cells were cultured with 10% FBS/RPMI medium and
harvested with 0.05% Trypsin and Cell Stripper (MediaTech). 5
million H358 cells in 0.05 ml Hank's Balanced Salt Solution (HBSS)
with Matrigel (1:1) were injected subcutaneously (SC) into the
upper right flank of each mouse. Tumor growth was monitored by
tumor volume measurement using a digital caliper starting on day 7
after inoculation, and followed 2 times per week until average
tumor volume reaches >100 & .ltoreq.300 mm.sup.3. Once
tumors were staged to the desired volume (average from 100 to 300
mm.sup.3), animals were randomized and mice with very large or
small tumors were culled. Mice were divided into the required
groups and randomized by tumor volume. Mice were then treated as
described in the individual experiments.
[0600] For the Hep3B orthotropic liver tumor model, tumor cells
were inoculated and tumors were established according to the
following methods. Female NCr nu/nu mice were identified by ear-tag
the day before, mice will be anesthetized with isoflurane. The mice
were then placed in a supine position on a water circulating
heating pad to maintain body temperature. A small transverse
incision below the sternum will be made to expose the liver. Cancer
cells were slowly injected into the upper left lobe of the liver
using a 28-gauge needle. The cells were injected at a 30-degree
angle into the liver, so that a transparent bleb of cells can be
seen through the liver capsule. Hep 3B2.1 7 cells were prepared by
suspending in cold PBS (0.1-5.times.10.sup.6 cells) and mixing with
diluted matrigel (30.times. in PBS). 30-50 ul of the cell/matrigel
was inoculated. After injection, a small piece of sterile gauze was
placed on the injection site, and light pressure was applied for 1
min to prevent bleeding. The abdomen was then closed with a 6-0
silk suture. After tumor cell implantation, animals were kept in a
warm cage, observed for 1-2 h, and subsequently returned to the
animal room after full recovery from the anesthesia. 7-10 days
after tumor implantation animals were randomized, divided into the
required groups and then treated as described in the individual
experiments.
[0601] LNCap Subcutaneous Flank Tumor Model
[0602] LNCaP cells (ATCC.RTM. CRL-1740.TM.) were grown in RPMI+10%
FBS supplemented with non-essential amino acids and sodium pyruvate
to a confluency of about 80%. Cells were mixed 1:1 with matrigel
and 5-7*106 cells injected subcutaneously into male SCID mice (6-8
weeks). Tumors usually developed within 3-5 weeks to a size of
100-350 mm.sup.3. Animals bearing tumors within this range were
randomized and treated with ASCs by injections into the tail vein.
For PD studies animals were sacrificed 96 hours after injection and
organ fragments harvested, weighted, and frozen in liquid nitrogen.
For RNA isolation, organ samples were homogenized in Trizol and RNA
prepared using a Qiagen RNeasy 96 Plus kit following the
instructions by the manufacturer. RNA concentrations were
determined spectroscopically. RNAs were converted into cDNAs by
reverse transcription and expression of specific targets quantified
by qPCR using the .DELTA..DELTA.CT method and validated Taqman
assays (Thermofisher). Samples were standardize to the expression
levels of PPIB.
[0603] Cholesterol siRNA Conjugate Synthesis
[0604] All siRNA single strands were fully assembled on solid phase
using standard phospharamidite chemistry and purified over HPLC.
Purified single strands were duplexed to get the double stranded
siRNA. Structure of cholesterol conjugated to the passenger strand
is illustrated in FIG. 2. Table 15 shows KRAS, EGFR, and CTNNB1
siRNA sequences.
TABLE-US-00016 TABLE 15 MW SEQ ID siRNA Strand Sequence (5'-3')
observed NO: KRAS Passenger
Chol-iBusgAfcGfaUfaCfaGfcUfaAfuUfcAfusuiB 7813.6 2049 KRAS Guide
UfsGfsasAfuUfaGfcUfgUfaUfcGfuCfausu 6874.6 2050 EGFR Passenger
Chol-iBasgUfuUfgCfcAfaGfgCfaCfgAfgUfusuiB 7884.6 2051 EGFR Guide
asCfsusCfgUfgCfcUfuGfgCfaAfaCfuusu 6860.6 2052 CTNNB1 Passenger
Chol-iBcsuGfuUfgGfaUfuGfaUfuCfgAfaAfusuiB 7847.5 2053 CTNNB1 Guide
usUfsusCfgAfaUfcAfaUfcCfaAfcAfgusu 6852.6 2054
[0605] The siRNA chemical modifications include: [0606] upper case
(N)=2'-OH (ribo); [0607] lower case (n)=2'-O-Me (methyl); [0608]
dN=2'-H (deoxy); [0609] Nf=2'-F (fluoro); [0610] s=phosphorothioate
backbone modification; [0611] iB=inverted abasic
[0612] Peptide Synthesis
[0613] Peptides were synthesized on solid phase using standard Fmoc
chemistry. Both peptides have cysteine at the N-terminus and the
cleaved peptides were purified by HPLC and confirmed by mass
spectroscopy. INF7 peptide is as illustrated in FIG. 3 (SEQ ID NO:
2055). Melittin peptide is as illustrated in FIG. 4 (SEQ ID NO:
2060).
[0614] Anti-EGFR Antibody
[0615] Anti-EGFR antibody is a fully human IgG1.kappa. monoclonal
antibody directed against the human epidermal growth factor
receptor (EGFR). It is produced in the Chinese Hamster Ovary cell
line DJT33, which has been derived from the CHO cell line CHO-K1SV
by transfection with a GS vector carrying the antibody genes
derived from a human anti-EGFR antibody producing hybridoma cell
line (2F8). Standard mammalian cell culture and purification
technologies are employed in the manufacturing of anti-EGFR
antibody.
[0616] The theoretical molecular weight (MW) of anti-EGFR antibody
without glycans is 146.6 kDa. The experimental MW of the major
glycosylated isoform of the antibody is 149 kDa as determined by
mass spectrometry. Using SDS-PAGE under reducing conditions the MW
of the light chain was found to be approximately 25 kDa and the MW
of the heavy chain to be approximately 50 kDa. The heavy chains are
connected to each other by two inter-chain disulfide bonds, and one
light chain is attached to each heavy chain by a single inter-chain
disulfide bond. The light chain has two intra-chain disulfide bonds
and the heavy chain has four intra-chain disulfide bonds. The
antibody is N-linked glycosylated at Asn305 of the heavy chain with
glycans composed of N-acetyl-glucosamine, mannose, fucose and
galactose. The predominant glycans present are fucosylated
bi-antennary structures containing zero or one terminal galactose
residue.
[0617] The charged isoform pattern of the IgG1.kappa. antibody has
been investigated using imaged capillary IEF, agarose IEF and
analytical cation exchange HPLC. Multiple charged isoforms are
found, with the main isoform having an isoelectric point of
approximately 8.7.
[0618] The major mechanism of action of anti-EGFR antibody is a
concentration dependent inhibition of EGF-induced EGFR
phosphorylation in A431 cancer cells. Additionally, induction of
antibody-dependent cell-mediated cytotoxicity (ADCC) at low
antibody concentrations has been observed in pre-clinical cellular
in vitro studies.
Example 3
[0619] Synthesis, Purification and Analysis of Antibody-PEG-EGFR
and Antibody-EGFR Conjugates
[0620] Step 1: Antibody Conjugation with Maleimide-PEG-NHS Followed
by SH-EGFR
[0621] Anti-EGFR antibody (EGFR-Ab) was exchanged with 1.times.
Phosphate buffer (pH 7.4) and made up to 5 mg/ml concentration. To
this solution, 2 equivalents of SMCC linker or
maleimide-PEGxkDa-NHS (x=1, 5, 10, 20) was added and rotated for 4
hours at room temperature. Unreacted maleimide-PEG was removed by
spin filtration using 50 kDa MWCO Amicon spin filters and PBS pH
7.4. The antibody-PEG-Mal conjugate was collected and transferred
into a reaction vessel. SH-C6-EGFR (2 equivalents) was added at RT
to the antibody-PEG-maleimide in PBS and rotated overnight. The
reaction mixture was analyzed by analytical SAX column
chromatography and conjugate along with unreacted antibody and
siRNA was seen.
[0622] Step 2: Purification
[0623] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing the antibody-PEG-EGFR conjugate were pooled,
concentrated and buffer exchanged with PBS, pH 7.4. Antibody siRNA
conjugates with SMCC linker, PEG1 kDa, PEG5 kDa and PEG10 kDa were
separated based on the siRNA loading. Conjugates with PEG20 kDa
gave poor separation.
[0624] Step-3: Analysis of the Purified Conjugate
[0625] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using either anion exchange chromatography method-2 or anion
exchange chromatography method-3. Examples of all the conjugates
made using these methods are described in Table 16.
TABLE-US-00017 TABLE 16 List of AXCYB conjugates HPLC retention
time (minutes) with Anion exchange chromatography method-2
Conjugate DAR = 1 DAR = 2 DAR = >2 EGFR-Ab-EGFR 9.0 9.9 10.4
EGFR-Ab-PEG1kDa-EGFR 9.2 10.0 10.6 EGFR-Ab-PEG5kDa-EGFR 8.7 9.3 ND
EGFR-Ab-PEG10kDa-EGFR 8.6 8.8 to 10; mix of DAR 2-3
EGFR-Ab-PEG20kDa-EGFR 8.6; Mixture of DAR of 1-3 Holo-anti-B cell
9.2 9.5 Ab-PEG20kDa-EGFR
[0626] Anion Exchange Chromatography Method-1 [0627] 1. Column:
Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID.times.15 cm, 13 um
[0628] 2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM
TRIS, 1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min [0629] 3.
Gradient:
TABLE-US-00018 [0629] a. % A % B Column Volume b. 100 0 1.00 c. 60
40 18.00 d. 40 60 2.00 e. 40 60 5.00 f. 0 100 2.00 g. 100 0
2.00
[0630] Anion Exchange Chromatography Method-2 [0631] 1. Column:
Thermo Scientific, ProPac.TM. SAX-10, Bio LC.TM., 4.times.250 mm
[0632] 2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B:
80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min
[0633] 3. Gradient:
TABLE-US-00019 [0633] a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d.
11.00 40 60 e. 13.00 40 60 f. 15.00 90 10 g. 20.00 90 10
[0634] Anion Exchange Chromatography Method-3 [0635] 1. Column:
Thermo Scientific, ProPac.TM. SAX-10, Bio LCTM, 4.times.250 mm
[0636] 2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B:
80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl [0637] 3. Flow Rate:
0.75 ml/min [0638] 4. Gradient:
TABLE-US-00020 [0638] a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d.
11.00 40 60 e. 23.00 40 60 f. 25.00 90 10 g. 30.00 90 10
[0639] The analytical data for EGFR antibody-PEG20 kDa-EGFR are
illustrated in FIG. 5 and FIG. 6. FIG. 5 shows the analytical HPLC
of EGFR antibody-PEG20 kDa-EGFR. FIG. 6 shows a SDS-PAGE analysis
of EGFR antibody-PEG20 kDa-EGFR conjugate. The analytical
chromatogram of EGFR antibody-PEG10 kDa-EGFR is illustrated in FIG.
7. The analytical data for EGFR antibody-PEG5 kDa-EGFR are
illustrated in FIG. 8 and FIG. 9. FIG. 8 shows the analytical
chromatogram of EGFR antibody-PEG5 kDa-EGFR. FIG. 9 shows SDS PAGE
analysis of EGFR antibody-PEG10 kDa-EGFR and EGFR antibody-PEG5
kDa-EGFR conjugates. The analytical data for EGFR antibody-PEG1
kDa-EGFR conjugates with different siRNA loading is illustrated in
FIG. 10.
Example 4
[0640] Synthesis, Purification and Analysis of Antibody-siRNA-PEG
Conjugates
[0641] Step 1: Antibody Conjugation with SMCC Linker Followed by
SH-KRAS-PEG5 kDa
[0642] Anti-EGFR antibody was exchanged with 1.times. Phosphate
buffer (pH 7.4) and made up to 5 mg/ml concentration. To this
solution, 2 equivalents of SMCC linker (succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was added and
rotated for 4 hours at room temperature. Unreacted SMCC linker was
removed by spin filtration using 50 kDa MWCO Amicon spin filters
and PBS buffer pH 7.4. The retentate was collected and 2
equivalents of SH-C6-KRAS-PEG5 kDa was added at RT and rotated
overnight. The reaction mixture was analyzed by analytical SAX
column chromatography and the conjugate along with unreacted
antibody and siRNA was observed.
[0643] Step 2: Purification
[0644] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing the antibody-KRAS-PEG conjugate were pooled,
concentrated and buffer exchanged with PBS, pH 7.4.
[0645] Step-3: Analysis of the Purified Conjugate
[0646] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using anion exchange chromatography method-3 (described in
example 1). Examples of the conjugates made using the methods
described in Examples 4 and 5 are illustrated in Table 17.
TABLE-US-00021 TABLE 17 List of A-X-B-Y-C conjugates HPLC retention
time (minutes) with Anion exchange chromatography method-3
Conjugate DAR = 1 DAR = 2 DAR => 2 EGFR-Ab-KRAS-PEG5kDa 9.2
EGFR-Ab-S-S-KRAS- 9.0 PEG5kDa Holo-anti-B cell Ab-KRAS- 9.2 9.7
10.1 PEG5kDa Panitumumab-KRAS- 9.2 9.7 10.2 PEG5kDa
[0647] The HPLC chromatogram of EGFR Antibody-KRAS-PEG5 kDa is
illustrated in FIG. 11. The HPLC chromatogram of
Panitumumab-KRAS-PEG5 kDa is as shown in FIG. 12.
Example 5
[0648] Synthesis, Purification and Analysis of
Antibody-S--S-siRNA-PEG Conjugates
[0649] Step 1: Antibody Conjugation with SPDP Linker Followed by
SH-siRNA-PEG5 kDa
[0650] Anti-EGFR antibody was exchanged with 1.times. Phosphate
buffer (pH 7.4) and made up to 5 mg/ml concentration. To this
solution, 2 equivalents of SPDP linker (succinimidyl
3-(2-pyridyldithio)propionate) was added and rotated for 4 hours at
room temperature. Unreacted SPDP linker was removed by spin
filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS
buffer. The retentate was collected and 2 equivalents of
SH-C6-siRNA-PEG5 kDa was added at room temperature and rotated
overnight. The reaction mixture was analyzed by analytical SAX
column chromatography and conjugate along with unreacted antibody
and siRNA was seen.
[0651] Step 2: Purification
[0652] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing the antibody-PEG-siRNA conjugate were pooled,
concentrated and buffer exchanged with PBS, pH 7.4.
[0653] Step-3: Analysis of the Purified Conjugate
[0654] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using anion exchange chromatography method-2. The HPLC
chromatogram of EGFR Antibody-S-S-siRNA-PEG5 kDa (DAR=1) is as
shown in FIG. 13.
Example 6
[0655] Synthesis, Purification and Analysis of
Antibody-SMCC-Endosomal Escape Peptide Conjugates
[0656] Step 1: Antibody Conjugation with SMCC Linker or
Maleimide-PEG-NHS Followed by SH-Cys-Peptide-CONH.sub.2
[0657] Anti-EGFR antibody was exchanged with 1.times. Phosphate
buffer (pH 7.4) and made up to 10 mg/ml concentration. To this
solution, 3 equivalents of SMCC linker (succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or maleimide-PEG1
kDa-NHS was added and rotated for 1.5 hours at room temperature.
Unreacted SMCC linker or PEG linker was removed by spin filtration
using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4 (25 mM
MES pH=6.1 for Melittin conjugates). The retentate was collected
and 3 equivalents of SH-Cys-Peptide-CONH.sub.2 was added at RT and
rotated overnight. The reaction mixture was then purified by either
HIC chromatography or cation exchange chromatography to isolate the
anti-EGFR antibody-Peptide or anti-EGFR antibody-PEG1k-Peptide.
[0658] Step 2: Purification
[0659] The crude reaction mixture was purified by AKTA explorer
FPLC using either hydrophobic interaction chromatography (HIC)
method-1 or cation exchange chromatography method-1. Fractions
containing the antibody-peptide conjugates were pooled,
concentrated and buffer exchanged with PBS, pH 7.4 (10 mM Acetate
pH=6.0 for Melittin conjugates).
[0660] Step-3: Analysis of the Purified Conjugate
[0661] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. Purity and peptide loading was assessed by analytical
HPLC using either HIC method-2 or cation exchange chromatography
method-2. Examples of all the conjugates made using the method of
Example 6 are described in Tables 18 and 19.
TABLE-US-00022 TABLE 18 List of AXYD conjugates HPLC retention time
(minutes) with HIC method-2 Conjugate DAR = 1 DAR = 2 DAR => 2
EGFR-Ab-INF7 7.7 9.3 11.2 EGFR-Ab-PEG24-INF7 8.4 12.2 15.2
TABLE-US-00023 TABLE 19 List of AXYD conjugates HPLC retention time
(minutes) with cation exchange chromatography method-2 Conjugate
DAR = 1 DAR => 1 DAR => 2 EGFR-Ab-Melittin 40.9 54.8
EGFR-Ab-PEG1kDa- 48. 53.4 55.8 melittin
[0662] Cation Exchange Chromatography Method-1 [0663] 1. Column: GE
Healthcare HiPrep SP HP 16/10 [0664] 2. Solvent A: 50 mM MES
pH=6.0; Solvent B: 50 mM MES+0.5M NaCl pH=6.0; Flow Rate: 2.0
ml/min [0665] 3. Gradient:
TABLE-US-00024 [0665] a. % A % B Column Volume b. 100 0 0.1 c. 100
0 Flush loop 12 ml d. 100 0 2.5 e. 0 100 15 f. 0 100 5 g. 100 0 0.5
h. 100 0 5
[0666] Cation Exchange Chromatography Method-2 [0667] 1. Column:
Thermo Scientific, MAbPac.TM. SCX-10, Bio LC.TM., 4.times.250 mm
(product #074625) [0668] 2. Solvent A: 20 mM MES pH=5.5; Solvent B:
20 mM MES+0.3 M NaCl pH=5.5; Flow Rate: 0.5 ml/min [0669] 3.
Gradient:
TABLE-US-00025 [0669] a. Time % A % B b. 0.0 100 0 c. 5 100 0 d. 50
0 100 e. 80 0 100 f. 85 100 0 g. 90 100 0
[0670] Hydrophobic Interaction Chromatography Method-1 (HIC
Method-1) [0671] 1. Column: GE Healthcare Butyl Sepharose High
Performance (17-5432-02) 200 ml [0672] 2. Solvent A: 50 mM Sodium
Phosphate+0.8M ammonium sulfate (pH=7.0); Solvent B: 80% 50 mM
Sodium Phosphate (pH=7.0), 20% IPA; Flow Rate: 3.0 ml/min [0673] 3.
Gradient:
TABLE-US-00026 [0673] a. % A % B Column Volume b. 100 0 0.1 c. 0
100 3 d. 0 100 1.35 e. 100 0 0.1 f. 100 0 0.5
[0674] Hydrophobic Interaction Chromatography Method-2 (HIC
Method-2) [0675] 1. Column: Tosoh Bioscience TSKgel Butyl-NPR 4.6
mm ID.times.10 cm 2.5 .mu.m [0676] 2. Solvent A: 100 mM Sodium
phosphate+1.8 M ammonium sulfate (pH=7.0); Solvent B: 80% 100 mM
sodium phosphate (pH=7.0), 20% IPA; Flow Rate: 0.5 ml/min [0677] 3.
Gradient:
TABLE-US-00027 [0677] a. Time % A % B b. 0 100 0 c. 3 50 50 d. 21 0
100 e. 23 0 100 f. 25 100 0
[0678] FIG. 14 illustrates the HPLC chromatogram of EGFR
antibody-PEG24-Melittin (loading=.about.1). FIG. 15 illustrates the
HPLC chromatogram of EGFR antibody-Melittin (n=.about.1). FIG. 16
shows the mass spectrum of EGFR antibody-Melittin (n=1). FIG. 17
shows the HIC chromatogram of EGFR antibody-PEG1kDa-INF7 (Peptide
loading=.about.1). FIG. 18 shows the HPLC chromatogram of EGFR
antibody-INF7 (Peptide Loading=.about.1).
Example 7
[0679] Synthesis, Purification and Analysis of
EEP-Antibody-siRNA-PEG Conjugates
[0680] Step 1: Conjugation of PEG24 Linker Followed by
SH-Cys-Peptide-CONH.sub.2 to EGFR-Ab-siRNA-PEG
[0681] EGFR-Ab-siRNA-PEG conjugate with a siRNA loading of 1 was
conjugated with 4 equivalents of PEG1k linker (succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) in PBS, pH 7.4
buffer and rotated for 1.5 hours at room temperature. Unreacted
PEG1k linker was removed by spin filtration using 50 kDa MWCO
Amicon spin filters and PBS buffer pH 7.4. The retentate was
collected and 4 equivalents of SH-Cys-Peptide-CONH.sub.2 was added
at RT and rotated overnight.
[0682] Step 2: Purification
[0683] The reaction mixture was then purified by repeated spin
filtration using PBS buffer pH7.4 and 50 kDa Amicon spin filters
until the unreacted peptide was removed as monitored by HPLC. The
product contains a mixture of conjugates with 0, 1, 2, 3 or more
peptides conjugated to the antibody backbone.
[0684] Step-3: Analysis of the Purified Conjugate
[0685] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity and the peptide loading of the conjugate
was assessed by analytical HPLC using either HIC method-2 or cation
exchange chromatography method-2. Examples of the conjugates made
using the method described in Example 7 are shown in Table 20.
TABLE-US-00028 TABLE 20 List of (A-X-B-Y-Cn)-L-D conjugates HPLC
retention time (minutes) with cation exchange chromatography
method-2 Conjugate DAR = 0 DAR = 1 DAR = 2 DAR = 3 (EGFR-Ab-siRNA-
24 38 27 9 PEG5kDa)-PEG1k-INF7 (EGFR-Ab-siRNA- 24 11.79 (broad
peak) PEG5kDa)-PEG1k-melittin
[0686] FIG. 19 shows INF7-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).
FIG. 20 shows Melittin-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).
Example 8
In Vivo Pharmacokinetics Study of a EGFR Antibody-siRNA-PEG
Conjugate (PK-055)
[0687] Groups (n=3) of female NCr nu/nu mice bearing subcutaneous
flank H358 tumors 100-150 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=4) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups that received EGFR
antibody-siRNA-PEG conjugates were dosed at 0.5 mg/kg (based on the
weight of siRNA) and groups that received cholesterol-siRNA
conjugates were dosed at 15 mg/kg. All groups (treatments and
controls) were administered a dose volume of 5 mL/kg. Non-terminal
blood samples were collected at 2, 15, or 60 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Table 21 describes the
study design in more detail and provides a cross-reference to the
conjugate synthesis and characterization. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. 50 mg pieces of tumor, liver, kidney, and
lung were collected and snap-frozen in liquid nitrogen. mRNA
knockdown analysis and siRNA quantitation were performed as
described in Examples 2-7.
TABLE-US-00029 TABLE 21 Study design for a EGFR antibody-siRNA-PEG
Conjugate (PK-055) with a cross-reference to the synthesis and
characterization of the conjugates tested. siRNA:EGFR- siRNA Ab
melittin:siRNA Survival Terminal Harvest Cross-reference Test Dose
Ratio Ratio Dose Bleed Bleed Time to synthesis and Group Article N
(mg/kg) (mol/mol) (mol/mol) ROA Schedule (min) (h) (h)
characterization 4 EGFR- 3 0.5 1.4 -- IV t = 0 2 24 24 Example 3 5
Ab- 3 0.5 1.4 -- IV t = 0 15 96 96 6 PEG10k- 3 0.5 1.4 -- IV t = 0
60 168 168 EGFR 7 EGFR- 3 0.5 1.25 -- IV t = 0 2 24 24 Example 3 8
Ab- 3 0.5 1.25 -- IV t = 0 15 96 96 9 PEG5k- 3 0.5 1.25 -- IV t = 0
60 168 168 EGFR 10 EGFR- 3 0.5 1.25 -- IV t = 0 2 24 24 Example 3
11 Ab- 3 0.5 1.25 -- IV t = 0 15 96 96 12 PEG1k- 3 0.5 1.25 -- IV t
= 0 60 168 168 EGFR 13 EGFR- 3 0.5 1.3 -- IV t = 0 2 24 24 Example
3 14 Ab- 3 0.5 1.3 -- IV t = 0 15 96 96 15 EGFR 3 0.5 1.3 -- IV t =
0 60 168 168 16 EGFR- 3 0.5 2.6 -- IV t = 0 2 24 24 Example 4 17
Ab- 3 0.5 2.6 -- IV t = 0 15 96 96 18 KRAS- 3 0.5 2.6 -- IV t = 0
60 168 168 PEG5k (n = 2 siRNAs per EGFR- Ab) 19 EGFR- 3 0.5 1.0 --
IV t = 0 2 24 24 Example 4 20 Ab- 3 0.5 1.0 -- IV t = 0 15 96 96 21
KRAS- 3 0.5 1.0 -- IV t = 0 60 168 168 PEG5k (n = 1 siRNA per EGFR-
Ab) 22 EGFR- 3 0.5 1.0 1:1 IV t = 0 2 24 24 Example 4 and 6 23 Ab-
3 0.5 1.0 1:1 IV t = 0 15 96 96 24 KRAS- 3 0.5 1.0 1:1 IV t = 0 60
168 168 PEG5k (n = 1) + EGFR- Ab- melittin 25 Chol- 3 15 -- -- IV t
= 0 2 24 24 General 26 EGFR- 3 15 -- -- IV t = 0 15 96 96
experimental 27 333mfm 3 15 -- -- IV t = 0 60 168 168 (Example 2)
28 Chol- 3 15 -- -- IV t = 0 2 24 24 General 29 KRAS- 3 15 -- -- IV
t = 0 15 96 96 experimental 30 237ffm 3 15 -- -- IV t = 0 60 168
168 (Example 2) 31 Vehicle 4 -- -- -- IV t = 0 -- -- 24 32 4 -- --
-- IV t = 0 -- -- 96 33 4 -- -- -- IV t = 0 -- -- 168
[0688] PEG linkers of various molecular weights and a small
molecule linker were used to attach EGFR siRNA to an EGFR antibody
(EGFR-Ab) and the PK was assessed to determine the effect of the
linker molecular weight on the behavior of the mAb-siRNA conjugate
in plasma. As illustrated in FIG. 21, the molecular weight of the
PEG linker does not have a large impact on the plasma PK, except
for the 10 kDa PEG leads to a faster siRNA clearance (i.e. lower
plasma concentrations at later times). The orientation of the siRNA
and PEG relative to the EGFR-Ab was also explored. As illustrated
in FIG. 22, having the siRNA in between the EGFR-Ab and the PEG5k
(EGFR antibody-KRAS-PEG5k) results in significantly higher plasma
concentrations than the alternative conjugate where PEG5k is in
between the EGFR-Ab and the siRNA (EGFR antibody-PEG5k-EGFR). In
some instances, the use of two different siRNAs on these conjugates
does not impact the plasma kinetics.
[0689] The drug loading on the EGFR-Ab was also investigated, with
n=1 and n=2 siRNAs per EGFR-Ab. As illustrated in FIG. 23, having
only one siRNA per EGFR-Ab resulted in much higher plasma
concentrations, whereas the higher loading of n=2 siRNA per EGFR-Ab
resulted in faster clearance from plasma. The impact of adding an
endosomal escape peptide (melittin) was assessed. EGFR
antibody-KRAS-PEG5k and EGFR antibody-melittin were mixed together
in solution and co-injected. As illustrated in FIG. 24, the
presence of EGFR antibody-melittin increases the clearance from
plasma of EGFR antibody-KRAS-PEG5k at later times.
[0690] The plasma PK of cholesterol-siRNA conjugates was next
compared to the mAb-siRNA conjugates after intravenous
administration via tail vein injection. As illustrated in FIG. 25,
the chol-siRNA conjugates are cleared much faster from plasma than
the mAb-siRNA conjugates. As illustrated from the PK profile,
having either EGFR or KRAS siRNA on the conjugate did not affect
the plasma kinetics.
[0691] In addition to the plasma PK analysis, siRNA concentrations
were determined in tissues at various times post-dose to determine
the tissue PK. Tissue concentrations were measured pmol/g and then
converted to pmol/mL by assuming the density of tissue equals 1
g/mL. In FIG. 26, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1
pmol/g tissue. As illustrated in FIG. 26A, a single i.v. dose of
0.5 mg/kg of EGFR antibody-siRNA resulted in approximately 100 nM
concentrations of siRNA in tumor at 24 h post-dose for virtually
all of the conjugates. In the case of these EGFR
antibody-linker-siRNA conjugates, the molecular weight of the
linker between the EGFR-Ab and the EGFR siRNA does not seem to
alter the PK of these conjugates in the s.c. flank H358 tumors. As
illustrated in FIG. 26B, the concentration of siRNA in liver
following a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNA is
approximately 100 nM at 24 h post-dose, similar to that seen in
tumor. Only the small molecule linker at 24 h post-dose produces a
siRNA concentration in liver approximately half of what is seen
with longer PEG linkers. siRNA concentrations decrease over time in
both tumor and liver tissue with these EGFR antibody-linker-siRNA
conjugates.
[0692] The orientation of the siRNA and PEG relative to the EGFR-Ab
was also explored relative to the tissue PK profiles. As
illustrated in FIG. 27, both the EGFR antibody-KRAS-PEG5k and the
EGFR antibody-PEG5k-EGFR conjugates deliver approximately 100 nM
siRNA into both tumor and liver following a single i.v. dose of 0.5
mg/kg. However, while the EGFR antibody-KRAS-PEG5k maintains the
siRNA concentration in tumor at approximately 100 nM until 168 h
post-dose, the other 3 curves decline in concentration over time.
Next, the tissue PK as a function of drug loading was assessed. As
illustrated from FIG. 28, n=1 siRNA per EGFR-Ab delivered higher
amounts of siRNA into tumor compared to liver. However, increasing
the siRNA loading to n=2 siRNA per EGFR-Ab increased the amount of
siRNA delivered to liver and decreased the amount of siRNA
delivered to tumor. Additionally, EGFR antibody-melittin was mixed
with some formulations in order to introduce endosomal escape
functionality. As illustrated from FIG. 29, mixing and
co-administering EGFR antibody-melittin with EGFR antibody-siRNA
did not have a large impact on the tissue PK. The addition of
melittin decreased uptake of siRNA in tumor and increased the
uptake of siRNA in liver.
[0693] The tissue PK profiles of cholesterol-siRNA conjugates
(using both EGFR and KRAS siRNA) in liver and in s.c. flank H358
tumors was also assessed. As illustrated from FIG. 30, both
chol-siRNA conjugates delivered approximately 5 .mu.NI
concentrations of siRNA into liver 24 h following a single i.v.
dose of 15 mg/kg. In liver, the chol-KRAS appears to clear slightly
faster than the chol-EGFR on the 1-week time scale. The two
different chol-siRNA conjugates further show different PK profiles
in tumor. Both cholesterol conjugates deliver less siRNA into tumor
compared to liver, but the chol-EGFR delivers more siRNA into tumor
when compared to the chol-KRAS conjugate. Both chol-siRNA
conjugates are cleared from tumor over time and with a similar
slope.
[0694] A PD analysis followed the PK analysis. As illustrated in
FIG. 31A, the chol-KRAS conjugate produced only marginal
(.about.25%) mRNA knockdown of the KRAS target gene in tumor
following a single i.v. dose of 15 mg/kg. However, as illustrated
in FIG. 31B, the same 15 mg/kg dose of chol-KRAS was able to
produce >50% mRNA knockdown in the mouse liver. The chol-EGFR
conjugate was able to produce >50% mRNA knockdown in tumor, as
illustrated in FIG. 32. In some instances, the higher knockdown
with chol-EGFR in tumor compared to chol-KRAS is due to the higher
siRNA concentrations observed in tumor with chol-EGFR compared to
chol-KRAS (FIG. 30). Finally, as illustrated in FIGS. 33 and 34,
most of the EGFR antibody-siRNA conjugates resulted in
approximately 25-50% EGFR or KRAS mRNA knockdown in tumors after a
single IV dose, but at a much lower dose (0.5 mg/kg) compared to
the chol-siRNA conjugates.
Example 9
[0695] Synthesis, Purification and Analysis of Additional
Antibody-siRNA Conjugates
[0696] Scheme-6: Antibody-Lys-siRNA-PEG Conjugates Via Antibody
Lysine Conjugation of SMCC Linker
[0697] Step 1: Antibody Conjugation with SMCC Linker Followed by
SH-siRNA
[0698] Antibody was buffer exchanged with 1.times. Phosphate buffer
(pH 7.4) and made up to 10 mg/ml concentration. To this solution, 2
equivalents of SMCC linker dissolved in DMSO was added and rotated
for 4 hours at room temperature. Unreacted SMCC linker was removed
by spin filtration using 50 kDa MWCO Amicon spin filters and PBS pH
7.4. The antibody-maleimide conjugate was collected into a reaction
vessel and SH-C6-siRNA or SH-C6-siRNA-C6-NHCO-PEG-XkDa (2
equivalents) (X=0.5 kDa to 10 kDa) was added at RT in pH 7.4 PBS
with 5 mM EDTA and rotated overnight. Analysis of the reaction
mixture by analytical SAX column chromatography method-2 showed
antibody siRNA conjugate along with unreacted antibody and
siRNA.
[0699] Step 2: Purification
[0700] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
[0701] Step-3: Analysis of the Purified Conjugate
[0702] The isolated conjugates were characterized by SAX
chromatography, SEC chromatography and SDS-PAGE analysis. The
purity of the conjugate was assessed by analytical HPLC using
either anion exchange chromatography method-2. All DAR1 conjugate
generally eluted at 9.0.+-.0.4 minutes while the DAR2 and DAR3
conjugates generally eluted at 9.7.+-.0.2 minutes. Typical DAR1
conjugate is greater than 90% pure after purification while typical
DAR>2 lysine conjugates contains 70-80% DAR2 and 20-30%
DAR3.
[0703] Step 1: Antibody Interchain Disulfide Reduction with
TCEP
[0704] Antibody was buffer exchanged with borax buffer (pH 8) and
made up to 10 mg/ml concentration. To this solution, 2 equivalents
of TCEP in water was added and rotated for 2 hours at RT. The
resultant reaction mixture was buffer exchanged with pH 7.4 PBS
containing 5 mM EDTA and added to a solution of SMCC-C6-siRNA or
SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2 equivalents) (X=0.5 kDa to 10
kDa) in pH 7.4 PBS containing 5 mM EDTA at RT and rotated
overnight. Analysis of the reaction mixture by analytical SAX
column chromatography showed antibody siRNA conjugate along with
unreacted antibody and siRNA.
[0705] Step 2: Purification
[0706] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-PEG-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
[0707] Step-3: Analysis of the Purified Conjugate
[0708] The isolated conjugates were characterized by SEC, SAX
chromatography and SDS-PAGE. The purity of the conjugate was
assessed by analytical HPLC using either anion exchange
chromatography method-2 or anion exchange chromatography method-3.
Isolated DAR1 conjugates are typically eluted at 9.0+0.3 min on
analytical SAX method-2 and are greater than 90% pure. The typical
DAR>2 cysteine conjugate contains more than 85% DAR2 and less
than 15% DAR3.
[0709] Step 1: Antibody Interchain Disulfide Reduction with
TCEP
[0710] Antibody was buffer exchanged with borax buffer (pH 8) and
made up to 10 mg/ml concentration. To this solution, 2 equivalents
of TCEP in water was added and rotated for 2 hours at RT. The
resultant reaction mixture was buffer exchanged with pH 7.4 PBS
containing 5 mM EDTA and added to a solution of
CBTF-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBS
containing 5 mM EDTA at RT and rotated overnight. Analysis of the
reaction mixture by analytical SAX column chromatography showed
antibody siRNA conjugate along with unreacted antibody and
siRNA.
[0711] Step 2: Purification
[0712] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 cysteine conjugate contains greater than 85% DAR2
and less than 15% DAR3 or higher.
[0713] Step-3: Analysis of the Purified Conjugate
[0714] The isolated conjugates were characterized by either mass
spec or SDS-PAGE. The purity of the conjugate was assessed by
analytical HPLC using either anion exchange chromatography method-2
or anion exchange chromatography method-3.
[0715] Step 1: Antibody Reduction with TCEP
[0716] Antibody was buffer exchanged with borax buffer (pH 8) and
made up to 5 mg/ml concentration. To this solution, 2 equivalents
of TCEP in water was added and rotated for 2 hours at RT. The
resultant reaction mixture was exchanged with pH 7.4 PBS containing
5 mM EDTA and added to a solution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa
(2 equivalents) in pH 7.4 PBS containing 5 mM EDTA at RT and
rotated overnight. Analysis of the reaction mixture by analytical
SAX column chromatography showed antibody siRNA conjugate along
with unreacted antibody and siRNA.
[0717] Step 2: Purification
[0718] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 cysteine conjugate contains greater than 85% DAR2
and less than 15% DAR3 or higher.
[0719] Step-3: Analysis of the Purified Conjugate
[0720] The isolated conjugates were characterized by either mass
spec or SDS-PAGE. The purity of the conjugate was assessed by
analytical HPLC using either anion exchange chromatography method-2
or anion exchange chromatography method-3.
[0721] Step 1: Antibody Reduction with TCEP
[0722] Antibody was buffer exchanged with borax buffer (pH 8) and
made up to 5 mg/ml concentration. To this solution, 2 equivalents
of TCEP in water was added and rotated for 2 hours at RT. The
resultant reaction mixture was exchanged with pH 7.4 PBS containing
5 mM EDTA and added to a solution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa
(2 equivalents) in pH 7.4 PBS containing 5 mM EDTA at RT and
rotated overnight. Analysis of the reaction mixture by analytical
SAX column chromatography showed antibody siRNA conjugate along
with unreacted antibody and siRNA.
[0723] Step 2: Purification
[0724] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 cysteine conjugate contains greater than 85% DAR2
and less than 15% DAR3 or higher.
[0725] Step-3: Analysis of the Purified Conjugate
[0726] The isolated conjugates were characterized by either mass
spec or SDS-PAGE. The purity of the conjugate was assessed by
analytical HPLC using either anion exchange chromatography method-2
or anion exchange chromatography method-3.
[0727] Step 1: Antibody Conjugation with SPDP Linker Followed by
SH-siRNA-PEG5 kDa
[0728] Antibody was buffer exchanged with pH 7.4 1.times.PBS and
made up to 10 mg/ml concentration. To this solution, 2 equivalents
of SPDP linker [succinimidyl 3-(2-pyridyldithio)propionate] or its
methylated version was added and rotated for 4 hours at room
temperature. Unreacted SPDP linker was removed by spin filtration
using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS buffer. The
retentate was collected and 2 equivalents of SH-C6-siRNA-PEG5 kDa
in pH 7.4 PBS was added at room temperature and rotated overnight.
The reaction mixture was analyzed by analytical SAX column
chromatography and the conjugate along with unreacted antibody and
siRNA was seen.
[0729] Step 2: Purification
[0730] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 lysine conjugate contains 70 to 80% DAR2 and 20 to
30% DAR3 or higher.
[0731] Step-3: Analysis of the Purified Conjugate
[0732] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using anion exchange chromatography method-2.
[0733] Step 1: Antibody Reduction and Conjugation with
Pyridyldithio-siRNA-PEG5 kDa
[0734] Antibody was buffer exchanged with pH 8.0 borax buffer and
made up to 10 mg/ml concentration. To this solution, 1.5
equivalents of TCEP was added and the reaction mixture was rotated
for 1 hour at room temperature. Unreacted TCEP was removed by spin
filtration using 50 kDa MWCO Amicon spin filters and buffer
exchanged with pH 7.4 PBS buffer. The retentate was collected and 2
equivalents of pyridyldithio-C6-siRNA-PEG5 kDa in pH 7.4 PBS was
added at room temperature and rotated overnight. The reaction
mixture was analyzed by analytical SAX column chromatography and
conjugate along with unreacted antibody and siRNA was seen.
[0735] Step 2: Purification
[0736] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
[0737] Step-3: Analysis of the Purified Conjugate
[0738] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using anion exchange chromatography method-2. Typical DAR>2
cysteine conjugate contains 90% DAR2 and 10% DAR3 or higher.
[0739] Step 1: Antibody Reduction and Conjugation with
Maleimide-ECL-siRNA-PEG5 kDa
[0740] Antibody was buffer exchanged with pH 8.0 borax buffer and
made up to 10 mg/ml concentration. To this solution, 1.5
equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)
reagent was added and rotated for 1 hour at room temperature.
Unreacted TCEP was removed by spin filtration using 50 kDa MWCO
Amicon spin filters and pH 7.4 PBS buffer with 5 mM EDTA. The
retentate was collected and 1.5 equivalents of
maleimide-ECL-C6-siRNA-PEG5 kDa in pH 7.4 PBS was added at room
temperature and rotated overnight. The reaction mixture was
analyzed by analytical SAX column chromatography and conjugate
along with unreacted antibody and siRNA was seen.
[0741] Step 2: Purification
[0742] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
[0743] Step-3: Analysis of the Purified Conjugate
[0744] The isolated conjugate was characterized by either mass spec
or SDS-PAGE. The purity of the conjugate was assessed by analytical
HPLC using anion exchange chromatography method-2. Typical DAR>2
lysine conjugate contains 70 to 80% DAR2 and 20 to 30% DAR3 or
higher.
[0745] Step 1: Antibody Conjugation with NHS-PEG4-TCO Followed by
Methyltetrazine-PEG4-siRNA-PEG5 kDa
[0746] Antibody was buffer exchanged with pH 7.4 PBS and made up to
5 mg/ml concentration. To this solution, 2 equivalents of
NHS-PEG4-TCO linker was added and rotated for 4 hours at room
temperature. Unreacted linker was removed by spin filtration using
50 kDa MWCO Amicon spin filters and pH 7.4 PBS. The retentate was
collected and 2 equivalents of methyltetrazine-PEG4-siRNA-PEG5 kDa
in pH 7.4 PBS was added at room temperature. The reaction mixture
was analyzed by analytical SAX column chromatography and the
antibody-siRNA conjugate was seen along with the unreacted antibody
and siRNA.
[0747] Step 2: Purification
[0748] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 lysine conjugate contains 70-80% DAR2 and 20-30%
DAR3 or higher.
[0749] Step-3: Analysis of the Purified Conjugate
[0750] The characterization and purity of the isolated conjugate
was characterized by either mass spec or SDS-PAGE. The purity of
the conjugate was assessed by analytical HPLC using anion exchange
chromatography method-2.
[0751] Step 1: Antibody Glycan Modification and Gal-N.sub.3
Addition
[0752] Antibody was buffer exchanged with pH 6.0, 50 mM sodium
phosphate buffer and treated with EndoS2 at 37.degree. C. for 16
hrs. The reaction mixture was buffer exchanged into TBS buffer (20
mM Tris, 0.9% NaCl, pH 7.4) and UDP-GalNAz was added followed by
MnCl.sub.2, and Gal-T(Y289L) in 50 mM Tris, 5 mM EDTA (pH 8). The
final solution contained concentrations of 0.4 mg/mL antibody, 10
mM MnCl.sub.2, 1 mM UDP-GalNAz, and 0.2 mg/mL Gal-T(Y289L) and was
incubated overnight at 30.degree. C.
[0753] Step 2: DIBO-PEG-TCO Conjugation to Azide Modified
Antibody
[0754] The reaction mixture from step-1 was buffer exchanged with
PBS and 2 equivalents of DIBO-PEG4-TCO linker was added and rotated
for 6 hours at room temperature. Unreacted linker was removed by
spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4
PBS. The retentate was collected and used as is in step-3.
[0755] Step 3: Methyl Tetrazine-siRNA Conjugation to TCO Labeled
Antibody
[0756] 2 equivalents of methyltetrazine-PEG4-siRNA-PEG5 kDa in pH
7.4 PBS was added to the retentate from step-2 and rotated at room
temperature for 1 hour. The reaction mixture was analyzed by
analytical SAX column chromatography and the antibody-siRNA
conjugate was seen along with the unreacted antibody and siRNA.
[0757] Step 4: Purification
[0758] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR>2 antibody-siRNA conjugates were
separated, concentrated and buffer exchanged with pH 7.4 PBS.
Typical DAR>2 lysine conjugate contains 70-80% DAR2 and 20-30%
DAR3 or higher.
[0759] Step-5: Analysis of the Purified Conjugate
[0760] The characterization and purity of the isolated conjugate
was characterized by either mass spec or SDS-PAGE. The purity of
the conjugate was assessed by analytical HPLC using anion exchange
chromatography method-2.
[0761] Step 1: Antibody Digestion with Pepsin
[0762] Antibody was buffer exchanged with pH 4.0, 20 mM sodium
acetate/acetic acid buffer and made up to 5 mg/ml concentration.
Immobilized pepsin (Thermo Scientific, Prod#20343) was added and
incubated for 3 hours at 37.degree. C. The reaction mixture was
filtered using 30 kDa MWCO Amicon spin filters and pH 7.4 PBS. The
retentate was collected and purified using size exclusion
chromatography to isolate F(ab')2. The collected F(ab')2 was then
reduced by 10 equivalents of TCEP and conjugated with
SMCC-C6-siRNA-PEG5 at room temperature in pH 7.4 PBS. Analysis of
reaction mixture on SAX chromatography showed Fab-siRNA conjugate
along with unreacted Fab and siRNA-PEG.
[0763] Step 2: Purification
[0764] The crude reaction mixture was purified by AKTA explorer
FPLC using anion exchange chromatography method-1. Fractions
containing DAR1 and DAR2 Fab-siRNA conjugates were separated,
concentrated and buffer exchanged with pH 7.4 PBS.
[0765] Step-3: Analysis of the Purified Conjugate
[0766] The characterization and purity of the isolated conjugate
was assessed by SDS-PAGE and analytical HPLC using anion exchange
chromatography method-2.
[0767] Purification and Analytical Methods
[0768] Anion Exchange Chromatography Method-1.
[0769] Column: Tosoh Bioscience, TSKGel SuperQ-SPW, 21.5 mm
ID.times.15 cm, 13 um
[0770] Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS,
1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min
[0771] Gradient:
TABLE-US-00030 a. % A % B Column Volume b. 100 0 1.00 c. 60 40
18.00 d. 40 60 2.00 e. 40 60 5.00 f. 0 100 2.00 g. 100 0 2.00
[0772] Anion Exchange Chromatography Method-2
[0773] Column: Thermo Scientific, ProPac.TM. SAX-10, Bio LC.TM.,
4.times.250 mm
[0774] Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%
10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min
[0775] Gradient:
TABLE-US-00031 a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00
40 60 e. 13.00 40 60 f. 15.00 90 10 g. 20.00 90 10
[0776] Anion Exchange Chromatography Method-3
[0777] Column: Thermo Scientific, ProPac.TM. SAX-10, Bio LC.TM.,
4.times.250 mm
[0778] Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%
10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl
[0779] Flow Rate: 0.75 ml/min
[0780] Gradient:
TABLE-US-00032 a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00
40 60 e. 23.00 40 60 f. 25.00 90 10 g. 30.00 90 10
[0781] Size Exclusion Chromatography Method-1
[0782] Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8.times.300
mm, 5 .mu.M
[0783] Mobile phase: 150 mM phosphate buffer
[0784] Flow Rate: 1.0 ml/min for 20 mins
[0785] siRNA Synthesis
[0786] All siRNA single strands were fully assembled on solid phase
using standard phospharamidite chemistry and purified over HPLC.
Purified single strands were duplexed to get the double stranded
siRNA.
[0787] Each siRNA passenger strand contains two conjugation
handles, C6-NH.sub.2 and C6-SH, one at each end of the strand. The
passenger strand with C6-NH.sub.2 handle at 5' end contains C6-SH
at its 3' end and the strand that contains C6-NH.sub.2 handle at 3'
end contains C6-SH at its 5' end. Both conjugation handles are
connected to siRNA passenger strand via inverted abasic
phosphodiester or phosphorothioate.
[0788] A representative structure of siRNA with C6-NH.sub.2
conjugation handle at the 5' end and C6-SH at 3'end of the
passenger strand.
[0789] ASC Architectures Described in Examples 10-41
[0790] ASC Architecture-1:
[0791] Antibody-Lys-SMCC-S-3'-Passenger strand. This conjugate was
generated by antibody lysine-SMCC conjugation to thiol at the 3'
end of passenger strand.
[0792] ASC Architecture-2:
[0793] Antibody-Cys-SMCC-3'-Passenger strand. This conjugate was
generated by antibody inter-chain cysteine conjugation to SMCC at
the 3' end of passenger strand.
[0794] ASC Architecture-3:
[0795] Antibody-Lys-SMCC-S-5'-passenger strand. This conjugate was
generated by antibody lysine-SMCC conjugation to C6-thiol at the 5'
end of passenger strand.
[0796] ASC Architecture-4:
[0797] Antibody-Cys-SMCC-5'-passenger strand. This conjugate was
generated by antibody inter-chain cysteine conjugation to SMCC at
the 5' end of passenger strand.
[0798] ASC Architecture-5:
[0799] Antibody-Lys-PEG-5'-passenger strand. This conjugate was
generated by antibody PEG-TCO conjugation to tetrazine at the 5'
end of passenger strand.
[0800] ASC Architecture-6:
[0801] Antibody-Lys-PEG-5'-passenger strand. This conjugate was
generated by antibody PEG-TCO conjugation to tetrazine at the 5'
end of passenger strand.
[0802] ASC Architecture-7:
[0803] Antibody-Cys-PEG-5'-passenger strand without inverted abasic
at 5' end. This conjugate was generated using procedure similar to
architecture-2. The antibody was conjugated directly to the amine
on passenger strand 5' end sugar.
[0804] Zalutumumab (EGFR-Ab)
[0805] Zalutumumab is a fully human IgG1.kappa. monoclonal antibody
directed against the human epidermal growth factor receptor (EGFR).
It is produced in the Chinese Hamster Ovary cell line DJT33, which
has been derived from the CHO cell line CHO-K1SV by transfection
with a GS vector carrying the antibody genes derived from a human
anti-EGFR antibody producing hybridoma cell line (2F8). Standard
mammalian cell culture and purification technologies are employed
in the manufacturing of zalutumumab.
[0806] The theoretical molecular weight (MW) of zalutumumab without
glycans is 146.6 kDa. The experimental MW of the major glycosylated
isoform of the antibody is 149 kDa as determined by mass
spectrometry. Using SDS-PAGE under reducing conditions the MW of
the light chain was found to be approximately 25 kDa and the MW of
the heavy chain to be approximately 50 kDa. The heavy chains are
connected to each other by two inter-chain disulfide bonds, and one
light chain is attached to each heavy chain by a single inter-chain
disulfide bond. The light chain has two intra-chain disulfide bonds
and the heavy chain has four intra-chain disulfide bonds. The
antibody is N-linked glycosylated at Asn305 of the heavy chain with
glycans composed of N-acetyl-glucosamine, mannose, fucose and
galactose. The predominant glycans present are fucosylated
bi-antennary structures containing zero or one terminal galactose
residue. The charged isoform pattern of the IgG1.kappa. antibody
has been investigated using imaged capillary IEF, agarose IEF and
analytical cation exchange HPLC. Multiple charged isoforms are
found, with the main isoform having an isoelectric point of
approximately 8.7.
[0807] The major mechanism of action of zalutumumab is a
concentration dependent inhibition of EGF-induced EGFR
phosphorylation in A431 cancer cells. Additionally, induction of
antibody-dependent cell-mediated cytotoxicity (ADCC) at low
antibody concentrations has been observed in pre-clinical cellular
in vitro studies.
[0808] Panitumumab (EGFR2-Ab)
[0809] Panitumumab is a clinically approved, fully human IgG2
subclass monoclonal antibody specific to the epidermal growth
factor receptor (EGFR). Panitumumab has two gamma heavy chains and
two kappa light chains. Glycosylated panitumumab has a total
molecular weight of approximately 147 kDa. Panitumumab is expressed
as a glycoprotein with a single consensus N-linked glycosylation
site located on the heavy chain. Panitumumab is produced from
Chinese Hamster Ovary (CHO) cells and purified by a series of
chromatography steps, viral inactivation step, viral filtration
step and ultrafiltration/diafiltration steps.
[0810] Panitumumab acts as a competitive antagonist at the ligand
binding site of EGFR to inhibit binding and signaling mediated by
EGF and transforming growth factor .alpha., the natural ligands for
this receptor. The affinity of binding panitumumab to the EGFR was
determined be 3.5 and 5.7.times.10'.sup.2M in recombinant EGFR
using BIAcore methods. Inhibition of binding of EGF was shown in
A431 cells, a human epidermal carcinoma cell line that expresses
EGFR. Intracellular acidification, phosphorylation and
internalization of the EGFR were blocked in a dose-dependent manner
by panitumumab in A431 cells. Panitumumab was also shown to inhibit
cell growth in vitro and in vivo in the same cell line.
[0811] Herceptin (EGFR3-Ab)
[0812] Herceptin is a clinically approved, humanized IgG1 subclass
monoclonal antibody specific to the epidermal growth factor
receptor2 (EGFR2) also known as Her2. Herceptin has human Fc
.gamma.1 isotype along with kappa light chains.
[0813] PSMA-Ab
[0814] PSMA-Ab is a humanized IgG1 subclass monoclonal antibody
specific to prostate specific membrane antigen (PSMA).
[0815] ASGR1-Ab
[0816] ASGR mAb-Sino103 is a rabbit IgG monoclonal antibody that
binds mouse asialoglycoprotein receptor1 (ASGPR1). It is supplied
by Sino Biologicals Inc. (Cat #50083-R103).
[0817] ASGR2-Ab
[0818] ASGR mAb-R&D is a rat IgG.sub.2A subclass monoclonal
antibody that binds mouse asialoglycoprotein receptor 1 (ASGPR1).
It is purified by protein A or G from hybridoma culture supernatant
and supplied by R&D Systems (Cat #MAB2755)
[0819] siRNA-TriGalNAc Conjugate
[0820] The siRNA triGalNAc conjugate was synthesized using Lys-Lys
dipeptide. Protected triGalNAc was coupled with dipeptide PEG
linker and purified. After the removal of carboxylic acid
protection group on the triGalNAc-dipeptide was conjugated to the
5' end of siRNA passenger strand.
Example 10: 2016-PK-163-LNCap
[0821] siRNA Design and Synthesis
[0822] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to obtain the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0823] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0824] ASC Synthesis and Characterization
[0825] The AXBYC conjugate used in groups 3-4 were made and
purified as a DAR1 (n=1) using ASC architecture-4, as described in
Example 9. The AXB and AXCYB conjugates were made as described in
Example 9.
[0826] In Vivo Study Design
[0827] Groups (n=5) of female SCID SHO mice bearing subcutaneous
flank LNCaP tumors 100-350 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=5) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups 1-6 were dosed at 1.0
or 0.5 mg/kg (based on the weight of siRNA) as per the study design
below. All groups (treatments and controls) were administered a
dose volume of 5 mL/kg. Mice were sacrificed by CO.sub.2
asphyxiation at 96 hours post-dose. Table 22 describes the study
design in more detail. 50 mg pieces of tumor and liver, were
collected and snap-frozen in liquid nitrogen. mRNA knockdown in
target tissue was determined using a comparative qPCR assay as
described in Example 2. Total RNA was extracted from the tissue,
reverse transcribed and mRNA levels were quantified using TaqMan
qPCR, using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00033 TABLE 22 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
PSMA-Ab(Cys)-EGFR (n = 1) 5 1 IV 5.0 1 96 2 PSMA-Ab(Cys)-EGFR (n =
1) 5 0.5 IV 5.0 1 96 3 PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1
96 4 PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 5
PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 1 IV 5.0 1 96 6
PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 0.5 IV 5.0 1 96 7
PSMA-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control 5
-- IV 5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP
tumors
[0828] The orientation of the siRNA and PEG relative to the PSMA-Ab
was explored in an in vivo mouse tumor model. As illustrated in
FIG. 50A, having the siRNA in between the PSMA-Ab and the PEG5k
(PSMA-Ab(Cys)-EGFR-PEG5k or the AXBYC format) resulted in higher
levels of EGFR mRNA knockdown in the tumor relative to the
alternative conjugate where PEG5k is in between the PSMA-Ab and the
siRNA (PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach
(AXBYC) also resulted in higher levels of EGFR mRNA knockdown in
the tumor relative to the conjugate without PEG5K
(PSMA-Ab(Cys)-EGFR or AXB format).
[0829] The orientation of the siRNA and PEG relative to the PSMA-Ab
was also explored relative to the tissue PK profiles. Tissue
concentrations were measured pmol/g and then converted to pmol/mL
by assuming the density of tissue equals 1 g/mL (a concentration of
1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue). As illustrated in FIG.
50B, having the siRNA in between the PSMA-Ab and the PEG5k (AXBYC)
resulted in higher levels of siRNA delivery to the tumor relative
to the alternative conjugate where PEG5k is in between the PSMA-Ab
and the siRNA (AXCYB). This approach (AXBYC) resulted in higher
levels of EGFR siRNA delivery to the tumor relative to the
conjugate without PEG5K (AXB).
[0830] In a mouse LNCaP subcutaneous xenograph model, it was
demonstrated that the AXBYC format for the antibody siRNA conjugate
resulted in higher levels of siRNA accumulation in the tumor tissue
and a greater magnitude of EGFR mRNA knockdown, relative to the
AXCYB and AXB formats. The LNCap tumor expresses human PSMA,
resulting in tumor tissue specific accumulation of the PSMA
targeted siRNA conjugates after i.v. administration, receptor
mediate uptake and siRNA facilitated knockdown of the target
gene.
Example 11: 2016-PK-202-LNCap
[0831] siRNA Design and Synthesis
[0832] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0833] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0834] ASC Synthesis and Characterization
[0835] The AXBYC conjugate used in groups 3-5 and 7 was made and
purified as a DAR1 (n=1) using ASC architecture-4, as described in
Example 9. The AXB (groups 1-2) and AXCYB (group 6) conjugates were
made as described in Example 9.
[0836] In Vivo Study Design
[0837] Groups (n=5) of female SCID SHO mice bearing subcutaneous
flank LNCaP tumors 100-350 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=5) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups 1-6 were dosed at 1.0
or 0.5 mg/kg (based on the weight of siRNA) as per the study design
below. All groups (treatments and controls) were administered a
dose volume of 5 mL/kg. Mice were sacrificed by CO.sub.2
asphyxiation at 96 hours post-dose. Table 23 describes the study
design in more detail. 50 mg pieces of tumor and liver, were
collected and snap-frozen in liquid nitrogen. mRNA knockdown in
target tissue was determined using a comparative qPCR assay as
described in Example 2. Total RNA was extracted from the tissue,
reverse transcribed and mRNA levels were quantified using TaqMan
qPCR, using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00034 TABLE 23 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
PSMA-Ab(Cys)-EGFR (n = 1) 5 1 IV 5.0 1 96 2 PSMA-Ab(Cys)-EGFR (n =
1) 5 0.5 IV 5.0 1 96 3 PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1
96 4 PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 5
PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 6
PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 0.5 IV 5.0 1 96 7
PSMA-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 8 PBS Control 5
-- IV 5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP
tumors
[0838] The orientation of the siRNA and PEG relative to the PSMA-Ab
was also explored in an in vivo mouse tumor model. As illustrated
in FIG. 51A, having the siRNA in between the PSMA-Ab and the PEG5k
(PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC format)) resulted in higher
levels of EGFR mRNA knockdown in the tumor relative to the
alternative conjugate where PEG5k is in between the PSMA-Ab and the
siRNA (PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach
(AXBYC) also resulted in higher levels of EGFR mRNA knockdown in
the tumor relative to the conjugate without PEG5K
(PSMA-Ab(Cys)-EGFR or AXB format).
[0839] The orientation of the siRNA and PEG relative to the PSMA-Ab
was also explored relative to the tissue PK profiles. Tissue
concentrations were measured pmol/g and then converted to pmol/mL
by assuming the density of tissue equals 1 g/mL (a concentration of
1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue). As illustrated in FIG.
51B, having the siRNA in between the PSMA-Ab and the PEG5k
(PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC) resulted in higher levels of
siRNA delivery to the tumor relative to the alternative conjugate
where PEG5k is in between the PSMA-Ab and the siRNA
(PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB). This approach (AXBYC) also
resulted in higher levels of EGFR siRNA delivery to the tumor
relative to the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or
AXB).
[0840] In a mouse LNCaP subcutaneous xenograph model, it was
demonstrated that the AXBYC format for the antibody siRNA conjugate
results in higher levels of siRNA accumulation in the tumor tissue
and a greater magnitude of EGFR mRNA knockdown, relative to the
AXCYB and AXB formats. The LNCap tumor expresses human PSMA,
resulting in tumor tissue specific accumulation of the PSMA
targeted siRNA conjugates after i.v. administration, receptor
mediate uptake and siRNA facilitated knockdown of the target
gene.
Example 12: 2016-PK-219-WT
[0841] siRNA Design and Synthesis
[0842] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)).
Base, sugar and phosphate modifications were used to optimize the
potency of the duplex and reduce immunogenicity. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphorothioate-inverted abasic-phosphorothioate linker, see
Example 9 for the chemical structure.
[0843] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0844] ASC Synthesis and Characterization
[0845] The AXBYC conjugate used in groups 4-6 was made and purified
as a DAR1 (n=1) using ASC architecture-4, as described in Example
9. The AXB (groups 1-3) and AXCYB (groups 7-9) and BYC (groups
10-12) conjugates were made as described in Example 9.
[0846] In Vivo Study Design
[0847] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
24 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00035 TABLE 24 siRNA Survival Terminal Dose # of Dose
Bleed Bleed Group Test Article N (mg/kg) ROA Doses Schedule (min)
(h) 1 EGFR-Ab(Cys)-EGFR 4 0.5 IV 1 t = 0 5 24 2 (n = 1) 4 0.5 IV 1
t = 0 30 96 3 4 0.5 IV 1 t = 0 180 168 4 EGFR-Ab(Cys)-EGFR- 4 0.5
IV 1 t = 0 5 24 5 PEG5k (n = 1) 4 0.5 IV 1 t = 0 30 96 6 4 0.5 IV 1
t = 0 180 168 7 EGFR-Ab(Cys)-PEG5k- 4 0.5 IV 1 t = 0 5 24 8 EGFR (n
= 1) 4 0.5 IV 1 t = 0 30 96 9 4 0.5 IV 1 t = 0 180 168 10 EGFR
Alone (aka 4 0.5 IV 1 t = 0 5 24 11 EGFR-PEG5k) 4 0.5 IV 1 t = 0 30
96 12 4 0.5 IV 1 t = 0 180 168 Total # of Animals: 48 WT mice
CD-1
[0848] In this in vivo PK experiment the orientation of the siRNA
and PEG relative to the EGFR-Ab was explored to determine the
behavior of the mAb-siRNA conjugate in plasma. As illustrated in
FIG. 52, all the mAb-siRNA conjugates (AXB, AXBYC and AXCYB
formats) had comparable plasma PK with approximately 10% of the
siRNA remaining in the systemic circulation after 168 hours (7
days), compared to the siRNA-PEG5K (BYC format) which was rapidly
cleared from the plasma.
[0849] The AXBYC format for the antibody siRNA conjugate has
improved PK properties relative the siRNA-PEG conjugate (BYC) which
was rapidly cleared from the plasma.
Example 13: 2016-PK-199-HCC827
[0850] siRNA Design and Synthesis
[0851] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)).
Base, sugar and phosphate modifications were used to optimize the
potency of the duplex and reduce immunogenicity. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA.
[0852] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and
phosphate modifications that were used for the active EGFR siRNA
duplex were used in the negative control siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA.
[0853] ASC Synthesis and Characterization
[0854] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0855] In Vivo Study Design
[0856] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control groups (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Treatment groups 1-3 and 4-6
were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA)
as per the study design below. As described in Example 9, groups
1-3 contained the same targeting antibody, but groups 4-6 had a
different EGFR targeting antibody, while the rest of the conjugate
components (linker, siRNA and PEG) were identical. Group 7 received
an antibody conjugate with a negative control siRNA sequence
(scramble) as a control for groups 1. All groups (treatments and
controls) were administered a dose volume of 5 mL/kg. Mice were
sacrificed by CO.sub.2 asphyxiation at 96 hours post-dose. Table 25
describes the study design in more detail. 50 mg pieces of tumor
and liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in Example 2. Total RNA was extracted from the
tissue, reverse transcribed and mRNA levels were quantified using
TaqMan qPCR, using the appropriately designed primers and probes.
PPIB (housekeeping gene) was used as an internal RNA loading
control, results were calculated by the comparative Ct method,
where the difference between the target gene Ct value and the PPIB
Ct value (.DELTA.Ct) is calculated and then further normalized
relative to the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00036 TABLE 25 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 2
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4
EGFR2-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5
EGFR2-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 6
EGFR2-Ab(Cys)-EGFR-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 7
EGFR-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control 5
-- IV 5.0 1 96 Total # of Animals: 40 nu/nu mice with HCC827
tumors
[0857] siRNA concentrations were determined 96 hours in the tumor
and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg.
Tissue concentrations were measured pmol/g and then converted to
pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG.
53A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As
illustrated in FIG. 53A, both antibody conjugates were capable of
delivering higher levels of siRNA to the tumor relative to the
liver, and a dose response was observed. The EGFR antibody
conjugate was capable of delivering more siRNA to the tumor tissue,
at all the doses tested, relative to the EGFR2 antibody. See FIG.
53B. Both conjugates were capable of EGFR gene specific mRNA
knockdown at 96 hours post-administration. The control conjugate
that contained the scrambled siRNA and the PBS vehicle control did
not produce significant EGFR gene specific mRNA knockdown.
[0858] As highlighted in FIG. 54, biological activity was
demonstrated with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated that tumor specific accumulation of 2
conjugates targeted with two different EGFR antibodies conjugated
to an siRNA designed to down regulate EGFR mRNA. The HCC827 tumor
expresses high levels of human EGFR and both conjugates have a
human specific EGFR antibody to target the siRNA, resulting in
tumor tissue specific accumulation of the conjugates. Receptor
mediate uptake resulted in siRNA mediated knockdown of the target
gene.
Example 14: 2016-PK-236-HCC827
[0859] siRNA Design and Synthesis
[0860] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0861] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0862] ASC Synthesis and Characterization
[0863] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0864] In Vivo Study Design
[0865] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group 6 (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Treatment groups 1-3 were
dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA),
groups 4 and 5 at 1.0 mg/kg, as per the study design below. As
described in Example 9, groups 1-3 contained the same targeting
antibody, but groups 4 had a different EGFR targeting antibody,
while the rest of the conjugate components (linker, siRNA and PEG)
were identical. Group 6 received an antibody conjugate with a
negative control siRNA sequence (scramble) as a control for groups
5. All groups (treatments and controls) were administered a dose
volume of 5 mL/kg. Mice were sacrificed by CO.sub.2 asphyxiation at
96 hours post-dose. Table 26 describes the study design in more
detail. 50 mg pieces of tumor and liver, were collected and
snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was
determined using a comparative qPCR assay as described in Example
2. Total RNA was extracted from the tissue, reverse transcribed and
mRNA levels were quantified using TaqMan qPCR, using the
appropriately designed primers and probes. PPIB (housekeeping gene)
was used as an internal RNA loading control, results were
calculated by the comparative Ct method, where the difference
between the target gene Ct value and the PPIB Ct value (.DELTA.Ct)
is calculated and then further normalized relative to the PBS
control group by taking a second difference (.DELTA..DELTA.Ct).
TABLE-US-00037 TABLE 26 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR3-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 2
EGFR3-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 PEG5k (n = 1) 3
EGFR3-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 PEG5k (n = 1) 4
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 5
EGFR-Ab(Cys)-scramble- 5 1 IV 5.0 1 96 PEG5k (n = 1) 6 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 30 nu/nu mice with HCC827
tumors
[0866] In this in vivo PD experiment, it was demonstrated that dose
dependent EGFR gene specific mRNA knockdown (FIG. 55) at 96 hour's
post-administration with a third example of an EGFR antibody
targeting agent (EGFR3). The control conjugate that contained the
scrambled siRNA and the PBS vehicle control did not produce
significant EGFR gene specific mRNA knockdown.
[0867] As highlighted in FIG. 54, it was demonstrated that
biological activity with the A-X-B-Y-C conjugate with a range of
different antibodies and siRNA cargos that are capable of in vivo
biological activity in a range of different tissue targets. In this
example, it was demonstrated that tumor specific down regulation of
EGFR mRNA using a third EGFR antibody targeting ligand. The HCC827
tumor expresses human EGFR and both conjugates have a human
specific EGFR antibody (EGFR and EGFR3) to target the siRNA,
resulting in tumor tissue specific accumulation of the conjugates.
Receptor mediate uptake resulted in siRNA mediated knockdown of the
target gene.
Example 15: 2016-PK-234-HCC827
[0868] siRNA Design and Synthesis
[0869] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence (5' to 3') of the guide/antisense strand was
TCUCGUGCCUUGGCAAACUUU (SEQ ID NO: 2117) and it was design to be
complementary to the gene sequence starting a base position 333 for
the human mRNA transcript for EGFR. Base, sugar and phosphate
modifications were used to optimize the potency of the duplex and
reduce immunogenicity. All siRNA single strands were fully
assembled on solid phase using standard phospharamidite chemistry
and purified over HPLC. Purified single strands were duplexed to
get the double stranded siRNA. The passenger strand contained two
conjugation handles, a C6-NH.sub.2 at the 5' end and a C6-SH at the
3' end. Both conjugation handles were connected to siRNA passenger
strand via phosphorothioate-inverted abasic-phosphorothioate
linker, see Example 9 for the chemical structure.
[0870] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0871] ASC Synthesis and Characterization
[0872] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0873] In Vivo Study Design
[0874] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group 10 (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Treatment groups 1-3, 4-6
and 7-9 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight
of siRNA), as per the study design below. As described in Example
9, groups 1-3 contained the same targeting antibody (EGFR3) but
groups 4-9 had a different EGFR targeting antibody, while the rest
of the conjugate components (linker, siRNA and PEG) were identical.
Group 7-9 received an antibody conjugate with a negative control
siRNA sequence (scramble) as a control for groups 1-6. All groups
(treatments and controls) were administered a dose volume of 5
mL/kg. Mice were sacrificed by CO.sub.2 asphyxiation at 96 hours
post-dose. Table 27 describes the study design in more detail. 50
mg pieces of tumor and liver, were collected and snap-frozen in
liquid nitrogen. mRNA knockdown in target tissue was determined
using a comparative qPCR assay as described in Example 2. Total RNA
was extracted from the tissue, reverse transcribed and mRNA levels
were quantified using TaqMan qPCR, using the appropriately designed
primers and probes. PPIB (housekeeping gene) was used as an
internal RNA loading control, results were calculated by the
comparative Ct method, where the difference between the target gene
Ct value and the PPIB Ct value (.DELTA.Ct) is calculated and then
further normalized relative to the PBS control group by taking a
second difference (.DELTA..DELTA.Ct). Quantitation of tissue siRNA
concentrations were determined using a stem-loop qPCR assay as
described in Example 2. The antisense strand of the siRNA was
reverse transcribed using a TaqMan MicroRNA reverse transcription
kit using a sequence-specific stem-loop RT primer. The cDNA from
the RT step was then utilized for real-time PCR and Ct values were
transformed into plasma or tissue concentrations using the linear
equations derived from the standard curves.
TABLE-US-00038 TABLE 27 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR3-Ab(Cys)-N3'-EGFR-5'S-PEG5k 5 1 IV 5.0 1 96 (n = 1) 2
EGFR3-Ab(Cys)-N3'-EGFR-5'S-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 3
EGFR3-Ab(Cys)-N3'-EGFR-5'S-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 4
EGFR-Ab(Cys)-N5'-EGFR-3'S-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5
EGFR-Ab(Cys)-N5'-EGFR-3'S-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 6
EGFR-Ab(Cys)-N5'-EGFR-3'S-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 7
EGFR-Ab(Cys)-N5'-scramble-3'S- 5 1 IV 5.0 1 96 PEG5k (n = 1) 8
EGFR-Ab(Cys)-N5'-scramble-3'S- 5 0.5 IV 5.0 1 96 PEG5k (n = 1) 9
EGFR-Ab(Cys)-N5'-scramble-3'S- 5 0.25 IV 5.0 1 96 PEG5k (n = 1) 10
PBS Control 5 -- IV 5.0 1 96 Total # of Animals: 50 nu/nu mice with
HCC827 tumors
[0875] siRNA concentrations were determined 96 hours in the tumor
and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg.
Tissue concentrations were measured pmol/g and then converted to
pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG.
56A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As
illustrated in FIG. 56A, both antibody conjugates were capable of
delivering higher levels of siRNA to the tumor relative to the
liver, and a dose response was observed. Both conjugates were
capable of EGFR gene specific mRNA knockdown at 96 hours
post-administration relative to the scramble and vehicle control.
See FIG. 56B.
[0876] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated tumor specific accumulation of 2 conjugates
targeted with two different EGFR antibodies conjugated to an siRNA
designed to down regulate EGFR mRNA. The HCC827 tumor expresses
high levels of human EGFR and both conjugates have a human specific
EGFR antibody to target the siRNA, resulting in tumor tissue
specific accumulation of the conjugates. Receptor mediate uptake
resulted in siRNA mediated knockdown of the target gene
Example 16: 2016-PK-237-HCC827
[0877] siRNA Design and Synthesis
[0878] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-SH at the 3' end, which was
connected to siRNA passenger strand via via phosphodiester-inverted
abasic-phosphorothioate linker. The C6-SH was connected through the
phosphodiester, see Example 9 for the chemical structure. In
addition, the 5' end of the passenger strand had the inverted
abasic removed and the antibody was conjugated directly to the
amine on passenger strand 5' end sugar on a T base using a
procedure similar to architecture 2, see Example 9.
[0879] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0880] ASC Synthesis and Characterization
[0881] Conjugates in groups 1-3 were made and purified as a DAR1
(n=1) using ASC architecture-7, as described in Example 9.
[0882] Conjugates in groups 4-6 were made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9.
[0883] In Vivo Study Design
[0884] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group 7 (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Treatment groups 1-3, 4-6
were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of
siRNA), as per the study design below. As described in Example 9,
groups 1-6 contained the same targeting antibody (EGFR) but groups
1-3 had an siRNA designed to downregulate KRAS and groups 4-6 had
an siRNA designed to downregulate EGFR. All groups (treatments and
controls) were administered a dose volume of 5 mL/kg. Mice were
sacrificed by CO.sub.2 asphyxiation at 96 hours post-dose. Table 28
describes the study design in more detail. 50 mg pieces of tumor
and liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in the methods section. Total RNA was extracted
from the tissue, reverse transcribed and mRNA levels were
quantified using TaqMan qPCR, using the appropriately designed
primers and probes. PPIB (housekeeping gene) was used as an
internal RNA loading control, results were calculated by the
comparative Ct method, where the difference between the target gene
Ct value and the PPIB Ct value (.DELTA.Ct) is calculated and then
further normalized relative to the PBS control group by taking a
second difference (.DELTA..DELTA.Ct). Quantitation of tissue siRNA
concentrations were determined using a stem-loop qPCR assay as
described in Example 2. The antisense strand of the siRNA was
reverse transcribed using a TaqMan MicroRNA reverse transcription
kit using a sequence-specific stem-loop RT primer. The cDNA from
the RT step was then utilized for real-time PCR and Ct values were
transformed into plasma or tissue concentrations using the linear
equations derived from the standard curves. Plasma concentrations
of the antibody component of the conjugate were determined using an
ELISA assay.
TABLE-US-00039 TABLE 28 siRNA Dose Survival Terminal Harvest Dose
Volume # of Bleed Bleed Time Group Test Article N (mg/kg) ROA
(mL/kg) Doses (min) (h) (h) 1 EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0
1 0.25 72 72 (n = 1) 2 EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0 1 3 96
96 (n = 1) 3 EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0 1 24 168 168 (n =
1) 4 EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 0.25 72 72 (n = 1) 5
EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 3 96 96 (n = 1) 6
EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 24 168 168 (n = 1) 7 PBS
Control 5 -- IV 5.0 1 -- -- 96 Total # of Animals: 35 nu/nu mice
with HCC827 tumors
[0885] siRNA concentrations were determined 96 hours in the tumor
and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg.
Tissue concentrations were measured pmol/g and then converted to
pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG.
57A and FIG. 57B, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1
pmol/g tissue. As illustrated in FIG. 57A and FIG. 57B, both
antibody conjugates were capable of delivering higher levels of
siRNA to the tumor relative to the liver. The conjugate that
contained the siRNA designed to downregulate KRAS was capable of
KRAS gene specific mRNA knockdown (FIG. 57C) at 96 hours
post-administration relative to the conjugate that contained the
siRNA designed to down regulate EGFR or the PBS vehicle control.
Both antibody conjugate constructs had similar PK properties (see
FIG. 58A and FIG. 58B) indicating the alternative conjugation
strategy used on the 5' guide strand for the antibody had no impact
on this biological parameter.
[0886] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated tumor specific accumulation and siRNA mediated
mRNA knockdown of a EGFR antibody conjugated to an siRNA designed
to down regulate KRAS mRNA. The HCC827 tumor expresses high levels
of human EGFR and the conjugate has a human specific EGFR antibody
to target the siRNA, resulting in tumor tissue specific
accumulation of the conjugates. Receptor mediate uptake resulted in
siRNA mediated knockdown of the KRAS gene.
Example 17: 2016-PK-187-Hep3B
[0887] siRNA Design and Synthesis
[0888] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0889] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0890] ASC Synthesis and Characterization
[0891] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0892] In Vivo Study Design
[0893] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank Hep-3B2 1-7 tumors 100-300 mm.sup.3 in volume were
treated with one intravenous (i.v.) tail vein injection of siRNA
conjugate, while control group 5 (n=5) of the same mice received
one i.v. injection of PBS as a vehicle control. Treatment groups
1-3 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of
siRNA), group 4 (scramble control) was dosed at 1.0 mg/kg, as per
the study design below. Group 4 received an antibody conjugate with
a negative control siRNA sequence (scramble) as a control for group
1. All groups (treatments and controls) were administered a dose
volume of 5 mL/kg. Mice were sacrificed by CO.sub.2 asphyxiation at
96 hours post-dose. Table 29 describes the study design in more
detail. 50 mg pieces of tumor and liver, were collected and
snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was
determined using a comparative qPCR assay as described in Example
2. Total RNA was extracted from the tissue, reverse transcribed and
mRNA levels were quantified using TaqMan qPCR, using the
appropriately designed primers and probes. PPIB (housekeeping gene)
was used as an internal RNA loading control, results were
calculated by the comparative Ct method, where the difference
between the target gene Ct value and the PPIB Ct value (.DELTA.Ct)
is calculated and then further normalized relative to the PBS
control group by taking a second difference (.DELTA..DELTA.Ct).
Quantitation of tissue siRNA concentrations were determined using a
stem-loop qPCR assay as described in Example 2. The antisense
strand of the siRNA was reverse transcribed using a TaqMan MicroRNA
reverse transcription kit using a sequence-specific stem-loop RT
primer. The cDNA from the RT step was then utilized for real-time
PCR and Ct values were transformed into plasma or tissue
concentrations using the linear equations derived from the standard
curves.
TABLE-US-00040 TABLE 29 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 2
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4
EGFR-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5
-- IV 5.0 1 96 Total # of Animals: 25 nu/nu mice with Hep3B
tumors
[0894] siRNA concentrations were determined 96 hours in the tumor
and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg.
Tissue concentrations were measured pmol/g and then converted to
pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG.
59A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As
illustrated in FIG. 59A, the antibody conjugate was capable of
delivering siRNA to the tumor. The conjugate was capable of EGFR
gene specific mRNA knockdown (FIG. 59B) at 96 hours
post-administration relative to the conjugate that contained the
negative control siRNA sequence or the PBS vehicle control.
[0895] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated tumor specific accumulation and siRNA mediated
mRNA knockdown of an EGFR antibody conjugated to an siRNA designed
to down regulate EGFR mRNA. The Hep-3B2 1-7 tumor cells express
human EGFR and the conjugate has a human specific EGFR antibody to
target the siRNA, resulting in tumor tissue specific accumulation
of the conjugates. Receptor mediate uptake resulted in siRNA
mediated knockdown of the EGFR gene.
Example 18: 2016-PK-257-WT
[0896] siRNA Design and Synthesis
[0897] R1442: N5-CTNNB1-3'S
[0898] CTNNB1: A 21mer duplex with 19 bases of complementarity and
3' dinucleotide overhangs was designed against human CTNNB1. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 1248 for the human mRNA
transcript for CTNNB1 (UAAUGAGGACCUAUACUUAUU; SEQ ID NO: 2095).
Base, sugar and phosphate modifications were used to optimize the
potency of the duplex and reduce immunogenicity. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to the siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[0899] ASC Synthesis and Characterization
[0900] The antibody conjugate was made and purified as a DAR1 (n=1)
using ASC architecture-1, as described in Example 9. The
tri-GalNAc-CTNNB1 conjugate was made as described in Example 9.
[0901] In Vivo Study Design
[0902] Groups 1-3 (n=4) of wild-type female CD-1 mice were treated
with one intravenous (i.v.) tail vein injections of siRNA
conjugates, the GalNAc targeted control was doses by subcutaneous
injection. Treatment groups 1-3 received doses of 2.0 1.0 and 0.5
mg/kg (based on the weight of siRNA) and the GalNAc targeted
control conjugate was doses at 2 mg/kg. All groups were
administered a dose volume of 5.0 mL/kg. Table 30 illustrates the
study design in more detail. 50 mg pieces of liver were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct).
TABLE-US-00041 TABLE 30 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
ASGR1-Ab(Lys)-CTNNB1- 4 2 IV 5.0 1 96 PEG5k (n = 1) 2
ASGR1-Ab(Lys)-CTNNB1- 4 1 IV 5.0 1 96 PEG5k (n = 1) 3
ASGR1-Ab(Lys)-CTNNB1- 4 0.5 IV 5.0 1 96 PEG5k (n = 1) 4
3GalNAc-CTNNB1 Control 5 2 s.c. 5.0 1 96 5 PBS Control 5 -- IV 5.0
1 96 Total # of Animals: 22 WT mice (CD-1)
[0903] CTNNB1 gene knockdown was determined 96 hours post
administration. As illustrated in FIG. 60, the GalNac-conjugated
siRNA was capable of gene specific knockdown after a single s.c
injection, as has been well described by others in the field. The
same siRNA conjugated to an ASGR antibody was also capable of
CTNNB1 gene specific downregulation and in a dose dependent
manner
[0904] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated liver delivery with an ASGR antibody conjugated to
an siRNA designed to down regulate CTNNB1 mRNA. Mouse Liver cells
express the asialoglycoprotein receptor (ASGR) and the conjugate
has a mouse specific ASGR antibody to target the siRNA, resulting
in siRNA mediated knockdown of the CTNNB1 in the liver.
Example 19: 2016-PK-253-WT
[0905] siRNA Design and Synthesis
[0906] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphodiester-inverted
abasic-phosphorothioate linker. The C6-NH.sub.2 and C6-SH were
connected through the phosphodiester, see Example 9 for the
chemical structure.
[0907] ASC Synthesis and Characterization
[0908] The antibody conjugate was made and purified as a DAR1 (n=1)
using ASC architecture-1, as described in Example 9. The
tri-GalNAc-CTNNB1 conjugate was made as described in Example 9.
[0909] In Vivo Study Design
[0910] Groups 1-3 (n=4) of wild-type female CD-1 mice were treated
with one intravenous (i.v.) tail vein injections of siRNA
conjugates, the GalNAc targeted control was doses by subcutaneous
injection. Treatment groups 1-3 received doses of 2.0 1.0 and 0.5
mg/kg (based on the weight of siRNA) and the GalNAc targeted
control conjugate was doses at 2 mg/kg. All groups were
administered a dose volume of 5.0 mL/kg. Table 31 illustrates the
study design in more detail. 50 mg pieces of liver were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct).
TABLE-US-00042 TABLE 31 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
ASGR2-Ab(Lys)-KRAS- 4 2 IV 5.0 1 96 PEG5k (n = 1) 2
ASGR2-Ab(Lys)-KRAS- 4 1 IV 5.0 1 96 PEG5k (n = 1) 3
ASGR2-Ab(Lys)-KRAS- 4 0.5 IV 5.0 1 96 PEG5k (n = 1) 4 3GalNAc-KRAS
Control 5 2 s.c. 5.0 1 96 5 PBS Control 5 -- IV 5.0 1 96 Total # of
Animals: 22 WT mice (CD-1)
[0911] KRAS gene knockdown was determined 96 hours post
administration. As illustrated in FIG. 61, the GalNac-conjugated
siRNA was capable of gene specific knockdown after a single s.c
injection, as has been well described by others in the field. The
same siRNA conjugated to an ASGR antibody was also capable of KRAS
gene specific downregulation and in a dose dependent manner
[0912] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated liver delivery with an ASGR antibody conjugated to
an siRNA designed to down regulate KRAS mRNA. Mouse Liver cells
express the asialoglycoprotein receptor (ASGR) and the conjugate
has a mouse specific ASGR antibody to target the siRNA, resulting
in siRNA mediated knockdown of the KRAS in the liver
Example 20: 2016-PK-129-WT-Plasma
[0913] siRNA Design and Synthesis
[0914] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via a phosphodiester-inverted
abasic-phosphodiester linker, see Example 9 for the chemical
structure.
[0915] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0916] ASC Synthesis and Characterization
[0917] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-1, as described in Example 9.
[0918] In Vivo Study Design
[0919] Groups (n=3) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
32 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves. Plasma concentrations of antibody
were determined using an ELISA assay.
TABLE-US-00043 TABLE 32 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Group Test Article N (mg/kg) ROA (mL/kg) Doses (min)
(h) 1 EGFR2-Ab(Lys)- 3 0.5 IV 5.0 1 5 24 2 KRAS-PEG5k (N = 1) 3 0.5
IV 5.0 1 30 96 3 3 0.5 IV 5.0 1 180 168 4 PSMA-Ab(Lys)- 3 0.5 IV
5.0 1 5 24 5 EGFR-PEG5k (N = 1) 3 0.5 IV 5.0 1 30 96 6 3 0.5 IV 5.0
1 180 168 Total # of Animals: 18 WT mice CD-1
[0920] In this in vivo PK experiment the plasma clearance of two
different conjugates was explored. As illustrated in FIG. 62, both
the mAb-siRNA conjugates had comparable plasma PK when comparing
the plasma levels of the siRNA (KRAS vs EGFR) or the antibody
(EGFR2 vs PSMA).
[0921] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated that two different conjugates with different
antibody targeting ligands and different siRNA cargos have
comparable plasma PK properties.
Example 21: 2016-PK-123-LNCaP
[0922] siRNA Design and Synthesis
[0923] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0924] ASC Synthesis and Characterization
[0925] All conjugates were made and purified as a DAR1 (n=1) or
DAR2 (n=2) using ASC architecture-1, as described in Example 9.
[0926] In Vivo Study Design
[0927] Groups (n=5) of female SCID SHO mice bearing subcutaneous
flank LNCaP tumors 100-350 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=5) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups were dosed as per the
study design in Table 33. All groups (treatments and controls) were
administered a dose volume of 5.71 mL/kg. Mice were sacrificed by
CO.sub.2 asphyxiation at 72 hours post-dose. 50 mg pieces of tumor
and liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in Example 2. Total RNA was extracted from the
tissue, reverse transcribed and mRNA levels were quantified using
TaqMan qPCR, using the appropriately designed primers and probes.
PPIB (housekeeping gene) was used as an internal RNA loading
control, results were calculated by the comparative Ct method,
where the difference between the target gene Ct value and the PPIB
Ct value (.DELTA.Ct) is calculated and then further normalized
relative to the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00044 TABLE 33 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Lys)-
5 2 IV 5.71 1 72 2 EGFR-PEG5k (n = 1) 5 1 IV 5.71 1 72 3 5 0.5 IV
5.71 1 72 4 PSMA-Ab(Lys)- 5 4 IV 5.71 1 72 5 EGFR-PEG5k (n = 2) 5 2
IV 5.71 1 72 6 5 1 IV 5.71 1 72 7 PSMA-Ab(Lys)- 5 2 IV 5.71 1 72
Scramble-PEG5k (n = 1) 8 EGFR siRNA Alone 5 2 IV 5.71 1 72 9
Vehicle 5 -- IV 5.71 1 72 Total # of Animals: 45 SCID SHO mice with
LNCaP tumors
[0928] siRNA concentrations were determined 72 hours in the tumor
and liver after a single i.v. injection, see FIG. 63A. As
illustrated in FIG. 63A, the antibody conjugate with a drug to
antibody ratio of 1 (n=1) was capable of delivering siRNA to the
tumor in a dose dependent manner, at levels greater than measured
in the liver and produced EGFR gene specific mRNA knockdown
relative to the scrambled and PBS controls. This is in contrast to
the antibody conjugate with a drug to antibody ratio of 2 (n=2),
which achieved lower concentrations of siRNA in the tumor at an
equivalent dose, liver and tumor concentrations which were of the
same magnitude and a lower levels of EGFR knockdown. The
unconjugated siRNA had poor tumor and liver accumulation and no
measurable EGFR mRNA knockdown. FIG. 63B illustrates relative
percentage of EGFR mRNA in LNCaP Tumor.
[0929] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated that the DAR1 conjugate is able to achieve greater
siRNA tumor concentrations, relative to the DAR 2 and unconjugated
siRNA. In addition, the DAR1 conjugate is able to achieve greater
levels of siRNA mediate knockdown of EGFR, relative to the DAR 2
and unconjugated siRNA.
Example 22: 2016-PK-258-WT
[0930] siRNA Design and Synthesis
[0931] HPRT: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human HPRT. The
sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU
(SEQ ID NO: 2102) and design to be complementary to the gene
sequence starting a base position 425 for the human mRNA transcript
for HPRT. Base, sugar and phosphate modifications were used to
optimize the potency of the duplex and reduce immunogenicity. All
siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[0932] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and
phosphate modifications that were used for the active EGFR siRNA
duplex were used in the negative control siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0933] ASC Synthesis and Characterization
[0934] Conjugates in groups 1-3 and 7-9 were made and purified as a
DAR1 (n=1) using ASC architecture-4, as described in Example 9.
Conjugates in groups 4-6 were made and purified as a DAR1 (n=1)
using ASC architecture-1, as described in Example 9.
[0935] In Vivo Study Design
[0936] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates,
while the control group (n=4) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 34 illustrates the
study design in more detail. 50 mg pieces of tissue, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00045 TABLE 34 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell
Ab(Cys)-HPRT-PEG5k 4 3 IV 5.0 1 96 (n = 1) 2 Anti-B cell
Ab(Cys)-HPRT-PEG5k 4 1 IV 5.0 1 96 (n = 1) 3 Anti-B cell
Ab(Cys)-HPRT-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 4 Anti-B cell
Ab(Lys)-HPRT-PEG5k 4 3 IV 5.0 1 96 (n = 1) 5 Anti-B cell
Ab(Lys)-HPRT-PEG5k 4 1 IV 5.0 1 96 (n = 1) 6 Anti-B cell
Ab(Lys)-HPRT-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 7 Anti-B cell
Ab(Cys)-scramble-PEG5k 4 3 IV 5.0 1 96 (n = 1) 8 Anti-B cell
Ab(Cys)-scramble-PEG5k 4 1 IV 5.0 1 96 (n = 1) 9 Anti-B cell
Ab(Cys)-scramble-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 10 PBS Control 4
-- IV 5.0 1 96 Total # of Animals: 77 WT mice (CD-1)
[0937] As illustrated on FIG. 64A-FIG. 64C, after a single i.v.
administration of an ASC dose dependent knockdown of HPRT in heart
muscle, gastroc skeletal muscle and liver were measured. There was
no measurable knockdown of HPRT in the lung tissue (FIG. 64D). In
addition, dose dependent accumulation of the siRNA in all four
tissue compartments was observed (FIG. 64E). There was no
significant difference in the biological activity (KD and tissue
concentration) between the lysine and cysteine conjugates.
[0938] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated that an anti-B cell antibody can be used to target
an siRNA to heart muscle, gastroc skeletal muscle and liver and
achieve gene specific downregulation of the reporter gene HPRT.
There was no measurable difference in the biological activity of
the ASC constructs when a lysine or cysteine conjugation strategy
was use to attach to the antibody.
Example 23: 2016-PK-254-WT
[0939] siRNA Design and Synthesis
[0940] HPRT: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human HPRT. The
sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU
(SEQ ID NO: 2102) and design to be complementary to the gene
sequence starting a base position 425 for the human mRNA transcript
for HPRT. Base, sugar and phosphate modifications that are well
described in the field of RNAi were used to optimize the potency of
the duplex and reduce immunogenicity. All siRNA single strands were
fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphodiester-inverted
abasic-phosphorothioate linker. The C6-NH.sub.2 and C6-SH were
connected through the phosphodiester, see Example 9 for the
chemical structure.
[0941] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and
phosphate modifications that were used for the active EGFR siRNA
duplex were used in the negative control siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0942] ASC Synthesis and Characterization
[0943] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0944] In Vivo Study Design
[0945] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates,
while the control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 35 illustrates the
study design in more detail. 50 mg pieces of tissue, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00046 TABLE 35 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell
Fab(Cys)-HPRT-PEG5k 4 10 IV 5.1 1 96 (n = 1) 2 Anti-B cell
Fab(Cys)-HPRT-PEG5k 4 3 IV 5.1 1 96 (n = 1) 3 Anti-B cell
Fab(Cys)-HPRT-PEG5k 4 1 IV 5.1 1 96 (n = 1) 4 Anti-B cell
Fab(Cys)-scramble-PEG5k 4 10 IV 5.1 1 96 (n = 1) 5 Anti-B cell
Fab(Cys)-scramble-PEG5k 4 3 IV 5.1 1 96 (n = 1) 6 Anti-B cell
Fab(Cys)-scramble-PEG5k 4 1 IV 5.1 1 96 (n = 1) 7 PBS Control 5 --
IV 5.1 1 96 Total # of Animals: 29 WT mice (CD-1)
[0946] As illustrated on FIG. 65A-FIG. 65C, after a single i.v.
administration of an ASC containing an anti-B cell Fab targeting
ligand, dose dependent knockdown of HPRT in heart muscle, gastroc
skeletal muscle and liver were measured. There was no measurable
knockdown of HPRT in the lung tissue (FIG. 65D). In addition, dose
dependent accumulation of the siRNA in all four tissue compartments
was observed (FIG. 65E).
[0947] As highlighted in FIG. 54, biological activity was
demonstrated with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example it
was demonstrated that an anti-B cell Fab is used to target an siRNA
to heart muscle, gastroc skeletal muscle and liver and achieve gene
specific downregulation of the reporter gene HPRT.
Example 24: 2016-PK-245-WT
[0948] siRNA Design and Synthesis
[0949] CTNNB1: A 21mer duplex with 19 bases of complementarity and
3' dinucleotide overhangs was designed against human CTNNB1. The
sequence of the guide/antisense strand was TUUCGAAUCAAUCCAACAGUU
(SEQ ID NO: 2096), design to target the gene sequence starting a
base position 1797 for the human mRNA transcript for CTNNB1. Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphodiester-inverted
abasic-phosphorothioate linker. The C6-NH.sub.2 and C6-SH were
connected through the phosphodiester, see Example 9 for the
chemical structure.
[0950] ASC Synthesis and Characterization
[0951] Conjugates in groups 3-4 were made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9.
Conjugates in groups 1-2 were made and purified as a DAR1 (n=1)
using ASC architecture-1, as described in Example 9.
[0952] In Vivo Study Design
[0953] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates,
while the control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 36 illustrates the
study design in more detail. 50 mg pieces of tissue, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00047 TABLE 36 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell
Ab(Lys)-CTNNB1-PEG5k 4 3 IV 5.0 1 96 (n = 1) 2 Anti-B cell
Ab(Lys)-CTNNB1-PEG5k 4 1 IV 5.0 1 96 (n = 1) 3 Anti-B cell
Ab(Cys)-CTNNB1-PEG5k 4 3 IV 5.0 1 96 (n = 1) 4 Anti-B cell
Ab(Cys)-CTNNB1-PEG5k 4 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5 -- IV
5.0 1 96 Total # of Animals: 21 WT mice (CD-1)
[0954] As illustrated on FIG. 66A and FIG. 66B, after a single i.v.
administration of an ASC containing an anti-B cell antibody
targeting ligand (anti-B cell-Ab), HPRT knockdown and dose
dependent tissue siRNA accumulation in heart muscle were elicited.
As illustrated on FIG. 66C and FIG. 66D, after a single i.v.
administration of an ASC containing an anti-B cell antibody
targeting ligand, HPRT knockdown and dose dependent tissue siRNA
accumulation in gastroc skeletal muscle were elicited. There was no
significant difference in the biological activity (KD and tissue
concentration) between the lysine and cysteine conjugates.
[0955] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated that an anti-B cell antibody is used to target
an siRNA to heart muscle and gastroc skeletal muscle and achieve
gene specific downregulation of CTNNB1 mRNA.
Example 25: 2016-PK-160-LNCaP
[0956] siRNA Design and Synthesis
[0957] AR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human AR. The sequence
of the guide/antisense strand was complementary to the gene
sequence starting a base position 2822 for the human mRNA
transcript for AR (Guide strand sequence: GAGAGCUCCAUAGUGACACUU;
SEQ ID NO: 2108). Base, sugar and phosphate modifications were used
to optimize the potency of the duplex and reduce immunogenicity.
All siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphorothioate-inverted abasic-phosphorothioate linker, see
Example 9 for the chemical structure.
[0958] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0959] ASC Synthesis and Characterization
[0960] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-1, as described in Example 9.
[0961] In Vivo Study Design
[0962] Groups (n=5) of female SCID SHO mice bearing subcutaneous
flank LNCaP tumors 100-350 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control group (n=5) of the same mice received one i.v. injection of
PBS as a vehicle control. The table below describes the study
design. Mice were sacrificed by CO.sub.2 asphyxiation at 96 hours
post-dose. 50 mg pieces of tumor and liver, were collected and
snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was
determined using a comparative qPCR assay as described in Example
2. Total RNA was extracted from the tissue, reverse transcribed and
mRNA levels were quantified using TaqMan qPCR, using the
appropriately designed primers and probes. PPIB (housekeeping gene)
was used as an internal RNA loading control, results were
calculated by the comparative Ct method, where the difference
between the target gene Ct value and the PPIB Ct value (.DELTA.Ct)
is calculated and then further normalized relative to the PBS
control group by taking a second difference (.DELTA..DELTA.Ct).
Quantitation of tissue siRNA concentrations were determined using a
stem-loop qPCR assay as described in Example 2. The antisense
strand of the siRNA was reverse transcribed using a TaqMan MicroRNA
reverse transcription kit using a sequence-specific stem-loop RT
primer. The cDNA from the RT step was then utilized for real-time
PCR and Ct values were transformed into plasma or tissue
concentrations using the linear equations derived from the standard
curves.
TABLE-US-00048 TABLE 37 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
ANT4044(Lys)-AR-PEG5k (n = 1) 5 1 IV 5.0 1 96 2
ANT4044(Lys)-AR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3
ANT4044(Lys)-AR-PE5k (n = 1) 5 0.25 IV 5.0 1 96 4
ANT4044(Lys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5
-- IV 5.0 1 96 Total # of Animals: 30 castrated SCID SHO mice with
LNCaP tumors
[0963] As illustrated in FIG. 67A, after a single i.v.
administration of an ASC containing a PSMA antibody targeting
ligand and siRNA designed to downregulate AR, AR knockdown in the
LNCaP tumor tissue at all the doses tested relative to the
scrambled control was elicited. As illustrated FIG. 67B, there was
measurable accumulation of siRNA in the tumor tissue and at levels
higher than those measured in the liver tissue.
[0964] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated delivery to an LNCaP prostate tumor with a PSMA
antibody conjugated to an siRNA designed to down regulate AR mRNA.
LNCaP cells express human PSMA on cell surface, the conjugate has a
human specific PSMA antibody that binds to the antigen and allows
internalization of the siRNA, resulting in siRNA mediated knockdown
of AR in the tumor tissue.
Example 26: In Vitro Uptake and Knockdown in B Cells
[0965] siRNA Design and Synthesis
[0966] HPRT: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human HPRT. The
sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU
(SEQ ID NO: 2102) and design to be complementary to the gene
sequence starting a base position 425 for the human mRNA transcript
for HPRT. Base, sugar and phosphate modifications were used to
optimize the potency of the duplex and reduce immunogenicity. All
siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[0967] ASC Synthesis and Characterization
[0968] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0969] In Vitro Study Design
[0970] Mouse spleens were harvested and kept in PBS with 100 u/ml
penicillin and streptomycin on ice. Spleens were smashed with clean
glass slides, cut into small pieces, homogenized with 18G needles,
and filtered (70 um nylon membrane). Dead cells were removed with
the dead cell removal kit from Milteny biotec (Catalog#130-090101)
according to manufacturer instruction. To isolate mouse B cells, B
cell isolation kit Milteny biotec (Catalog#130-090-862) was used
following manufacturer instruction. Briefly, live spleen cells were
resuspended with 200 .mu.l of MACS buffer per mouse spleen. Non-B
cells were depleted with biotin-conjugated monoclonal antibodies
against CD43 (Ly48), CD4, and Ter-119, coupled with anti-biotin
magnetic microbeads. From one mouse spleen, 30 million live B cells
can be obtained. To activate isolated mouse B cells
(2.times.10.sup.6/m1 in 10% FBS RPMI-1640 with 100 u/ml penicillin
and streptomycin), a cocktail of 10 .mu.g/ml LPS, 5 .mu.g/ml
anti-IgM, 1 .mu.g/ml anti-CD40, 0.05 .mu.g/ml IL-4, and 0.05
.mu.g/ml INF.gamma. was added. After four hours of activation, ASCs
(1 pM to 10 nM) were added to 10.sup.6 cells per well in 24 (0.5 ml
media) or 12 (1 ml media) well plates. After 48 hours of ASC
treatments, cells were harvested and isolated RNAs were analyzed
for mRNA knockdown.
TABLE-US-00049 TABLE 38 Group Test Article 1 Anti-B cell
Ab(Cys)-HPRT-PEG5k (n = 1) 2 Anti-B cell Ab (Cys)-scramble-PEG5k (n
= 1) 3 Anti-B cell Fab(Cys)-HPRT-PEG5k (n = 1) 4 Anti-B cell
Fab(Cys)-scramble-PEG5k (n = 1) 5 Anti-B cell Ab 6 Vehicle
Control
[0971] In this in vitro experiment in activated primary mouse B
cells, the ability of an anti-B cell antibody and Fab ASCs to
deliver an siRNA design to downregulate Hypoxanthine-guanine
phosphoribosyltransferase (HPRT) was measured. As illustrated in
FIG. 68A, the Fab conjugate was able to downregulate HPRT relative
to the vehicle or scramble controls. As illustrated in FIG. 68B,
the antibody conjugate was able to downregulate HPRT relative to
the antibody, vehicle, and scramble controls.
[0972] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated delivery to an activated mouse B cell with a
mouse anti-B cell antibody or anti-B cell Fab conjugated to an
siRNA designed to down regulate HPRT mRNA. Activated mouse B cells
recognize and internalize the antibody-siRNA conjugate via surface
receptors that recognize the anti-B cell antibody, resulting in
siRNA mediated knockdown of HPRT.
Example 27: 2016-PK-249-WT
[0973] siRNA Design and Synthesis
[0974] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0975] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0976] ASC Synthesis and Characterization
[0977] The conjugate for groups 1-2 were made and purified as a
DAR1 (n=1) using ASC architecture-4, as described in Example 9. The
conjugate for groups 3-4 were made and purified as a DAR2 (n=2)
using ASC architecture-4, as described in Example 9. The conjugate
for groups 5-6 were made and purified as a DAR1 (n=1) using ASC
architecture-5, as described in Example 9. The conjugate for groups
7-8 were made and purified as a DAR2 (n=2) using ASC
architecture-5, as described in Example 9. The conjugate for groups
9-10 were made and purified as a DAR1 (n=1) using ASC
architecture-6, as described in Example 9. The conjugate for groups
11-12 were made and purified as a DAR2 (n=2) using ASC
architecture-6, as described in Example 9.
[0978] In Vivo Study Design
[0979] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates
(groups 1-12) or antibody alone (groups 13-14). Table 39
illustrates the study design. Non-terminal blood samples were
collected at 0.25, and 3 hours post-dose via puncture of the
retro-orbital plexus and centrifuged to generate plasma for PK
analysis. Mice were sacrificed by CO.sub.2 asphyxiation at 24 and
72 hours post-dose. Terminal blood samples were collected via
cardiac puncture and processed to generate plasma for PK analysis.
Quantitation of plasma siRNA concentrations were determined using a
stem-loop qPCR assay as described in Example 2. The antisense
strand of the siRNA was reverse transcribed using a TaqMan MicroRNA
reverse transcription kit using a sequence-specific stem-loop RT
primer. The cDNA from the RT step was then utilized for real-time
PCR and Ct values were transformed into plasma or tissue
concentrations using the linear equations derived from the standard
curves. Plasma concentrations of antibody were determined using an
ELISA assay.
TABLE-US-00050 TABLE 39 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Gr Test Article N (mg/kg) (mL/kg) Doses (h) (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 4 0.5 5.0 1 0.25 24 2
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 4 0.5 5.0 1 3 72 3
EGFR-Ab(Cys)-EGFR-PEG5k (n = 2) 4 0.5 5.0 1 0.25 24 4
EGFR-Ab(Cys)-EGFR-PEG5k (n = 2) 4 0.5 5.0 1 3 72 5
EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 0.25 24 (n = 1) 6
EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 3 72 (n = 1) 7
EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 0.25 24 (n = 2) 8
EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 3 72 (n = 2) 9
EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 0.25 24 PEG5k (n = 1) 10
EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 3 72 PEG5k (n = 1) 11
EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 0.25 24 PEG5k (n = 2) 12
EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 3 72 PEG5k (n = 2) 13
EGFR-Ab 4 0.5 5.0 1 0.25 24 14 EGFR-Ab 4 0.5 5.6 1 3 72 Total # of
Animals: 56 WT mice CD-1
[0980] In this in vivo PK study it was demonstrated the utility of
site specific conjugation. As illustrated in FIG. 69A, the DAR1
(n=1) test article (group 9) had a comparable siRNA plasma
clearance profile to the two controls (groups 1 and 5), with
approximately 10% of the siRNA remaining in the plasma after 168
hours. All the DAR2 (n=2) conjugates had much faster clearance of
the siRNA from the plasma relative to the DAR1 conjugates. As
illustrated in FIG. 69B, the DAR1 (n=1) test article (group 9) had
a comparable EGFR-Ab plasma clearance profile to the two controls
(groups 1 and 5). All the DAR2 (n=2) conjugates had much faster
clearance of the antibody from the plasma relative to the DAR1
conjugates.
[0981] In the above Examples, it was demonstrated the use of lysine
and cysteine conjugation strategies to attach the siRNA to the
antibody. In this example, it was demonstrated the utility of a
site specific conjugation strategy and demonstrate the conjugate
has comparable PK properties to non-specific conjugation
strategies.
Example 28: 2016-PK-180-HCC827
[0982] siRNA Design and Synthesis
[0983] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0984] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0985] ASC Synthesis and Characterization
[0986] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0987] In Vivo Study Design
[0988] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 40 describes the study
design. Mice were sacrificed by CO.sub.2 asphyxiation at 96 hours
post-dose. 50 mg pieces of tumor and liver, were collected and
snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was
determined using a comparative qPCR assay as described in Example
2. Total RNA was extracted from the tissue, reverse transcribed and
mRNA levels were quantified using TaqMan qPCR, using the
appropriately designed primers and probes. PPIB (housekeeping gene)
was used as an internal RNA loading control, results were
calculated by the comparative Ct method, where the difference
between the target gene Ct value and the PPIB Ct value (.DELTA.Ct)
is calculated and then further normalized relative to the PBS
control group by taking a second difference (.DELTA..DELTA.Ct).
Quantitation of plasma and tissue siRNA concentrations were
determined using a stem-loop qPCR assay as described in Example 2.
The antisense strand of the siRNA was reverse transcribed using a
TaqMan MicroRNA reverse transcription kit using a sequence-specific
stem-loop RT primer. The cDNA from the RT step was then utilized
for real-time PCR and Ct values were transformed into plasma and
tissue concentrations using the linear equations derived from the
standard curves.
TABLE-US-00051 TABLE 40 siRNA Dose Harvest Dose Volume # of Time Gr
Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 2
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4
EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 5
EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 6
EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 7
EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 1 IV 5.0 1 96 8
EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 9
EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 10
EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 1 IV 5.0 1 96 (n = 1) 11
EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 12
EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 15
EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 16 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 80 nu/nu mice with HCC827
tumors
[0989] In this in vivo PK study, replacing the SMCC linker between
the antibody and siRNA with an enzymatically cleavable linker and
the introduction of a cleavable disulfide linker between the siRNA
and PEG, or the combination of both were tested. As illustrated in
FIG. 70A, all the linker combination were capable of EGFR mRNA
knockdown in the HCC827 tumor cells relative to the scrambled
control. As illustrated in FIG. 70B, all the linker combinations
produced comparable siRNA tissue accumulation in the tumor and
liver. As illustrated in FIG. 70C, all the conjugates were capable
of maintaining high levels of siRNA in the plasma, with
approximately 10% remaining in the plasma after 168 hours.
[0990] In this AXBYC example, it was demonstrated that different
linker combinations ("X" and/or "Y") can be used to conjugate the
siRNA to the antibody and PEG.
Example 29: 2016-PK-162-LNCaP
[0991] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[0992] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[0993] ASC Synthesis and Characterization
[0994] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[0995] In Vivo Study Design
[0996] Groups 1-7 (n=5) of female SCID SHO mice bearing
subcutaneous flank LNCaP tumors 100-350 mm.sup.3 in volume were
treated with one intravenous (i.v.) tail vein injection of siRNA
conjugate, while control group 8 (n=5) of the same mice received
one i.v. injection of PBS as a vehicle control. The table below
describes the study design. Mice were sacrificed by CO.sub.2
asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and
liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in Example 2. Total RNA was extracted from the
tissue, reverse transcribed and mRNA levels were quantified using
TaqMan qPCR, using the appropriately designed primers and probes.
PPIB (housekeeping gene) was used as an internal RNA loading
control, results were calculated by the comparative Ct method,
where the difference between the target gene Ct value and the PPIB
Ct value (.DELTA.Ct) is calculated and then further normalized
relative to the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00052 TABLE 41 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
PSMA-Ab(Lys)-SS-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) 2
PSMA-Ab(Lys)-SS-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 3
PSMA-Ab(Cys)-ECL-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) 4
PSMA-Ab(Cys)-ECL-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 5
PSMA-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) FRESH 6
PSMA-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) FROZEN 7
PSMA-Ab(Cys)-svcramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP
tumors
[0997] In this in vivo PK study, a disulfide (SS), enzymatically
cleavable (ECL) or SMCC linker was used between the antibody and
siRNA. As illustrated in graph 1 on slide 42, the tumor tissue
accumulation of the siRNA was reduced when the cleavable disulfide
leaker was used instead of the ECL or SMCC linkers. As illustrated
on graph 2 on slide 42, the ECL linker strategy produced EGFR mRNA
knockdown in the LNCaP tumor cells relative to the scrambled
control. However, the SS linker failed to produce EGFR mRNA
knockdown in the LNCaP tumor cells relative to the scrambled
control. In addition to these linker experiments, the feasibility
of -80.degree. C. storage of the ASC was examined. The Formulation
was snap-frozen in liquid nitrogen at 5 mg/ml antibody
concentration, thawed at room temperature after 30 days storage at
-80.degree. C. and diluted to the required dosing concentration
prior to administration. As illustrated on graph 3 on slide 42, the
construct stored at -80.degree. C., thawed prior to administration,
retained its ability to produce EGFR mRNA knockdown in the LNCaP
tumor cells relative to the scrambled control.
[0998] In this AXBYC example, it was demonstrated that an ECL
linker ("X") can be used to conjugate the antibody to the siRNA and
that an ASC can be stored at -80.degree. C. for 1 month and thawed
prior to administration.
Example 30: 2016-PK-181-HCC827
[0999] siRNA Design and Synthesis
[1000] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications that are well described in the
field of RNAi were used to optimize the potency of the duplex and
reduce immunogenicity. All siRNA single strands were fully
assembled on solid phase using standard phospharamidite chemistry
and purified over HPLC. Purified single strands were duplexed to
get the double stranded siRNA. The passenger strand contained two
conjugation handles, a C6-NH.sub.2 at the 5' end and a C6-SH at the
3' end. Both conjugation handles were connected to siRNA passenger
strand via phosphorothioate-inverted abasic-phosphorothioate
linker, see Example 9 for the chemical structure.
[1001] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[1002] ASC Synthesis and Characterization
[1003] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1004] In Vivo Study Design
[1005] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 42 describes the study
design. Mice were sacrificed by CO.sub.2 asphyxiation at 96 hours
post-dose. 50 mg pieces of tumor and liver, were collected and
snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was
determined using a comparative qPCR assay as described in Example
2. Total RNA was extracted from the tissue, reverse transcribed and
mRNA levels were quantified using TaqMan qPCR, using the
appropriately designed primers and probes. PPIB (housekeeping gene)
was used as an internal RNA loading control, results were
calculated by the comparative Ct method, where the difference
between the target gene Ct value and the PPIB Ct value (.DELTA.Ct)
is calculated and then further normalized relative to the PBS
control group by taking a second difference (.DELTA..DELTA.Ct).
Quantitation of tissue siRNA concentrations were determined using a
stem-loop qPCR assay as described in the methods section. The
antisense strand of the siRNA was reverse transcribed using a
TaqMan MicroRNA reverse transcription kit using a sequence-specific
stem-loop RT primer. The cDNA from the RT step was then utilized
for real-time PCR and Ct values were transformed into tissue
concentrations using the linear equations derived from the standard
curves.
TABLE-US-00053 TABLE 42 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 2
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3
EGFR-Ab(Cys)-SS-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 4
EGFR-Ab(Cys)-SS-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 6
EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 7
EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 8 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 80 nu/nu mice with HCC827
tumors
[1006] In this in vivo PK study, a disulfide or SMCC linker was
used between the antibody and siRNA. As illustrated in FIG. 72A,
the tumor tissue accumulation of the siRNA was reduced when the
cleavable disulfide leaker was used instead of the more stable SMCC
linker. As illustrated in FIG. 72B, both linker strategies were
capable of producing EGFR mRNA knockdown in the HCC827 tumor cells
relative to the scrambled control.
[1007] In this AXBYC example, it was demonstrated the use of a
cleavable disulfide linker ("X") between the antibody and
siRNA.
Example 31: 2016-PK-220-WT
[1008] siRNA Design and Synthesis
[1009] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU;
SEQ ID NO: 2088). Base, sugar and phosphate modifications were used
to optimize the potency of the duplex and reduce immunogenicity.
All siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphorothioate-inverted abasic-phosphorothioate linker, see
Example 9 for the chemical structure.
[1010] ASC Synthesis and Characterization
[1011] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1012] In Vivo Study Design
[1013] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
43 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 hours post-dose. Terminal blood
samples were collected via cardiac puncture and processed to
generate plasma for PK analysis. Quantitation of plasma siRNA
concentrations were determined using a stem-loop qPCR assay as
described in Example 2. The antisense strand of the siRNA was
reverse transcribed using a TaqMan MicroRNA reverse transcription
kit using a sequence-specific stem-loop RT primer. The cDNA from
the RT step was then utilized for real-time PCR and Ct values were
transformed into plasma or tissue concentrations using the linear
equations derived from the standard curves. Plasma concentrations
of antibody were determined using an ELISA assay.
TABLE-US-00054 TABLE 43 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Group Test Article N (mg/kg) ROA (mL/kg) Doses (min)
(h) 1 EGFR-Ab(Lys)-SPDP- 4 0.5 IV 5.0 1 5 24 2 KRAS-PEG5k (n = 1) 4
0.5 IV 5.0 1 30 96 3 4 0.5 IV 5.0 1 180 168 4 EGFR-Ab(Cys)-SPDP- 4
0.5 IV 5.0 1 5 24 5 KRAS-PEG5k (n = 1) 4 0.5 IV 5.0 1 30 96 6 4 0.5
IV 5.0 1 180 168 7 EGFR-Ab(Cys)-SMPT- 4 0.5 IV 5.0 1 5 24 8
KRAS-PEG5k (n = 1) 4 0.5 IV 5.0 1 30 96 9 4 0.5 IV 5.0 1 180 168 10
EGFR-Ab(Cys)-SS(methyl)- 4 0.5 IV 5.0 1 5 24 11 KRAS-PEG5k (n = 1)
4 0.5 IV 5.0 1 30 96 12 4 0.5 IV 5.0 1 180 168 13 EGFR-Ab(Cys)- 4
0.5 IV 5.0 1 5 24 14 SS(dimethyl)-KRAS-PEG5k 4 0.5 IV 5.0 1 30 96
15 (n = 1) 4 0.5 IV 5.0 1 180 168 Total # of Animals: 60 WT mice
CD-1
[1014] In this in vivo PK study, different disulfide linkers were
explored, with varying degrees of steric hindrance, to understand
how the rate of disulfide cleavage impacts ASC plasma PK. As
illustrated in FIG. 73A, the clearance of the siRNA from the plasma
was modulated by varying the degree of steric hindrance of the
disulfide linker. FIG. 73B illustrates the clearance of the
antibody zalutumumab from the plasma.
[1015] In this example, it was demonstrated biological activity
with a range of different AXBYC conjugates in which a range of
different disulfide linkers ("X") can be used to conjugate the
siRNA to the antibody.
Example 32: 2016-PK-256-WT
[1016] siRNA Design and Synthesis
[1017] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU;
SEQ ID NO: 2088). Base, sugar and phosphate modifications were used
to optimize the potency of the duplex and reduce immunogenicity.
All siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphorothioate-inverted abasic-phosphorothioate linker, see
Example 9 for the chemical structure.
[1018] ASC Synthesis and Characterization
[1019] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1020] In Vivo Study Design
[1021] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
44 illustrates the study design in more detail. Non-terminal blood
samples were collected at 0.25, 3, and 24 hours post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 72, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves. Plasma concentrations of antibody
were determined using an ELISA assay.
TABLE-US-00055 TABLE 44 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Group Test Article N (mg/kg) ROA (mL/kg) Doses (h)
(h) 1 EGFR-Ab(Cys)-SMCC- 4 0.5 IV 5.0 1 0.25 72 KRAS-PEG5k (n = 1)
2 EGFR-Ab(Cys)-SMCC- 4 0.5 IV 5.0 1 3 96 KRAS-PEG5k (n = 1) 3
EGFR-Ab(Cys)-SMCC- 4 0.5 IV 5.0 1 24 168 KRAS-PEG5k (n = 1) 4
EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 0.25 72 KRAS-PEG5k (n = 1) 5
EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 3 96 KRAS-PEG5k (n = 1) 6
EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 24 168 KRAS-PEG5k (n = 1) 7
EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 0.25 72 KRAS-PEG5k (n = 1) 8
EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 3 96 KRAS-PEG5k (n = 1) 9
EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 24 168 KRAS-PEG5k (n = 1) Total #
of Animals: 60 WT mice CD-1
[1022] In this in vivo PK study a range of different linkers
between the antibody and siRNA were tested to determine the effect
on plasma clearance. As illustrated on the graph on slide 45, all
the conjugates were capable of maintaining high levels of siRNA in
the plasma, with greater than 10% remaining in the plasma after 168
hours.
[1023] In this example, it was demonstrated biological activity
with a range of different AXBYC conjugates in which a range of
different linkers ("Y") can be used to conjugate the siRNA to the
antibody while maintaining the improved plasma kinetics over those
historically observed for unconjugated siRNA.
Example 33: 2016-PK-237-HCC827
[1024] siRNA Design and Synthesis
[1025] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (Guide strad sequence: ACUCGUGCCUUGGCAAACUUU;
SEQ ID NO: 2082). Base, sugar and phosphate modifications were used
to optimize the potency of the duplex and reduce immunogenicity.
All siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA.
[1026] Two different passenger strands were made containing two
conjugation handles (C6-NH.sub.2 and C6-SH) in two different
orientations (S5'-EGFR-3'N and N5'-EGFR-3'S). In the N5'-EGFR-3'S
passenger strand both conjugation handles were connected to siRNA
passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure. In the S5'-EGFR-3'N passenger strand both conjugation
handles were connected to siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[1027] ASC Synthesis and Characterization
[1028] The conjugate for groups 1-3 was made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9. The
conjugate for groups 4-6 was made and purified as a DAR1 (n=1)
using ASC architecture-2, as described in Example 9.
[1029] In Vivo Study Design
[1030] Groups (n=5) of female NCr nu/nu mice bearing subcutaneously
(SC) flank HCC827 tumors 100-300 mm.sup.3 in volume were treated
with one intravenous (i.v.) tail vein injection of siRNA conjugate,
while control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 45 describes the study
design. Mice were sacrificed by CO.sub.2 asphyxiation at 72, 96,
and 168 hours post-dose. 50 mg pieces of tumor and liver, were
collected and snap-frozen in liquid nitrogen. mRNA knockdown in
target tissue was determined using a comparative qPCR assay as
described in Example 2. Total RNA was extracted from the tissue,
reverse transcribed and mRNA levels were quantified using TaqMan
qPCR, using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue and plasma siRNA
concentrations were determined using a stem-loop qPCR assay as
described in Example 2. The antisense strand of the siRNA was
reverse transcribed using a TaqMan MicroRNA reverse transcription
kit using a sequence-specific stem-loop RT primer. The cDNA from
the RT step was then utilized for real-time PCR and Ct values were
transformed into plasma or tissue concentrations using the linear
equations derived from the standard curves.
TABLE-US-00056 TABLE 45 siRNA Dose Survival Terminal Harvest Dose
Volume # of Bleed Bleed Time Gr Test Article N (mg/kg) ROA (mL/kg)
Doses (min) (h) (h) 1 EGFR-Ab(Cys)-S5'- 5 0.5 IV 5.0 1 0.25 72 72
EGFR-3'N-PEG5k (n = 1) 2 EGFR-Ab(Cys)-S5'- 5 0.5 IV 5.0 1 3 96 96
EGFR-3'N-PEG5k (n = 1) 3 EGFR-Ab(Cys)-S5'- 5 0.5 IV 5.0 1 24 168
168 EGFR-3'N-PEG5k (n = 1) 4 EGFR-Ab(Cys)-N5'- 5 0.5 IV 5.0 1 0.25
72 72 EGFR-3'S-PEG5k (n = 1) 5 EGFR-Ab(Cys)-N5'- 5 0.5 IV 5.0 1 3
96 96 EGFR-3'S-PEG5k (n = 1) 6 EGFR-Ab(Cys)-N5'- 5 0.5 IV 5.0 1 24
168 168 EGFR-3'S-PEG5k (n = 1) 7 PBS Control 5 -- IV 5.0 1 -- -- 96
Total # of Animals: 65 nu/nu mice with HCC827 tumors
[1031] In this in vivo PK study the biological outcome of changes
in the orientation of the conjugation site of the antibody and PEG
(5' or 3') onto the siRNA were evaluated. In addition, the
biological outcome of using a lysine or cysteine to attach the
linker to the antibody was evaluated. As illustrated FIG. 75A, both
orientations of siRNA produced comparable EGFR tumor knockdown. As
illustrated FIG. 75B and FIG. 75C, both orientations produced
comparable siRNA tissue accumulation in the tumor and liver. As
illustrated in FIG. 75D, both orientations produce a comparable
plasma clearance kinetics.
[1032] As highlighted in FIG. 54, it was demonstrated biological
activity with the A-X-B-Y-C conjugate with a range of different
antibodies and siRNA cargos that are capable of in vivo biological
activity in a range of different tissue targets. In this example,
it was demonstrated that the antibody can be conjugated onto the 5'
and 3' ends of the passenger strand of the siRNA and while
maintaining the biological activity of the EGFR siRNA and tissue
distribution.
Example 34: 2016-PK-259-WT
[1033] siRNA Design and Synthesis
[1034] HPRT: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human HPRT. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 425 for the human mRNA
transcript for HPRT (guide strand sequence: UUAAAAUCUACAGUCAUAGUU;
SEQ ID NO: 2104). Base, sugar and phosphate modifications were used
to optimize the potency of the duplex and reduce immunogenicity.
All siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. Two
different passenger strands were made containing two conjugation
handles (C6-NH.sub.2 and C6-SH) in two different orientations
(S5'-HPRT-3'N and N5'-HPRT-3'S). Both conjugation handles were
connected to siRNA passenger strand via phosphodiester-inverted
abasic-phosphorothioate linker. The C6-NH.sub.2 and C6-SH were
connected through the phosphodiester, see Example 9 for the
chemical structure.
[1035] ASC Synthesis and Characterization
[1036] The conjugate for groups 1-3 was made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9. The
conjugate for groups 4-6 was made and purified as a DAR1 (n=1)
using ASC architecture-2, as described in Example 9. The conjugate
for groups 7-9 was made and purified as a DAR1 (n=1) using ASC
architecture-1, as described in Example 9. The conjugate for groups
10-12 was made and purified as a DAR1 (n=1) using ASC
architecture-3, as described in Example 9.
[1037] In Vivo Study Design
[1038] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates,
while the control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 46 illustrates the
study design in more detail. 50 mg pieces of tissue, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00057 TABLE 46 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell
Ab(Cys)-N5'-HPRT-3'S- 4 3 IV 5.0 1 96 PEG5k (n = 1) 2 Anti-B cell
Ab(Cys)-N5'-HPRT-3'S- 4 1 IV 5.0 1 96 PEG5k (n = 1) 3 Anti-B cell
Ab(Cys)-N5'-HPRT-3'S- 4 0.3 IV 5.0 1 96 PEG5k (n = 1) 4 Anti-B cell
Ab(Cys)-N3'-HPRT-5'S- 4 3 IV 5.0 1 96 PEG5k (n = 1) 5 Anti-B cell
Ab(Cys)-N3'-HPRT-5'S- 4 1 IV 5.0 1 96 PEG5k (n = 1) 6 Anti-B cell
Ab(Cys)-N3'-HPRT-5'S- 4 0.3 IV 5.0 1 96 PEG5k (n = 1) 7 Anti-B cell
Ab(Lys)-S3'-HPRT-5'N- 4 2 IV 5.0 1 96 PEG5k (n = 1) 8 Anti-B cell
Ab(Lys)-S3'-HPRT-5'N- 4 0.75 IV 5.0 1 96 PEG5k (n = 1) 9 Anti-B
cell Ab(Lys)-S3'-HPRT-5'N- 4 0.25 IV 5.0 1 96 PEG5k (n = 1) 10
Anti-B cell Ab(Lys)-S5'-HPRT-3'N- 4 2 IV 5.0 1 96 PEG5k (n = 1) 11
Anti-B cell Ab(Lys)-S5'-HPRT-3'N- 4 0.75 IV 5.0 1 96 PEG5k (n = 1)
12 Anti-B cell Ab(Lys)-S5'-HPRT-3'N- 4 0.25 IV 5.0 1 96 PEG5k (n =
1) 13 PBS Control 5 -- IV 5.0 1 96 Total # of Animals: 53 WT mice
(CD-1)
[1039] In the in vivo PK study the biological outcome of changes in
the orientation of the conjugation site of the antibody and PEG (5'
or 3') onto the siRNA were evaluated. In addition, the biological
outcome of using a lysine or cysteine to attach the linker to the
antibody was evaluated. As illustrated in FIG. 76A-FIG. 76D, all
the combinations of making the antibody conjugates produced
comparable HPRT knockdown in the four tissue compartments measured.
As illustrated in FIG. 77A-FIG. 77D, all the combinations of making
the antibody conjugates produced comparable siRNA tissue
accumulation in the different compartments measured.
[1040] In this example, it was demonstrated that a variety of
different conjugation strategies to the siRNA and antibody can be
used in the A-X-B-Y-C format while maintaining the biological
activity of the HPRT siRNA and tissue distribution.
Example 35: 2016-PK-267-WT
[1041] siRNA Design and Synthesis
[1042] CTNNB1: A 21mer duplex with 19 bases of complementarity and
3' dinucleotide overhangs was designed against human CTNNB1. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 1797 for the human mRNA
transcript for CTNNB1 (guide strand sequence:
UUUCGAAUCAAUCCAACAGUU; SEQ ID NO: 2098). Base, sugar and phosphate
modifications were used to optimize the potency of the duplex and
reduce immunogenicity. All siRNA single strands were fully
assembled on solid phase using standard phospharamidite chemistry
and purified over HPLC. Purified single strands were duplexed to
get the double stranded siRNA.
[1043] Two different passenger strands were made containing two
conjugation handles (C6-NH.sub.2 and C6-SH) in two different
orientations (S5'-CTNNB1-3'N and N5'-CTNNB1-3'S). Both conjugation
handles were connected to siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[1044] ASC Synthesis and Characterization
[1045] The conjugate for groups 1-3 was made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9. The
conjugate for groups 4-6 was made and purified as a DAR1 (n=1)
using ASC architecture-3, as described in Example 9. The conjugate
for groups 7-9 was made and purified as a DAR1 (n=1) using ASC
architecture-2, as described in Example 9. The conjugate for groups
10-12 was made and purified as a DAR1 (n=1) using ASC
architecture-1, as described in Example 9.
[1046] In Vivo Study Design
[1047] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates,
while the control group (n=5) of the same mice received one i.v.
injection of PBS as a vehicle control. Table 47 illustrates the
study design in more detail. 50 mg pieces of tissue, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct).
TABLE-US-00058 TABLE 47 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell
Ab(Cys)-N5'- 4 3 IV 5.0 1 96 CTNNB1-3'S-PEG5k (n = 1) 2 Anti-B cell
Ab(Cys)-N5'- 4 1 IV 5.0 1 96 CTNNB1-3'S-PEG5k (n = 1) 3 Anti-B cell
Ab(Cys)-N5'- 4 0.3 IV 5.0 1 96 CTNNB1-3'S-PEG5k (n = 1) 4 Anti-B
cell Ab(Lys)-S5'- 4 3 IV 5.0 1 96 CTNNB1-3'N-PEG5k (n = 1) 5 Anti-B
cell Ab(Lys)-S5'- 4 1 IV 5.0 1 96 CTNNB1-3'N-PEG5k (n = 1) 6 Anti-B
cell Ab(Lys)-S5'- 4 0.3 IV 5.0 1 96 CTNNB1-3'N-PEG5k (n = 1) 7
Anti-B cell Ab(Cys)-N3'- 4 3 IV 5.0 1 96 CTNNB1-5'S-PEG5k (n = 1) 8
Anti-B cell Ab(Cys)-N3'- 4 1 IV 5.0 1 96 CTNNB1-5'S-PEG5k (n = 1) 9
Anti-B cell Ab(Cys)-N3'- 4 0.3 IV 5.0 1 96 CTNNB1-5'S-PEG5k (n = 1)
10 Anti-B cell Ab(Lys)-S3'- 4 3 IV 5.0 1 96 CTNNB1-5'N-PEG5k (n =
1) 11 Anti-B cell Ab(Lys)-S3'- 4 1 IV 5.0 1 96 CTNNB1-5'N-PEG5k (n
= 1) 12 Anti-B cell Ab(Lys)-S3'- 4 0.3 IV 5.0 1 96 CTNNB1-5'N-PEG5k
(n = 1) 13 PBS Control 5 -- IV 5.0 1 96 Total # of Animals: 53 WT
mice (CD-1)
[1048] In this in vivo PK study, the biological outcome of changes
in the orientation of the conjugation site of the antibody and PEG
(5' or 3') onto the siRNA and the biological outcome of using a
lysine or cysteine to attach the linker to the antibody were
evaluated. As illustrated in FIG. 78A-FIG. 78D, all the
combinations of making the antibody conjugates produced comparable
CTNNB1 knockdown in the four tissue compartments measured.
[1049] In this example, it was demonstrated that a variety of
different conjugation strategies to the siRNA and antibody can be
used in the A-X-B-Y-C format while maintaining the biological
activity of the CTNNB1 siRNA.
Example 36: 2016-PK-188-PK
[1050] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1051] ASC Synthesis and Characterization
[1052] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1053] In Vivo Study Design
[1054] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
48 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00059 TABLE 48 Dose Survival Terminal Volume # of Bleed
Bleed Gr Test Article N ROA (mL/kg) Doses (min) (h) 1
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 4 IV 5.0 1 5 24 2 4 IV 5.0 1 30 96
3 4 IV 5.0 1 180 168 4 EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 4 IV 5.0
1 5 24 5 4 IV 5.0 1 30 96 6 4 IV 5.0 1 180 168 7
EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 4 IV 5.0 1 5 24 8 4 IV 5.0 1 30
96 9 4 IV 5.0 1 180 168 10 EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 4 IV 5.0
1 5 24 11 (n = 1) 4 IV 5.0 1 30 96 12 4 IV 5.0 1 180 168 Total # of
Animals: 48 WT mice CD-1
[1055] As illustrated in FIG. 79, all the ASC with the different
cleavable linker configurations achieved equivalent plasma PK
profiles, with approximately 10% of the siRNA remaining 168 hours
after administration.
[1056] In this example, it was demonstrated biological activity
with a range of A-X-B-Y-C conjugates in which a variety of
different linker strategies (component X and Y) were used to
conjugate the PEG and antibody to the siRNA passenger strand.
Example 37: 2016-PK-201-LNCaP
[1057] siRNA Design and Synthesis
[1058] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1059] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[1060] ASC Synthesis and Characterization
[1061] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1062] In Vivo Study Design
[1063] Groups 1-7 (n=5) of female SCID SHO mice bearing
subcutaneous flank LNCaP tumors 100-350 mm.sup.3 in volume were
treated with one intravenous (i.v.) tail vein injection of siRNA
conjugate, while control group 8 (n=5) of the same mice received
one i.v. injection of PBS as a vehicle control. Table 49 describes
the study design. Mice were sacrificed by CO.sub.2 asphyxiation at
96 hours post-dose. 50 mg pieces of tumor and liver, were collected
and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue
was determined using a comparative qPCR assay as described in
Example 2. Total RNA was extracted from the tissue, reverse
transcribed and mRNA levels were quantified using TaqMan qPCR,
using the appropriately designed primers and probes. PPIB
(housekeeping gene) was used as an internal RNA loading control,
results were calculated by the comparative Ct method, where the
difference between the target gene Ct value and the PPIB Ct value
(.DELTA.Ct) is calculated and then further normalized relative to
the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00060 TABLE 49 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Cys)-
5 1 IV 5.0 1 96 EGFR-SS-PEG5k (n = 1) 2 PSMA-Ab(Cys)- 5 0.5 IV 5.0
1 96 EGFR-SS-PEG5k (n = 1) 3 PSMA-Ab(Cys)- 5 1 IV 5.0 1 96
EGFR-ECL-PEG5k (n = 1) 4 PSMA-Ab(Cys)- 5 0.5 IV 5.0 1 96
EGFR-ECL-PEG5k (n = 1) 5 PSAM-Ab(Cys)- 5 1 IV 5.0 1 96 EGFR-PEG5k
(n = 1) 6 PSAM-Ab(Cys)- 5 0.5 IV 5.0 1 96 EGFR-PEG5k (n = 1) 7
PSMA-Ab(Cys)- 5 1 IV 5.0 1 96 scramble-PEG5k (n = 1) 8 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP
tumors
[1064] As illustrated in FIG. 80A, a variety of different linkers
were used between the siRNA and PEG, after i.v administration of a
single dose of siRNA measurable tumor tissue EGFR downregulation
was achieved relative to the negative control siRNA sequence or PBS
controls. In addition, as illustrated in FIG. 80B, the different
linker configurations resulted in tumor siRNA accumulation at
higher levels than the other tissue samples measured (liver,
spleen, lung and kidney).
[1065] In this example, it was demonstrated biological activity
with a range of A-X-B-Y-C conjugates in which a variety of
different linkers strategies (component Y) were used to conjugate
the PEG to the siRNA passenger strand.
Example 38: 2016-PK-198-HCC827
[1066] siRNA Design and Synthesis
[1067] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1068] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[1069] ASC Synthesis and Characterization
[1070] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1071] In Vivo Study Design
[1072] Groups 1-15 (n=5) of female NCr nu/nu mice bearing
subcutaneously (SC) flank HCC827 tumors 100-300 mm.sup.3 in volume
were treated with one intravenous (i.v.) tail vein injection of
siRNA conjugate, while control group 16 (n=5) of the same mice
received one i.v. injection of PBS as a vehicle control. Table 50
describes the study design. Mice were sacrificed by CO.sub.2
asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and
liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in Example 2. Total RNA was extracted from the
tissue, reverse transcribed and mRNA levels were quantified using
TaqMan qPCR, using the appropriately designed primers and probes.
PPIB (housekeeping gene) was used as an internal RNA loading
control, results were calculated by the comparative Ct method,
where the difference between the target gene Ct value and the PPIB
Ct value (.DELTA.Ct) is calculated and then further normalized
relative to the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00061 TABLE 50 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG2k (n = 1) 2
EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 PEG2k (n = 1) 3
EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 PEG2k (n = 1) 4
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 dPEG.sub.48 (n = 1) 5
EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 dPEG.sub.48 (n = 1) 6
EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 dPEG.sub.48 (n = 1) 7
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 dPEG.sub.24 (n = 1) 8
EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 dPEG.sub.24 (n = 1) 9
EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 dPEG.sub.24 (n = 1) 10
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 dPEG.sub.12 (n = 1) 11
EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 dPEG.sub.12 (n = 1) 12
EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 dPEG.sub.12 (n = 1) 13
EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 14
PSMA-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 15
EGFR-Ab(Cys)-scramble- 5 1 IV 5.0 1 96 PEG5k (n = 1) 16 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 80 nu/nu mice with HCC827
tumors
[1073] As illustrated in FIG. 81A, all the ASC with the different
configurations of linear PEG length achieved dose dependent EGFR
mRNA knockdown in the HCC827 tumor cells, relative to the negative
control siRNA sequence (scramble) and PBS controls. As illustrated
in FIG. 81B, all the ASC with the different configurations in
linear PEG length achieved equivalent dose dependent siRNA tumor
tissue accumulation. In addition to low liver, lung, kidney and
spleen accumulation relative to tumor.
[1074] In this example, it was demonstrated biological activity
with a range of A-X-B-Y-C conjugates in which a variety of
different PEG (component C) lengths were used.
Example 39: 2016-PK-194-WT
[1075] siRNA Design and Synthesis
[1076] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1077] ASC Synthesis and Characterization
[1078] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1079] In Vivo Study Design
[1080] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
51 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00062 TABLE 51 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Group Test Article N (mg/kg) ROA (mL/kg) Doses (min)
(h) 1 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 2 EGFR-PEG2k (n = 1) 4 0.5
IV 5.0 1 30 96 3 4 0.5 IV 5.0 1 180 168 4 EGFR-Ab(Cys)- 4 0.5 IV
5.0 1 5 24 5 EGFR -dPEG.sub.48 (n = 1) 4 0.5 IV 5.0 1 30 96 6 4 0.5
IV 5.0 1 180 168 7 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 8 EGFR
-dPEG.sub.24 (n = 1) 4 0.5 IV 5.0 1 30 96 9 4 0.5 IV 5.0 1 180 168
10 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 11 EGFR -dPEG.sub.12 (n = 1) 4
0.5 IV 5.0 1 30 96 12 4 0.5 IV 5.0 1 180 168 Total # of Animals: 48
WT mice CD-1
[1081] As illustrated on slide 54, all the ASC with the different
linear PEG lengths achieved equivalent plasma PK profiles, with
approximately 10% of the siRNA remaining 168 hours after
administration.
[1082] In this example, it was demonstrated equivalent plasma PK
properties with a range of A-X-B--Y-C conjugates in which a variety
of different PEG (component C) lengths were used.
Example 40: 2016-PK-195-WT
[1083] siRNA Design and Synthesis
[1084] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1085] ASC Synthesis and Characterization
[1086] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1087] In Vivo Study Design
[1088] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
52 illustrates the study design in more detail. Non-terminal blood
samples were collected at 5, 30, and 180 minutes post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis. Quantitation of plasma siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00063 TABLE 52 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Gr Test Article N (mg/kg) ROA (mL/kg) Doses (min)
(h) 1 EGFR-Ab(Cys)-EGFR-PEG10k 4 0.5 IV 5.0 1 5 24 2 (n = 1) 4 0.5
IV 5.0 1 30 96 3 4 0.5 IV 5.0 1 180 168 4 EGFR-Ab(Cys)-EGFR- 4 0.5
IV 5.0 1 5 24 5 (dPEG24).sub.3 (n = 1) 4 0.5 IV 5.0 1 30 96 6 4 0.5
IV 5.0 1 180 168 7 EGFR-Ab(Cys)-EGFR- 4 0.5 IV 5.0 1 5 24 8
(dPEG12).sub.3 (n = 1) 4 0.5 IV 5.0 1 30 96 9 4 0.5 IV 5.0 1 180
168 10 EGFR-Ab(Cys)-EGFR-(dPEG4).sub.3 4 0.5 IV 5.0 1 5 24 11 (n =
1) 4 0.5 IV 5.0 1 30 96 12 4 0.5 IV 5.0 1 180 168 Total # of
Animals: 48 WT mice CD-1
[1089] As illustrated in FIG. 83, all the ASC with the different
PEG configurations (length and branching) achieved equivalent
plasma PK profiles, with approximately 10% of the siRNA remaining
168 hours after administration.
[1090] In this example, it was demonstrated equivalent plasma PK
properties with a range of A-X-B--Y-C conjugates in which a variety
of different PEG (component C) lengths and branching were used.
Example 41: 2016-PK-236-HCC827
[1091] siRNA Design and Synthesis
[1092] EFGR: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human EGFR. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 333 for the human mRNA
transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,
sugar and phosphate modifications were used to optimize the potency
of the duplex and reduce immunogenicity. All siRNA single strands
were fully assembled on solid phase using standard phospharamidite
chemistry and purified over HPLC. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via phosphorothioate-inverted
abasic-phosphorothioate linker, see Example 9 for the chemical
structure.
[1093] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate
modifications were used to reduce immunogenicity and were
comparable to those used in the active siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[1094] ASC Synthesis and Characterization
[1095] All conjugates were made and purified as a DAR1 (n=1) using
ASC architecture-4, as described in Example 9.
[1096] In Vivo Study Design
[1097] Groups 1-12 (n=5) of female NCr nu/nu mice bearing
subcutaneously (SC) flank HCC827 tumors 100-300 mm.sup.3 in volume
were treated with one intravenous (i.v.) tail vein injection of
siRNA conjugate, while control group 13 (n=5) of the same mice
received one i.v. injection of PBS as a vehicle control. Table 53
describes the study design. Mice were sacrificed by CO.sub.2
asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and
liver, were collected and snap-frozen in liquid nitrogen. mRNA
knockdown in target tissue was determined using a comparative qPCR
assay as described in Example 2. Total RNA was extracted from the
tissue, reverse transcribed and mRNA levels were quantified using
TaqMan qPCR, using the appropriately designed primers and probes.
PPIB (housekeeping gene) was used as an internal RNA loading
control, results were calculated by the comparative Ct method,
where the difference between the target gene Ct value and the PPIB
Ct value (.DELTA.Ct) is calculated and then further normalized
relative to the PBS control group by taking a second difference
(.DELTA..DELTA.Ct). Quantitation of tissue siRNA concentrations
were determined using a stem-loop qPCR assay as described in
Example 2. The antisense strand of the siRNA was reverse
transcribed using a TaqMan MicroRNA reverse transcription kit using
a sequence-specific stem-loop RT primer. The cDNA from the RT step
was then utilized for real-time PCR and Ct values were transformed
into plasma or tissue concentrations using the linear equations
derived from the standard curves.
TABLE-US-00064 TABLE 53 siRNA Dose Harvest Dose Volume # of Time
Group Test Article N (mg/kg) ROA (mL/kg) Doses (h) 1
EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 1 IV 5.0 1 96 2
EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 0.5 IV 5.0 1 96 3
EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 0.25 IV 5.0 1 96 4
EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 1 IV 5.0 1 96 5
EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 0.5 IV 5.0 1 96 6
EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 0.25 IV 5.0 1 96 7
EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 1 IV 5.0 1 96 8
EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 0.5 IV 5.0 1 96 9
EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 0.25 IV 5.0 1 96 10
EGFR-Ab(Cys)-EGFR-(dPEG4)3 (n = 1) 5 1 IV 5.0 1 96 11
EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 12
EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 13 PBS Control
5 -- IV 5.0 1 96 Total # of Animals: 65 nu/nu mice with HCC827
tumors
[1098] As illustrated in FIG. 84, all the ASC with the different
configurations of PEG (length and branching) achieved equivalent
EGFR mRNA knockdown in the HCC827 tumor cells to the construct with
the linear PEG5K at the 1 mg/kg dose. Those constructs tested in a
dose response format, showed dose dependent knockdown of EGFR mRNA.
As illustrated in FIG. 85, all the ASC with the different
variations in linear PEG length and PEG branching achieved
equivalent siRNA tumor tissue accumulation to the construct with
the linear PEG5K at the 1 mg/kg dose. In addition to low liver
accumulation relative to tumor, those constructs tested in a dose
response format, showed dose dependent tumor tissue accumulation of
siRNA.
[1099] In this example, it was demonstrated biological activity
with a range of A-X-B-Y-C conjugates in which a variety of
different PEG (component C) lengths and branching were used.
Example 42: In Vitro Knockdown with ASCs with PEG Polymers
[1100] siRNA Design and Synthesis
[1101] HPRT: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human HPRT. The
sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU
(SEQ ID NO: 2082) and design to be complementary to the gene
sequence starting a base position 425 for the human mRNA transcript
for HPRT. Base, sugar and phosphate modifications were used to
optimize the potency of the duplex and reduce immunogenicity. All
siRNA single strands were fully assembled on solid phase using
standard phospharamidite chemistry and purified over HPLC. Purified
single strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via
phosphodiester-inverted abasic-phosphorothioate linker. The
C6-NH.sub.2 and C6-SH were connected through the phosphodiester,
see Example 9 for the chemical structure.
[1102] Negative control siRNA sequence (scramble): A published
(Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with
19 bases of complementarity and 3' dinucleotide overhangs was used.
The sequence (5' to 3') of the guide/antisense strand was
UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and
phosphate modifications that were used for the active EGFR siRNA
duplex were used in the negative control siRNA. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. Purified single
strands were duplexed to get the double stranded siRNA. The
passenger strand contained two conjugation handles, a C6-NH.sub.2
at the 5' end and a C6-SH at the 3' end. Both conjugation handles
were connected to siRNA passenger strand via a
phosphodiester-inverted abasic-phosphodiester linker, see Example 9
for the chemical structure.
[1103] ASC Synthesis and Characterization
[1104] Conjugates in groups 1-3 made and purified as a DAR1 (n=1)
using ASC architecture-1, as described in Example 9. Conjugates in
groups 4-6 were made and purified as a DAR1 (n=1) using ASC
architecture-4, as described in Example 9.
[1105] In Vitro Study Design
[1106] Mouse spleens were harvested and kept in PBS with 100 u/ml
penicillin and streptomycin on ice. Spleens were smashed with clean
glass slides, cut into small pieces, homogenized with 18G needles,
and filtered (70 um nylon membrane). Dead cells were removed with
the dead cell removal kit from Milteny biotec (Catalog#130-090101)
according to manufacturer instruction. To isolate mouse B cells, B
cell isolation kit Milteny biotec (Catalog#130-090-862) was used
following manufacturer instruction. Briefly, live spleen cells were
resuspended with 200 .mu.l of MACS buffer per mouse spleen. Non-B
cells were depleted with biotin-conjugated monoclonal antibodies
against CD43 (Ly48), CD4, and Ter-119, coupled with anti-biotin
magnetic microbeads. From one mouse spleen, 30 million live B cells
can be obtained. To activate isolated mouse B cells
(2.times.10.sup.6/m1 in 10% FBS RPMI-1640 with 100 u/ml penicillin
and streptomycin), a cocktail of 10 .mu.g/ml LPS, 5 .mu.g/ml
anti-IgM, 1 .mu.g/ml anti-CD40, 0.05 .mu.g/ml IL-4, and 0.05
.mu.g/ml INF.gamma. was added. After four hours of activation, ASCs
(1 pM to 10 nM) were added to 10.sup.6 cells per well in 24 (0.5 ml
media) or 12 (1 ml media) well plates. After 48 hours of ASC
treatments, cells were harvested and isolated RNAs were analyzed
for mRNA knockdown. See Table 54 for the study design.
TABLE-US-00065 TABLE 54 Group Test Article 1 Anti-B cell
Ab(Lys)-S3'-HPRT-5'N-pOEGMA8K 2 Anti-B cell Ab(Lys)-S3'-HPRT-5'N
-pHPMA5K 3 Anti-B cell Ab(Lys)-S3'-HPRT-5'N -pHPMA10K 4 Anti-B cell
Ab(Cys)-N5'-HPRT-3'S-pMAA10K 5 Anti-B cell
Ab(Cys)-N5'-HPRT-3'S-PEG5K 6 Anti-B cell
Ab(Cys)-N5'-scramble-3'S-PEG5K
[1107] In this in vitro experiment in activated primary mouse B
cells, the ability of an anti-B cell antibody ASCs to deliver an
siRNA design to downregulate Hypoxanthine-guanine
phosphoribosyltransferase (HPRT) with a range of alternative PEG
polymers were measured. As illustrated in FIG. 86, the range of ASC
with alternative PEGs were able to downregulate HPRT relative to
the scramble control.
[1108] In this example, the biological activity was demonstrated
with a range of A-X-B-Y-C conjugates in which a variety of polymer
alternatives to PEG (component C) were used.
Example 43: PK-236-WT
[1109] siRNA Design and Synthesis
[1110] KRAS: A 21mer duplex with 19 bases of complementarity and 3'
dinucleotide overhangs was designed against human KRAS. The
sequence of the guide/antisense strand was complementary to the
gene sequence starting a base position 237 for the human mRNA
transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU;
SEQ ID NO: 2088). Base, sugar and phosphate modifications that are
well described in the field of RNAi were used to optimize the
potency of the duplex and reduce immunogenicity. All siRNA single
strands were fully assembled on solid phase using standard
phospharamidite chemistry and purified over HPLC. The base at
position 11 on the passenger strand had a Cy5 fluorescent label
attached, as described in Example 9. Purified single strands were
duplexed to get the double stranded siRNA. The passenger strand
contained two conjugation handles, a C6-NH.sub.2 at the 5' end and
a C6-SH at the 3' end. Both conjugation handles were connected to
siRNA passenger strand via a phosphodiester-inverted
abasic-phosphodiester linker, see Example 9 for the chemical
structure.
[1111] ASC Synthesis and Characterization
[1112] Conjugates in groups 1-3 were made and purified as a DAR1
(n=1) using ASC architecture-4, as described in Example 9.
Conjugates in groups 4-6 were made and purified as a DAR1 (n=1)
using ASC architecture-4, but there was no PEG on the 3' end of the
passenger strand. Prior to conjugateion, the 3'thiol was end-capped
using N-ethylmaleimide. Conjugates in groups 7-9 were made and
purified as a DAR1 (n=1) using ASC architecture-1, as described in
Example 9. Conjugates in groups 10-12 made and purified as a DAR1
(n=1) using ASC architecture-1, but there was no PEG on the 5' end
of the passenger strand.
[1113] In Vivo Study Design
[1114] Groups (n=4) of wild-type female CD-1 mice were treated with
one intravenous (i.v.) tail vein injections of siRNA conjugates.
Treatment groups received 0.5 mg/kg (based on the weight of siRNA)
and all groups were administered a dose volume of 5.0 mL/kg. Table
55 illustrates the study design in more detail. Non-terminal blood
samples were collected at 0.25, 1, and 4 hours post-dose via
puncture of the retro-orbital plexus and centrifuged to generate
plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 24, 48, or 72 h post-dose. Terminal blood samples
were collected via cardiac puncture and processed to generate
plasma for PK analysis.
[1115] Plasma samples (K2 EDTA) were processed within 4 hours after
harvesting. Plasma samples were diluted with matching mouse plasma
(Bioreclamation) (2-400 fold) and the concentration of CY5-siRNA in
these plasma samples quantified spectroscopically using a TECAN
Infinite M200 Pro (Excitation 635 nm; Emission 675 nm). To release
macromolecular interactions that might quench the CY5 fluorescence,
all samples were diluted 2-fold into water containing 0.01% Tween
20 and 100 ug/ml heparin prior to quantification. To determine the
amount of intact ASCs in these plasma samples, plasma samples were
diluted with mouse plasma to 2-50 nM CY5-siRNA and incubated with
Protein G Dynabeads (Thermofisher) loaded with 150 nM of a purified
EGFR-Fc protein (Sino Biological). These binding reactions were
incubated at RT for 1 hour. Beads were washed twice with PBS
containing 0.01% Tween 20 and 0.05% BSA before ASCs bound to EGFR
were eluted by incubation in 0.1 M citric acid (pH 2.7). The amount
of CY5-siRNA contained in the input, unbound fraction, washes and
bead eluate was quantified by fluorescence as stated above.
TABLE-US-00066 TABLE 55 siRNA Dose Survival Terminal Dose Volume #
of Bleed Bleed Gr Test Article N (mg/kg) ROA (mL/kg) Doses (h) (h)
1 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 3'S-PEG5k (n =
1) 2 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 3'S-PEG5k (n =
1) 3 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 3'S-PEG5k (n =
1) 4 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 3'S--NEM (n
= 1) 5 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 3'S--NEM (n =
1) 6 EGFR-Ab(Cys)-N5'-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 3'S--NEM (n =
1) 7 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 5'N-PEG5k (n
= 1) 8 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 5'N-PEG5k (n
= 1) 9 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 5'N-PEG5k (n
= 1) 10 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24
5'NH.sub.2 (n = 1) 11 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1 1
48 5'NH.sub.2 (n = 1) 12 EGFR-Ab(Lys)-S3'-Cy5.KRAS- 4 0.5 IV 5.0 1
4 72 5'NH.sub.2 (n = 1) Total # of Animals: 96 WT mice CD-1
[1116] In this in vivo PK study, the in vivo plasma stability of
two AXBYC conjugates (cysteine and lysine conjugation to the
EGFR-Ab) relative to two AXB conjugates were compared. As
illustrated in FIG. 87, the concentration of the siRNA was
determined using two methods. The fluorescence of the plasma was
measured directly and the siRNA concentration determined using a
standard curve. Or a magnetic bead decorated with EGFR was used to
bind the antibody conjugates and then the fluorescence of the
sample was measured and the siRNA concentration determined using a
standard curve. All data were plotted as a percentage of the
injected dose. In both examples of the AXBYC conjugates (cysteine
and lysine conjugation to the EGFR-Ab) improved plasma PK were
observed relative to the corresponding AXB conjugate.
[1117] In this example, in vivo plasma PK for the Cys and Lys AXBYC
conjugates compared to the matching control AXB conjugates was
demonstrate.
Example 44: In Vivo Pharmacodynamics Study of a Cholesterol-KRAS
Conjugate (PD-058)
[1118] Groups (n=5) of female NCr nu/nu mice bearing intrahepatic
Hep 3B tumors one week after inoculation were treated with three
intravenous (i.v.) tail vein injections (separated by 48 h) of
cholesterol-siRNA conjugate, while control groups (n=5) of the same
mice received three i.v. injections of PBS as a vehicle control on
the same dosing schedule. Treatment groups that received chol-KRAS
were dosed at 10, 4, or 2 mg/kg. All groups (treatments and
controls) were administered a dose volume of 6.25 mL/kg. Table 56
describes the study design in more detail and gives a
cross-reference to the conjugate synthesis and characterization.
Mice were sacrificed by CO.sub.2 asphyxiation at 72 h post-final
dose. 50 mg pieces of tumor-bearing liver were collected and
snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA
quantitation were performed as described in Examples 2-7.
TABLE-US-00067 TABLE 56 Study design for a Cholesterol-KRAS
Conjugate (PD-058) with a cross-reference to the synthesis and
characterization of the conjugates tested. siRNA Dose # of
Cross-reference for synthesis and Group Test Article N (mg/kg) ROA
Doses characterization 1 Chol-KRAS 5 10 iv 3 General experimental
(Example 2) 2 Chol-KRAS 5 4 iv 3 General experimental (Example 2) 3
Chol-KRAS 5 2 iv 3 General experimental (Example 2) 4 Vehicle (PBS)
5 iv 3
[1119] The chol-KRAS conjugate was assessed for mRNA knockdown in a
3-dose study with a dose response. As illustrated in FIG. 35,
within the mouse liver tissue there was a clear dose-response for
mouse KRAS mRNA knockdown. The lowest dose of 2 mg/kg resulted in
45% knockdown of mouse KRAS, while the highest dose of 10 mg/kg
resulted in 65% knockdown of mouse KRAS in this 3-dose format.
However, there were not enough human tumor cells in the mouse liver
at the time of harvest to detect a signal from human KRAS
(potentially due to model development issues, it appeared that not
enough human cells were inoculated to produce fast-growing tumors).
As such, it was not possible to measure the knockdown in tumor.
Example 45: In Vivo Pharmacokinetics Study of a Cholesterol-siRNA
Conjugate (PK-063)
[1120] Groups (n=3) of wild-type female CD-1 mice were treated with
either one or two intravenous (i.v.) tail vein injections of
chol-siRNA conjugate. Treatment groups received chol-KRAS at 10
mg/kg (based on the weight of siRNA) and the 2-dose groups received
the second dose 48 h after the first dose. All groups were
administered a dose volume of 6.25 mL/kg. Table 57 illustrates the
study design in more detail and gives a cross-reference to the
conjugate synthesis and characterization. Non-terminal blood
samples were collected at 2, 15, 60 or 120 minutes post-final dose
via puncture of the retro-orbital plexus and centrifuged to
generate plasma for PK analysis. Mice were sacrificed by CO.sub.2
asphyxiation at 4, 24, 96, or 144 h post-final dose. Terminal blood
samples were collected via cardiac puncture and processed to
generate plasma for PK analysis. 50 mg pieces of tumor, liver,
kidney, and lung were collected and snap-frozen in liquid nitrogen.
mRNA knockdown analysis and siRNA quantitation were performed as
described in Examples 2-7.
TABLE-US-00068 TABLE 57 Study design for a Cholesterol-siRNA
Conjugate (PK-063) with a cross-reference to the synthesis and
characterization of the conjugates tested. siRNA Survival Terminal
Harvest Cross-reference Test Dose # of Bleed Bleed Time to
synthesis and Group Article N (mg/kg) ROA Doses (min) (h) (h)
characterization 1 Chol- 3 10 IV 1 2 4 4 General 2 KRAS 3 10 IV 1
15 24 24 experimental 3 3 10 IV 1 60 96 96 (Example 2) 4 3 10 IV 1
120 144 144 5 Chol- 3 10 IV 2 2 4 4 General 6 KRAS 3 10 IV 2 15 24
24 experimental 7 3 10 IV 2 60 96 96 (Example 2) 8 3 10 IV 2 120
144 144
[1121] The pharmacokinetic behavior of chol-siRNA was assessed in a
single-dose format compared to a 2-dose format. As illustrated from
FIG. 36, the plasma PK profiles for the first dose and a second
dose following 48 h later are nearly identical. The mechanism for
clearance from plasma has not saturated from the first dose and the
second dose behaves similarly. The tissue PK for 3 major tissues
(the liver, kidneys, and lungs) was similarly assessed. As
illustrated from FIG. 37, chol-KRAS was delivered to liver in the
highest concentrations, with kidneys and lungs having approximately
10-fold lower siRNA concentrations compared to liver. For all three
tissues, the siRNA concentrations following the second doses were
higher than the siRNA concentrations following the first dose,
demonstrating that there is accumulation of siRNA in tissues when
doses of chol-siRNA are spaced by 48 h.
[1122] In Vivo Study a Cholesterol-siRNA Conjugate (PK-067).
[1123] Groups (n=3) of female NCr nu/nu mice bearing subcutaneous
flank H358 tumors 100-150 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=4) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups that received
cholesterol-siRNA conjugates were dosed at 5 mg/kg (based on the
weight of siRNA). Some treatment groups also received
cholesterol-peptide conjugates at specified molar peptide:siRNA
ratios, where all chol-siRNA and chol-peptide conjugates were mixed
together in solution and co-injected. All groups (treatments and
controls) were administered a dose volume of 5 mL/kg. Table 58
shows the study design in more detail and gives a cross-reference
to the conjugate synthesis and characterization. Mice were
sacrificed by CO.sub.2 asphyxiation at 24, 72, or 144 h post-dose.
50 mg pieces of tumor, liver, kidneys, and lungs were collected and
snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA
quantitation were performed as described in Examples 2-7.
TABLE-US-00069 TABLE 58 Study design for a Cholesterol-siRNA
Conjugate (PK-067) with a cross-reference to the conjugate
synthesis and characterization mol EEP/ siRNA mol Harvest
Cross-reference Test Dose siRNA # of Time to synthesis and Group
Article N (mg/kg) Ratio ROA Doses (h) characterization 1 chol-KRAS
3 5 -- IV 1 24 General 2 3 5 -- IV 1 72 experimental 3 3 5 -- IV 1
144 (Example 2) 4 chol-KRAS + 3 5 1 IV 1 24 General 5 chol-Melittin
3 5 1 IV 1 72 experimental 6 3 5 1 IV 1 144 (Example 2) 7 chol-KRAS
+ 3 5 3 IV 1 24 General 8 chol-Melittin 3 5 3 IV 1 72 experimental
9 3 5 3 IV 1 144 (Example 2) 10 chol-KRAS + 3 5 10 IV 1 24 General
11 chol-Melittin 3 5 10 IV 1 72 experimental 12 3 5 10 IV 1 144
(Example 2) 13 chol-KRAS + 3 5 1 IV 1 24 General 14 chol-INF7 3 5 1
IV 1 72 experimental 15 3 5 1 IV 1 144 (Example 2) 16 chol-KRAS + 3
5 3 IV 1 24 General 17 chol-INF7 3 5 3 IV 1 72 experimental 18 3 5
3 IV 1 144 (Example 2) 19 chol-KRAS + 3 5 10 IV 1 24 General 20
chol-INF7 3 5 10 IV 1 72 experimental 21 3 5 10 IV 1 144 (Example
2) 22 Vehicle (PBS) 4 -- -- IV 1 24 General 23 4 -- -- IV 1 72
experimental 24 4 -- -- IV 1 144 (Example 2) Total # of 75 nu/nu
mice with H358 tumors Animals:
[1124] Endosomolytic moieties (EEPs) such as INF7 and melittin were
conjugated to cholesterol, mixed with chol-siRNA, and then
co-injected into mice to demonstrate an increase in siRNA potency
due to the improved endosomal escape. First, the effect of adding
the EEPs on the siRNA concentration in various tissues was
assessed. As illustrated in FIG. 38A, the addition of chol-INF7 at
any of the molar ratios of EEP:siRNA did not affect the siRNA tumor
PK. However, as illustrated in FIG. 38B, the addition of
chol-melittin at a 1:1 ratio did not affect the tumor PK but the
addition of chol-melittin at a 3:1 EEP:siRNA ratio decreased the
amount of siRNA in tumor. As illustrated in FIG. 39, neither
chol-INF7 nor chol-melittin had much of an impact on the liver PK.
Similarly, as illustrated in FIGS. 40 and 41, the chol-INF7 and
chol-melittin also did not have much of an impact on the PK profile
in kidneys and lungs. Finally, the effect of the chol-EEP
conjugates on mRNA KD was assessed and, as shown in FIG. 42, the
baseline level of knockdown for chol-KRAS alone was approximately
50%. The addition of 1:1 chol-melittin or 3:1 chol-INF7 improves
the knockdown at each time point, due to improved endosomal
escape.
[1125] In Vivo Study a Cholesterol-siRNA Conjugate (PK-076).
[1126] Groups (n=5) of female NCr nu/nu mice bearing subcutaneous
flank H358 tumors 100-150 mm.sup.3 in volume were treated with
three intravenous (i.v.) tail vein injections of siRNA conjugate
separated by 48 h, while control groups (n=5) of the same mice
received three i.v. injections of PBS as a vehicle control on the
same dosing schedule. Treatment groups that received
cholesterol-siRNA conjugates were dosed at 5 mg/kg (based on the
weight of siRNA). Some treatment groups also received
cholesterol-peptide conjugates at specified molar peptide:siRNA
ratios, where all chol-siRNA and chol-peptide conjugates were mixed
together in solution and co-injected. All groups (treatments and
controls) were administered a dose volume of 5 mL/kg. Table 59
describes the study design in more detail and gives a
cross-reference to the conjugate synthesis and characterization.
Mice were sacrificed by CO.sub.2 asphyxiation at 24 or 96 h
post-dose. 50 mg pieces of tumor, liver, kidneys, and lungs were
collected and snap-frozen in liquid nitrogen. mRNA knockdown
analysis and siRNA quantitation were performed as described in
Examples 2-7.
TABLE-US-00070 TABLE 59 Study design for a Cholesterol-siRNA
Conjugate (PK-076) with a cross-reference to the synthesis and
characterization of the conjugates tested. EEP/ siRNA siRNA Harvest
Cross-reference Test Dose Ratio # of Time to synthesis and Group
Article N (mg/kg) (mol/mol) ROA Doses (h) characterization 1
chol-KRAS 5 5 -- IV 3 24 General 2 5 5 -- IV 3 96 experimental
(Example 2) 3 chol-KRAS + 5 5 1 IV 3 24 General 4 chol-melittin
(1:1) 5 5 1 IV 3 96 experimental (Example 2) 5 chol-KRAS + 5 5 3 IV
3 24 General 6 chol-INF7 (3:1) 5 5 3 IV 3 96 experimental (Example
2) 7 Vehicle 5 -- -- IV 3 24 8 5 -- -- IV 3 96
[1127] The activity seen in the single-dose study with chol-siRNA
and chol-EEP was followed up with a three dose study. The 3:1 ratio
of EEP:siRNA was selected for INF7, and the 1:1 ratio was selected
for melittin. As illustrated in FIG. 43 and FIG. 44, the addition
of either chol-EEP to the chol-siRNA does not seem to greatly
affect the tissue PK following three doses. As for the knockdown,
FIG. 45 shows that addition of chol-melittin clearly improves tumor
knockdown 24 h post-dose. It also shows that chol-melittin improves
tumor knockdown at 96 h post-dose.
[1128] In vivo study a Cholesterol-siRNA Conjugate (PK-079).
[1129] Groups (n=5) of female NCr nu/nu mice bearing subcutaneous
flank H358 tumors 100-150 mm.sup.3 in volume were treated with one
intravenous (i.v.) tail vein injection of siRNA conjugate, while
control groups (n=5) of the same mice received one i.v. injection
of PBS as a vehicle control. Treatment groups that received EGFR
antibody-siRNA-PEG conjugates were dosed at 0.5 mg/kg (based on the
weight of siRNA) and groups that also received EGFR
antibody-melittin had the dose of EGFR-Ab matched between EGFR
antibody-siRNA and EGFR antibody-melittin. All groups (treatments
and controls) were administered a dose volume of 5 mL/kg. Table 60
describes the study design in more detail and gives a
cross-reference to the conjugate synthesis and characterization.
Mice were sacrificed by CO.sub.2 asphyxiation at 96 h post-dose. 50
mg pieces of tumor, liver, kidney, and lung were collected and
snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA
quantitation were performed as described in Examples 2-7.
TABLE-US-00071 TABLE 60 Study design for a Cholesterol-siRNA
Conjugate (PK-079) with a cross-reference to the synthesis and
characterization of the conjugates tested. siRNA siRNA:EGFR-
melittin:siRNA Harvest Cross-reference Test Dose Ab Ratio Ratio #
of Time to synthesis and Group Article N (mg/kg) (mol/mol)
(mol/mol) ROA Doses (h) characterization 1 EGFR-Ab- 5 0.5 1 -- IV 1
96 Example 4 PEG5k-EGFR 2 EGFR-Ab- 5 0.5 1 1:1 IV 1 96 Example 3
and 6 PEG5k-EGFR + EGFR-Ab- melittin 3 EGFR-Ab-KRAS- 5 0.5 1 1:1 IV
1 96 Example 3 and 6 PEG5k + EGFR-Ab- melittin 4 EGFR antibody 5 --
-- -- IV 1 96 General Alone experimental (Example 2) 5 Vehicle 5 --
-- -- IV 1 96
[1130] The PK/PD relationship for EGFR antibody-siRNA conjugates to
deliver siRNA to tumor and produce mRNA knockdown in tumor was
evaluated for reproducibility. As illustrated in FIG. 46, once
again a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNA
conjugate was able to deliver approximate 100 nM concentrations of
siRNA into tumor with both configurations of the conjugate. The
addition of EGFR antibody-melittin did not appear to impact the
tissue PK. Out of the four tissues analyzed, tumor had the highest
concentration and liver the second highest, with kidneys and lungs
showing low uptake of siRNA. As illustrated in FIG. 47, the strong
siRNA delivery to tumor once again translated into approximately
50% knockdown of EGFR or KRAS in the tumors. Free EGFR-Ab, run as a
control group, showed no mRNA knockdown as did the PBS control.
[1131] In Vivo Study a Cholesterol-siRNA Conjugate (PD-077).
[1132] Groups (n=11) of female NCr nu/nu mice bearing intrahepatic
Hep3B tumors one week after inoculation were treated with nine
intravenous (i.v.) or subcutaneous (s.c.) injections (TIW) of
cholesterol-siRNA conjugate, while control groups (n=11) of the
same mice received nine i.v. tail vein injections of PBS as a
vehicle control (also dosed TIW). Treatment groups that received
chol-CTNNB1 were dosed at 5 mg/kg. All groups (treatments and
controls) were administered a dose volume of 6.25 mL/kg. Table 61
describes the study design in more detail and gives a
cross-reference to the conjugate synthesis and characterization.
Non-terminal blood samples were collected once per week via
puncture of the retro-orbital plexus and processed to generate
serum for alpha-Fetoprotein (AFP) measurement. Mice were sacrificed
by CO.sub.2 asphyxiation at 24 h post-final dose. 50 mg pieces of
tumor-bearing liver were collected and snap-frozen in liquid
nitrogen. mRNA knockdown analysis was performed as described above.
AFP was quantified using the Human alpha-Fetoprotein DuoSet ELISA
kit (R&D Systems) according to the manufacturer's
instructions.
TABLE-US-00072 TABLE 61 Study design for a Cholesterol-siRNA
Conjugate (PK-077) with a cross-reference to the synthesis and
characterization of the conjugates tested. siRNA Terminal
Cross-reference Test Dose # of Survival Bleed to synthesis and
Group Article N (mg/kg) ROA Doses Bleed (h) characterization 5
Chol-CTNNB1 11 5 IV 9 Weekly 24 General experimental (Example 2) 8
Chol-CTNNB1 11 5 SC 9 Weekly 24 General experimental (Example 2) 11
Vehicle 11 IV 9 Weekly 24 Total # of Animals: 33
[1133] Since earlier studies demonstrated that it was possible for
a single dose of chol-siRNA to generate knockdown in normal liver,
it was hypothesized that knockdown could be achieved in orthotopic
liver tumors as well. Mice were inoculated with intrahepatic Hep3B
tumors that were allowed to grow for one week post-inoculation, and
then these mice were administered 5 mg/kg doses of chol-CTNNB1
(either i.v. or s.c.) three times a week for three weeks (9 total
doses). As illustrated in FIG. 48, the chol-CTNNB1 dosed s.c. was
able to produce >50% mRNA knockdown at the harvest time point of
24 h post-final dose. In contrast, the chol-CTNNB1 siRNA that was
dosed i.v. does not seem to show any mRNA knockdown at this time
point (although some mice did not have any measurable human CTNNB1
signal, it was hard to determine if the loss of signal was related
to knockdown or low tumor burden). The human Hep3B cells are also
known to secrete human alpha-Fetoprotein (AFP), and it is known
that the amount of secreted AFP correlates with the number of Hep3B
cells. Thus, the concentration of AFP in serum is taken as a marker
of tumor load in the mouse, and the increase in AFP over time
correlates with tumor growth. As illustrated in FIG. 49, the
chol-CTNNB1 dosed s.c. markedly reduced the AFP levels in those
mice, which provides evidence that the CTNNB1 mRNA knockdown led to
the inhibition of tumor growth.
Example 46. Liver PK/PD Study
[1134] Female wild-type CD-1 mice will be dosed with chol-siRNA-EEP
conjugates at 5 mg/kg (based on the weight of siRNA). In these
studies the siRNA used will be against the mouse Factor VII (FVII)
such that FVII knockdown can be determined by measuring the FVII
protein levels in plasma. Multiple EEPs (endosomolytic moieties)
will be used to determine the peptide sequence that demonstrates
optimal endosomal escape, resulting in the best knockdown of the
FVII target gene relative to the control.
Example 47. Tumor PK/PD Study
[1135] Female NCr nu/nu mice bearing subcutaneous flank H358 tumors
will be dosed with EGFR antibody-siRNA-EEP conjugates at 0.5 mg/kg
(based on siRNA). Multiple EEPs (endosomolytic moieties) will be
used to determine the peptide sequence that demonstrates optimal
endosomal escape, resulting in the best knockdown of the target
gene relative to the control.
Example 48. Formulation of an ABC Conjugate with Nanoparticles
[1136] An exemplary ABC conjugate is packaged into self-assembled
nanoparticles using cyclodextrin polymers (10 kDa) and an excess of
non-conjugated siRNAs (ED 40-60 nm, PDI 0.1-0.2). In these
particles, the exemplary ABC conjugate maintains its ability to
interact with the antibody target. The stability and target binding
competency of the particles in circulation in vivo is regulated
through modifications of the packaging siRNAs.
[1137] Nanoparticle Formation
[1138] Nanoparticles are prepared at a final siRNA concentration of
1.6 mg/mL. siRNA containing CY5-siRNA at a ratio of 1:20 is first
diluted to 2.times. final concentration in water. Cyclodextrin
polymer (CDP) is diluted to 2.times. final concentration necessary
to achieve a nitrogen to phosphorus ratio (N:P) of 3:1 in 10 mM
phosphate buffer at neutral pH. CDP is added quickly to siRNA and
is further mixed by pipetting. Particles are incubated for at least
15 minutes before dosing or analysis.
[1139] In Vitro EGFR Binding
[1140] Nanoparticles containing various amount of the exemplary ABC
conjugate are diluted into Fetal calf serum to a final
concentration of 10 nM and are incubated for 1h at RT with Protein
G Dynabeads (Thermofisher) loaded with 150 nM of a purified EGFR-Fc
protein (Sino Biological). Beads are washed twice with PBS
containing 0.01% Tween 20 and 0.05% BSA before bead-bound
nanoparticles are disrupted with water containing 0.01% Tween 20
and 100 ug/ml heparin. The amount of CY5-siRNA contained in the
input, unbound fraction, washes and bead eluate is quantified by
fluorescence using a TECAN Infinite M200 Pro (Excitation 635 nm;
Emission 675 nm).
[1141] CY5-ASC Plasma Quantification
[1142] Quantification of nanoparticles in mouse plasma is performed
as illustrated in Example 43. The CY5-siRNAs bound to EGFR beads
are released by using heparin to compete the electrostatic
interactions between CDP and siRNAs.
[1143] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein may be employed
in practicing the disclosure. It is intended that the following
claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170281795A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170281795A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References