U.S. patent application number 16/738014 was filed with the patent office on 2020-10-08 for rnai agents, compositions and methods of use thereof for treating transthyretin (ttr) associated diseases.
The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to Klaus Charisse, Satyanarayana Kuchimanchi, Martin A. Maier, Muthiah Manoharan, Kallanthottathil G. Rajeev, Tracy Zimmermann.
Application Number | 20200318111 16/738014 |
Document ID | / |
Family ID | 1000004914760 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200318111 |
Kind Code |
A1 |
Rajeev; Kallanthottathil G. ;
et al. |
October 8, 2020 |
RNAI AGENTS, COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING
TRANSTHYRETIN (TTR) ASSOCIATED DISEASES
Abstract
The present invention provides RNAi agents, e.g., double
stranded RNAi agents, that target the transthyretin (TTR) gene and
methods of using such RNAi agents for treating or preventing
TTR-associated diseases.
Inventors: |
Rajeev; Kallanthottathil G.;
(Wayland, MA) ; Zimmermann; Tracy; (Winchester,
MA) ; Manoharan; Muthiah; (Weston, MA) ;
Maier; Martin A.; (Belmont, MA) ; Kuchimanchi;
Satyanarayana; (Acton, MA) ; Charisse; Klaus;
(Acton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000004914760 |
Appl. No.: |
16/738014 |
Filed: |
January 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15188317 |
Jun 21, 2016 |
10570391 |
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16738014 |
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14358972 |
May 16, 2014 |
9399775 |
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PCT/US2012/065691 |
Nov 16, 2012 |
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15188317 |
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61680098 |
Aug 6, 2012 |
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61615618 |
Mar 26, 2012 |
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61561710 |
Nov 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/322 20130101;
C12N 15/1136 20130101; C12N 2310/14 20130101; A61K 31/713 20130101;
C12N 2310/343 20130101; C12N 2320/30 20130101; C12N 2310/351
20130101; C07H 21/02 20130101; C12N 15/113 20130101; C12N 2310/321
20130101; A61K 31/7125 20130101; C12N 2310/346 20130101; C12N
2310/315 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7125 20060101 A61K031/7125; A61K 31/713
20060101 A61K031/713; C07H 21/02 20060101 C07H021/02 |
Claims
1. A double stranded RNAi agent comprising a sense strand
complementary to an antisense strand, wherein said antisense strand
comprises a region complementary to part of an mRNA encoding
transthyretin (TTR), wherein each strand is independently 15 to 30
nucleotides in length, wherein said double stranded RNAi agent is
represented by formula (III): sense:
5'n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.sub.a-n.s-
ub.q3' antisense:
3'n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub.-
i-N.sub.a'-n.sub.q'5' (III) wherein: i, j, k, and l are each
independently 0 or 1; p, p', q, and q' are each independently 0-6;
each N.sub.a and N.sub.a' independently represents an
oligonucleotide sequence comprising 0-25 nucleotides which are
either modified or unmodified or combinations thereof, each
sequence comprising at least two differently modified nucleotides;
each N.sub.b and N.sub.b' independently represents an
oligonucleotide sequence comprising 0-10 nucleotides which are
either modified or unmodified or combinations thereof; each
n.sub.p, n.sub.p', n.sub.q, and n.sub.q' independently represents
an overhang nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z'
each independently represent one motif of three identical
modifications on three consecutive nucleotides; modifications on
N.sub.b differ from the modification on Y and modifications on
N.sub.b' differ from the modification on Y'; and wherein the sense
strand is conjugated to at least one ligand.
2. The RNAi agent of claim 1, wherein i is 1; j is 1; or both i and
j are 1; or wherein k is 1; l is 1; or both k and l are 1.
3. (canceled)
4. The RNAi agent of claim 1, wherein XXX is complementary to
X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to
Z'Z'Z'.
5.-10. (canceled)
11. The RNAi agent of claim 1, wherein the duplex region is 15-30
nucleotide pairs in length 17-23 nucleotide pairs in length; 17-25
nucleotide pairs in length; 23-27 nucleotide pairs in length 19-21
nucleotide pairs in length; or 21-23 nucleotide pairs in
length.
12.-17. (canceled)
18. The RNAi agent of claim 1, wherein the modifications on the
nucleotides are selected from the group consisting of LNA, HNA,
CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl,
2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.
19. The RNAi agent of claim 18, wherein the modifications on the
nucleotides are 2'-O-methyl, 2'-fluoro or both.
20. The RNAi agent of claim 1, wherein the ligand is one or more
GalNAc derivatives attached through a bivalent or trivalent
branched linker.
21. The RNAi agent of claim 1, wherein the ligand is ##STR00014##
and wherein the ligand is attached to the 3' end of the sense
strand.
22. (canceled)
23. (canceled)
24. The RNAi agent of claim 1 further comprising at least one
phosphorothioate or methylphosphonate internucleotide linkage.
25.-41. (canceled)
42. A pharmaceutical composition comprising the RNAi agent of claim
1.
43.-49. (canceled)
50. A method of inhibiting expression of a transthyretin (TTR) in a
cell comprising contacting said cell with the RNAi agent of claim 1
or with the pharmaceutical composition of claim 42 in an amount
effective to inhibit expression of said TTR in said cell, thereby
inhibiting expression of said transthyretin (TTR) in said cell.
51. (canceled)
52. (canceled)
53. The method of claim 50, wherein said cell is present within a
subject.
54. The method of claim 53, wherein said subject is a human.
55.-78. (canceled)
79. A method of treating or preventing a TTR-associated disease in
a subject, comprising administering to said subject a
therapeutically effective amount or a prophylactically effective
amount of the RNAi agent of claim 1 or the pharmaceutical
composition of claim 42, thereby treating or preventing said
TTR-associated disease in said subject.
80. (canceled)
81. The method of claim 79, wherein said subject is a human.
82. The method of claim 79, wherein said subject is a subject
suffering from a TTR-associated disease.
83. (canceled)
84. The method of claim 79, wherein said subject carries a TTR gene
mutation that is associated with the development of a
TTR-associated disease.
85. The method of claim 79, wherein said TTR-associated disease is
selected from the group consisting of senile systemic amyloidosis
(SSA), systemic familial amyloidosis, familial amyloidotic
polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC),
leptomeningeal/Central Nervous System (CNS) amyloidosis, and
hyperthyroxinemia.
86. (canceled)
87. (canceled)
88. The method of claim 79, wherein said RNAi agent is administered
to said subject via subcutaneous, intramuscular or intravenous
administration.
89.-96. (canceled)
97. The method of claim 79, further comprising assessing the level
of TTR mRNA expression or TTR protein expression in a sample
derived from the subject.
98.-113. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/188,317, filed on Jun. 21, 2016, which is a
continuation of U.S. patent application Ser. No. 14/358,972, filed
on May 16, 2014, now U.S. Pat. No. 9,399,775, issued on Jul. 26,
2016, which is a 35 U.S.C. .sctn. 371 national stage filing of
International Application No. PCT/US2012/065691, filed on Nov. 16,
2012, which claims priority to U.S. Provisional Application No.
61/561,710, filed on Nov. 18, 2011, U.S. Provisional Application
No. 61/615,618, filed on Mar. 26, 2012, and U.S. Provisional
Application No. 61/680,098, filed on Aug. 6, 2012. The entire
contents of each of the foregoing application are hereby
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 8, 2020, is named 121301_00105_SL.txt and is 541,680 bytes
in size.
BACKGROUND OF THE INVENTION
[0003] Transthyretin (TTR) (also known as prealbumin) is found in
serum and cerebrospinal fluid (CSF). TTR transports retinol-binding
protein (RBP) and thyroxine (T4) and also acts as a carrier of
retinol (vitamin A) through its association with RBP in the blood
and the CSF. Transthyretin is named for its transport of thyroxine
and retinol. TTR also functions as a protease and can cleave
proteins including apoA-I (the major HDL apolipoprotein), amyloid
.beta.-peptide, and neuropeptide Y. See Liz, M. A. et al. (2010)
IUBMB Life, 62(6):429-435.
[0004] TTR is a tetramer of four identical 127-amino acid subunits
(monomers) that are rich in beta sheet structure. Each monomer has
two 4-stranded beta sheets and the shape of a prolate ellipsoid.
Antiparallel beta-sheet interactions link monomers into dimers. A
short loop from each monomer forms the main dimer-dimer
interaction. These two pairs of loops separate the opposed, convex
beta-sheets of the dimers to form an internal channel.
[0005] The liver is the major site of TTR expression. Other
significant sites of expression include the choroid plexus, retina
(particularly the retinal pigment epithelium) and pancreas.
[0006] Transthyretin is one of at least 27 distinct types of
proteins that is a precursor protein in the formation of amyloid
fibrils. See Guan, J. et al. (Nov. 4, 2011) Current perspectives on
cardiac amyloidosis, Am J Physiol Heart Circ Physiol,
doi:10.1152/ajpheart.00815.2011. Extracellular deposition of
amyloid fibrils in organs and tissues is the hallmark of
amyloidosis. Amyloid fibrils are composed of misfolded protein
aggregates, which may result from either excess production of or
specific mutations in precursor proteins. The amyloidogenic
potential of TTR may be related to its extensive beta sheet
structure; X-ray crystallographic studies indicate that certain
amyloidogenic mutations destabilize the tetrameric structure of the
protein. See, e.g., Saraiva M. J. M. (2002) Expert Reviews in
Molecular Medicine, 4(12):1-11.
[0007] Amyloidosis is a general term for the group of amyloid
diseases that are characterized by amyloid deposits. Amyloid
diseases are classified based on their precursor protein; for
example, the name starts with "A" for amyloid and is followed by an
abbreviation of the precursor protein, e.g., ATTR for amloidogenic
transthyretin. Ibid.
[0008] There are numerous TTR-associated diseases, most of which
are amyloid diseases. Normal-sequence TTR is associated with
cardiac amyloidosis in people who are elderly and is termed senile
systemic amyloidosis (SSA) (also called senile cardiac amyloidosis
(SCA) or cardiac amyloidosis). SSA often is accompanied by
microscopic deposits in many other organs. TTR amyloidosis
manifests in various forms. When the peripheral nervous system is
affected more prominently, the disease is termed familial
amyloidotic polyneuropathy (FAP). When the heart is primarily
involved but the nervous system is not, the disease is called
familial amyloidotic cardiomyopathy (FAC). A third major type of
TTR amyloidosis is leptomeningeal amyloidosis, also known as
leptomeningeal or meningocerebrovascular amyloidosis, central
nervous system (CNS) amyloidosis, or amyloidosis VII form.
[0009] Mutations in TTR may also cause amyloidotic vitreous
opacities, carpal tunnel syndrome, and euthyroid hyperthyroxinemia,
which is a non-amyloidotic disease thought to be secondary to an
increased association of thyroxine with TTR due to a mutant TTR
molecule with increased affinity for thyroxine. See, e.g., Moses et
al. (1982) J. Clin. Invest., 86, 2025-2033.
[0010] Abnormal amyloidogenic proteins may be either inherited or
acquired through somatic mutations. Guan, J. et al. (Nov. 4, 2011)
Current perspectives on cardiac amyloidosis, Am J Physiol Heart
Circ Physiol, doi:10.1152/ajpheart.00815.2011. Transthyretin
associated ATTR is the most frequent form of hereditary systemic
amyloidosis. Lobato, L. (2003) J. Nephrol., 16:438-442. TTR
mutations accelerate the process of TTR amyloid formation and are
the most important risk factor for the development of ATTR. More
than 85 amyloidogenic TTR variants are known to cause systemic
familial amyloidosis. TTR mutations usually give rise to systemic
amyloid deposition, with particular involvement of the peripheral
nervous system, although some mutations are associated with
cardiomyopathy or vitreous opacities. Ibid.
[0011] The V30M mutation is the most prevalent TTR mutation. See,
e.g., Lobato, L. (2003) JNephrol, 16:438-442. The V122I mutation is
carried by 3.9% of the African American population and is the most
common cause of FAC. Jacobson, D. R. et al. (1997) N. Engl. J. Med.
336 (7): 466-73. It is estimated that SSA affects more than 25% of
the population over age 80. Westermark, P. et al. (1990) Proc.
Natl. Acad. Sci. U.S.A. 87 (7): 2843-5.
[0012] Accordingly, there is a need in the art for effective
treatments for TTR-associated diseases.
SUMMARY OF THE INVENTION
[0013] The present invention provides RNAi agents, e.g., double
stranded RNAi agents, targeting the Transthyretin (TTR) gene. The
present invention also provides methods of inhibiting expression of
TTR and methods of treating or preventing a TTR-associated disease
in a subject using the RNAi agents, e.g. double stranded RNAi
agents, of the invention. The present invention is based, at least
in part, on the discovery that RNAi agents that comprise particular
chemical modifications show a superior ability to inhibit
expression of TTR. Agents including a certain pattern of chemical
modifications (e.g., an alternating pattern) and a ligand are shown
herein to be effective in silencing the activity of the TTR gene.
Furthermore, agents including one or more motifs of three identical
modifications on three consecutive nucleotides, including one such
motif at or near the cleavage site of the agents, show surprisingly
enhanced TTR gene silencing activity. When a single such chemical
motif is present in the agent, it is preferred to be at or near the
cleavage region for enhancing of the gene silencing activity.
Cleavage region is the region surrounding the cleavage site, i.e.,
the site on the target mRNA at which cleavage occurs.
[0014] Accordingly, in one aspect, the present invention features
RNAi agents, e.g., double stranded RNAi agents, for inhibiting
expression of a transthyretin (TTR). The double stranded RNAi agent
includes a sense strand complementary to an antisense strand. The
antisense strand includes a region complementary to a part of an
mRNA encoding transthyretin. Each strand has 14 to 30 nucleotides,
and the double stranded RNAi agent is represented by formula
(III):
sense:
5'n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.s-
ub.a-n.sub.q3'
antisense:
3'n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub.-
i-N.sub.a'-n.sub.q'5' (III).
[0015] In Formula III, i, j, k, and l are each independently 0 or
1; p, p', q, and q' are each independently 0-6; each N.sub.a and
N.sub.a' independently represents an oligonucleotide sequence
including 0-25 nucleotides which are either modified or unmodified
or combinations thereof, each sequence including at least two
differently modified nucleotides; each N.sub.b and N.sub.b'
independently represents an oligonucleotide sequence including 0-10
nucleotides which are either modified or unmodified or combinations
thereof, each n.sub.p, n.sub.p', n.sub.q, and n.sub.q'
independently represents an overhang nucleotide; XXX, YYY, ZZZ,
X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif
of three identical modifications on three consecutive nucleotides;
modifications on N.sub.b differ from the modification on Y and
modifications on N.sub.b' differ from the modification on Y'. In
some embodiments, the sense strand is conjugated to at least one
ligand, e.g., at least one ligand, e.g., at least one ligand
attached to the 3' end of the sense strand. In other embodiments,
the ligand may be conjugated to the antisense strand.
[0016] In some embodiments, i is 1; j is 1; or both i and j are
1.
[0017] In some embodiments, k is 1; l is 1; or both k and l are
1.
[0018] In some embodiments, i is 0; j is 1.
[0019] In some embodiments, i is 1, j is 0.
[0020] In some embodiments, k is 0; l is 1.
[0021] In some embodiments, k is 1; l is 0.
[0022] In some embodiments, XXX is complementary to X'X'X', YYY is
complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.
[0023] In some embodiments, the YYY motif occurs at or near the
cleavage site of the sense strand.
[0024] In some embodiments, the Y'Y'Y' motif occurs at the 11, 12
and 13 positions of the antisense strand from the 5'-end.
[0025] In some embodiments, the Y' is 2'-O-methyl.
[0026] In some embodiments, the Y' is 2'-fluoro.
[0027] In some embodiments, formula (III) is represented as formula
(IIIa):
sense: 5'n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3'
antisense:
3'n.sub.p'-N.sub.a'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.a'n.sub.q'5'
(IIIa).
In formula IIIa, each N.sub.b and N.sub.b' independently represents
an oligonucleotide sequence including 1-5 modified nucleotides.
[0028] In some embodiments, formula (III) is represented as formula
(IIIb):
sense: 5'n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q3'
antisense:
3'n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.a'-n.sub.q'5'
(IIIb).
In formula IIIb each N.sub.b and N.sub.b' independently represents
an oligonucleotide sequence including 1-5 modified nucleotides.
[0029] In some embodiments, formula (III) is represented as formula
(IIIc):
sense:
5'n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3'
antisense:
3'n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.a'-n.sub-
.q'5' (IIIc).
In formula IIIc, each N.sub.b and -N.sub.b' independently
represents an oligonucleotide sequence including 1-5 modified
nucleotides and each N.sub.a and N.sub.a' independently represents
an oligonucleotide sequence including 2-10 modified
nucleotides.
[0030] In many embodiments, the duplex region is 15-30 nucleotide
pairs in length. In some embodiments, the duplex region is 17-23
nucleotide pairs in length, 17-25 nucleotide pairs in length, 23-27
nucleotide pairs in length, 19-21 nucleotide pairs in length, or
21-23 nucleotide pairs in length.
[0031] In certain embodiments, each strand has 15-30
nucleotides.
[0032] In some embodiments, the modifications on the nucleotides
are selected from the group consisting of LNA, HNA, CeNA,
2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro,
2'-deoxy, 2'-hydroxyl, and combinations thereof. In some preferred
embodiments, the modifications on the nucleotides are 2'-O-methyl
or 2'-fluoro.
[0033] In some embodiments, the ligand is one or more
N-acetylgalactosamine (GalNAc) derivatives attached through a
bivalent or trivalent branched linker. In particular embodiments,
the ligand is
##STR00001##
[0034] In some embodiments, the ligand is attached to the 3' end of
the sense strand.
[0035] In some embodiments, the RNAi agent is conjugated to the
ligand as shown in the following schematic
##STR00002##
[0036] wherein X is O or S.
[0037] In some embodiments, the RNAi agent is conjugated to the
ligand as shown in the following schematic
##STR00003##
[0038] In some embodiments, the RNAi agent further includes at
least one phosphorothioate or methylphosphonate internucleotide
linkage. In some embodiments, the phosphorothioate or
methylphosphonate internucleotide linkage is at the 3'-terminal of
one strand. In some embodiments, the strand is the antisense
strand. In other embodiments, the strand is the sense strand.
[0039] In certain embodiments, the base pair at the 1 position of
the 5'-end of the duplex is an AU base pair.
[0040] In some embodiments, the Y nucleotides contain a 2'-fluoro
modification.
[0041] In some embodiments, the Y' nucleotides contain a
2'-O-methyl modification.
[0042] In some embodiments, p'>0. In some such embodiments, each
n is complementary to the target mRNA. In other such embodiments,
each n is non-complementary to the target mRNA. In some
embodiments, p, p', q and q' are 1-6. In some preferred
embodiments, p'=1 or 2. In some preferred embodiments, p'=2. In
some such embodiments, q'=0, p=0, q=0, and p' overhang nucleotides
are complementary to the target mRNA. In other such embodiments,
q'=0, p=0, q=0, and p' overhang nucleotides are non-complementary
to the target mRNA.
[0043] In some embodiments, the sense strand has a total of 21
nucleotides and the antisense strand has a total of 23
nucleotides.
[0044] In certain embodiments, linkages between n.sub.p' include
phosphorothioate linkages. In some such embodiments, the linkages
between n.sub.p' are phosphorothioate linkages.
[0045] In some embodiments, the RNAi agent is selected from the
group of agents listed in Table 1.
[0046] In preferred embodiments, the RNAi agent is selected from
the group consisting of AD-51544, AD-51545, AD-51546, and
AD-51547.
[0047] In an even more preferred embodiment, the RNAi agent is
AD-51547 having the following structure:
TABLE-US-00001 sense: (SEQ ID NO: 2) 5'-
UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3' antisense: (SEQ ID NO: 3)
5'- uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3'
wherein lowercase nucleotides (a, u, g, c) indicate 2'-O-methyl
nucleotides; Nf (e.g., Af) indicates a 2'-fluoro nucleotide; s
indicates a phosphothiorate linkage; L96 indicates a GalNAc.sub.3
ligand.
[0048] In another aspect, the present invention features a cell
containing the RNAi agent for inhibiting expression of TTR.
[0049] In a further aspect, the present invention features a
pharmaceutical composition comprising an RNAi agent for inhibiting
expression of TTR. In some embodiments, the pharmaceutical
composition is a solution comprising the RNAi agent. In some
embodiments, the solution comprising the RNAi agent is an
unbuffered solution, e.g., saline solution or water. In other
embodiments, the solution is a buffered solution, e.g., a solution
of phosphate buffered saline (PBS). In other embodiments, the
pharmaceutical composition is a liposome or a lipid formulation. In
some embodiments, the lipid formulation comprises a XTC or MC3.
[0050] In yet another aspect, the present invention features
methods of inhibiting expression of transthyretin (TTR) in a cell.
The methods include contacting a cell with an RNAi agent, e.g., a
double stranded RNAi agent, in an amount effective to inhibit
expression of TTR in the cell, thereby inhibiting expression of TTR
in the cell.
[0051] In some embodiments, the expression of TTR is inhibited by
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, or at least about 90%.
[0052] In other embodiments, the cell is contacted in vitro with
the RNAi agent. In other embodiments, the cell is present within a
subject. In preferred embodiments, the subject is a human.
[0053] In further embodiments, the subject is a subject suffering
from a TTR-associated disease and the effective amount is a
therapeutically effective amount. In other embodiments, the subject
is a subject at risk for developing a TTR-associated disease and
the effective amount is a prophylactically effective amount. In
some embodiments, a subject at risk for developing a TTR-associated
disease is a subject who carries a TTR gene mutation that is
associated with the development of a TTR-associated disease.
[0054] In certain embodiments, the TTR-associated disease is
selected from the group consisting of senile systemic amyloidosis
(SSA), systemic familial amyloidosis, familial amyloidotic
polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC),
leptomeningeal/Central Nervous System (CNS) amyloidosis, and
hyperthyroxinemia.
[0055] In some embodiments, the subject has a TTR-associated
amyloidosis and the method reduces an amyloid TTR deposit in the
subject.
[0056] In other embodiments, the RNAi agent is administered to the
subject by an administration means selected from the group
consisting of subcutaneous, intravenous, intramuscular,
intrabronchial, intrapleural, intraperitoneal, intraarterial,
lymphatic, cerebrospinal, and any combinations thereof. In certain
embodiments, the RNAi agent is administered to the subject via
subcutaneous or intravenous administration. In preferred
embodiments, the RNAi agent is administered to the subject via
subcutaneous administration. In some such embodiments, the
subcutaneous administration includes administration via a
subcutaneous pump or subcutaneous depot.
[0057] In certain embodiments, the RNAi agent is administered to
the subject such that the RNAi agent is delivered to a specific
site within the subject. In some embodiments, the site is selected
from the group consisting of liver, choroid plexus, retina, and
pancreas. In preferred embodiments, the site is the liver. In some
embodiments, the delivery of the RNAi agent is mediated by
asialoglycoprotein receptor (ASGP-R) present in hepatocytes.
[0058] In some embodiments, the RNAi agent is administered at a
dose of between about 0.25 mg/kg to about 50 mg/kg, e.g., between
about 0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to
about 1 mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between
about 0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about
10 mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10
mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg,
between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to
about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
[0059] In some embodiments, the RNAi agent is administered at a
dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2
mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,
about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about
11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15
mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19
mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23
mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27
mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg,
about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg,
about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,
about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg,
about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg,
about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
[0060] In some embodiments, the RNAi agent is administered in two
or more doses. In particular embodiments, the RNAi agent is
administered at intervals selected from the group consisting of
once every about 2 hours, once every about 3 hours, once every
about 4 hours, once every about 6 hours, once every about 8 hours,
once every about 12 hours, once every about 24 hours, once every
about 48 hours, once every about 72 hours, once every about 96
hours, once every about 120 hours, once every about 144 hours, once
every about 168 hours, once every about 240 hours, once every about
336 hours, once every about 504 hours, once every about 672 hours
and once every about 720 hours.
[0061] In other embodiments, the method further includes assessing
the level of TTR mRNA expression or TTR protein expression in a
sample derived from the subject.
[0062] In preferred embodiments, administering the RNAi agent does
not result in an inflammatory response in the subject as assessed
based on the level of a cytokine or chemokine selected from the
group consisting of G-CSF, IFN-.gamma., IL-10, IL-12 (p70),
IL1.beta., IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1.alpha.,
MIP-1.beta., TNF.alpha., and any combinations thereof, in a sample
from the subject.
[0063] In some embodiments, the RNAi agent is administered using a
pharmaceutical composition
[0064] In preferred embodiments, the RNAi agent is administered in
a solution. In some such embodiments, the siRNA is administered in
an unbuffered solution. In one embodiment, the siRNA is
administered in water. In other embodiments, the siRNA is
administered with a buffer solution, such as an acetate buffer, a
citrate buffer, a prolamine buffer, a carbonate buffer, or a
phosphate buffer or any combination thereof. In some embodiments,
the buffer solution is phosphate buffered saline (PBS).
[0065] In another embodiment, the pharmaceutical composition is a
liposome or a lipid formulation comprising SNALP or XTC. In one
embodiment, the lipid formulation comprises an MC3.
[0066] In another aspect, the invention provides methods of
treating or preventing a TTR-associated disease in a subject. The
methods include administering to the subject a therapeutically
effective amount or prophylactically effective amount of an RNAi
agent, e.g., a double stranded RNAi agent, thereby treating or
preventing the TTR-associated disease in the subject.
[0067] In some embodiments, TTR expression in a sample derived from
the subject is inhibited by at least about 10%, at least about 20%,
at least about 30%, at least about 40%, at least about 50%, at
least about 60% or at least about 70% at least about 80%, or at
least about 90%.
[0068] In some embodiments, the subject is a human.
[0069] In some embodiments, the subject is a subject suffering from
a TTR-associated disease. In other embodiments, the subject is a
subject at risk for developing a TTR-associated disease.
[0070] In some embodiments, the subject is a subject who carries s
a TTR gene mutation that is associated with the development of a
TTR-associated disease.
[0071] In certain embodiments, the TTR-associated disease is
selected from the group consisting of senile systemic amyloidosis
(SSA), systemic familial amyloidosis, familial amyloidotic
polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC),
leptomeningeal/Central Nervous System (CNS) amyloidosis, and
hyperthyroxinemia.
[0072] In some embodiments, the subject has a TTR-associated
amyloidosis and the method reduces an amyloid TTR deposit in the
subject.
[0073] In some embodiments, the RNAi agent is administered to the
subject by an administration means selected from the group
consisting of subcutaneous, intravenous, intramuscular,
intrabronchial, intrapleural, intraperitoneal, intraarterial,
lymphatic, cerebrospinal, and any combinations thereof. In certain
embodiments, the RNAi agent is administered to the subject via
subcutaneous or intravenous administration. In preferred
embodiments, the RNAi agent is administered to the subject via
subcutaneous administration. In some such embodiments, the
subcutaneous administration includes administration via a
subcutaneous pump or subcutaneous depot.
[0074] In certain embodiments, the RNAi agent is administered to
the subject such that the RNAi agent is delivered to a specific
site within the subject. In some such embodiments, the site is
selected from the group consisting of liver, choroid plexus,
retina, and pancreas. In preferred embodiments, the site is the
liver. In some embodiments, the delivery of the RNAi agent is
mediated by asialoglycoprotein receptor (ASGP-R) present in
hepatocytes.
[0075] In some embodiments, the RNAi agent is administered at a
dose of between about 0.25 mg/kg to about 50 mg/kg, e.g., between
about 0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to
about 1 mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between
about 0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about
10 mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10
mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg,
between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to
about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
[0076] In some embodiments, the RNAi agent is administered at a
dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2
mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,
about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about
11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15
mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19
mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23
mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27
mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg,
about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg,
about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,
about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg,
about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg,
about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
[0077] In some embodiments, the RNAi agent is administered in two
or more doses. In particular embodiments, the RNAi agent is
administered at intervals selected from the group consisting of
once every about 2 hours, once every about 3 hours, once every
about 4 hours, once every about 6 hours, once every about 8 hours,
once every about 12 hours, once every about 24 hours, once every
about 48 hours, once every about 72 hours, once every about 96
hours, once every about 120 hours, once every about 144 hours, once
every about 168 hours, once every about 240 hours, once every about
336 hours, once every about 504 hours, once every about 672 hours
and once every about 720 hours.
[0078] In other embodiments, the method further includes assessing
the level of TTR mRNA expression or TTR protein expression in a
sample derived from the subject.
[0079] In preferred embodiments, administering the RNAi agent does
not result in an inflammatory response in the subject as assessed
based on the level of a cytokine or chemokine selected from the
group consisting of G-CSF, IFN-.gamma., IL-10, IL-12 (p70),
IL1.beta., IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1.alpha.,
MIP-1.beta., TNF.alpha., and any combinations thereof, in a sample
from the subject.
[0080] In some embodiments, the RNAi agent is administered using a
pharmaceutical composition, e.g., a liposome.
[0081] In some embodiments, the RNAi agent is administered in a
solution. In some such embodiments, the siRNA is administered in an
unbuffered solution. In one embodiment, the siRNA is administered
in saline or water. In other embodiments, the siRNA is administered
with a buffer solution, such as an acetate buffer, a citrate
buffer, a prolamine buffer, a carbonate buffer, or a phosphate
buffer or any combination thereof. In some embodiments, the buffer
solution is phosphate buffered saline (PBS).
[0082] In another aspect, the present invention provides a method
of inhibiting expression of transthyretin (TTR) in a cell,
including contacting a cell with an RNAi agent, e.g., a double
stranded RNAi agent, in an amount effective to inhibit expression
of TTR in the cell. In one aspect, the double stranded RNAi agent
is selected from the group of agents listed in Table 1, thereby
inhibiting expression of transthyretin (TTR) in the cell.
[0083] In another aspect, the present invention provides a method
of inhibiting expression of transthyretin (TTR) in a cell,
including contacting a cell with an RNAi agent, e.g., a double
stranded RNAi agent, in an amount effective to inhibit expression
of TTR in the cell. In one aspect, the double stranded RNAi agent
is selected from the group consisting of AD-51544, AD-51545,
AD-51546, and AD-51547, thereby inhibiting expression of
transthyretin (TTR) in the cell.
[0084] In a further aspect, the present invention provides a method
of treating or preventing a TTR-associated disease in a subject,
including administering to the subject a therapeutically effective
amount or a prophylactically effective amount of an RNAi agent,
e.g., a double stranded RNAi agent. In one aspect, the double
stranded RNAi agent is selected from the group of agents listed in
Table 1, thereby treating or preventing a TTR-associated disease in
the subject.
[0085] In yet another aspect, the present invention provides a
method of treating or preventing a TTR-associated disease in a
subject, including administering to the subject a therapeutically
effective amount or a prophylactically effective amount of an RNAi
agent, e.g., a double stranded RNAi agent. In one aspect, the
double stranded RNAi agent is selected from the group consisting of
AD-51544, AD-51545, AD-51546, and AD-51547, thereby treating or
preventing a TTR-associated disease in the subject.
[0086] In further aspects, the invention provides kits for
performing the methods of the invention. In one aspect, the
invention provides a kit for performing a method of inhibiting
expression of transthyretin (TTR) in a cell comprising contacting a
cell with an RNAi agent, e.g., a double stranded RNAi agent, in an
amount effective to inhibit expression of said TTR in said cell,
thereby inhibiting the expression of TTR in the cell. The kit
comprises an RNAi agent and instructions for use and, optionally,
means for administering the RNAi agent to the subject.
[0087] The present invention is further illustrated by the
following detailed description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] FIG. 1 is a graph depicting that administering to mice a
single subcutaneous dose of a GalNAc-conjugated RNAi agent
targeting TTR resulted in dose-dependent suppression of TTR
mRNA.
[0089] FIG. 2 is a graph depicting that administering to mice a
single subcutaneous dose of 7.5 mg/kg or 30 mg/kg of a GalNAc
conjugated RNAi agent targeting TTR resulted in long lasting
suppression of TTR mRNA.
[0090] FIG. 3 depicts the human TTR mRNA sequence.
[0091] FIG. 4 is a graph depicting improved silencing activity of
RNAi agents modified relative to the parent AD-45163.
[0092] FIG. 5 is a graph depicting improved silencing activity of
RNAi agents modified relative to the parent AD-45165.
[0093] FIG. 6 is a graph depicting improved free uptake silencing
following 4 hour incubation with RNAi agents modified relative to
the parent AD-45163.
[0094] FIG. 7 is a graph depicting improved free uptake silencing
following 24 hour incubation with RNAi agents modified relative to
the parent AD-45163.
[0095] FIG. 8 is a graph depicting improved free uptake silencing
following 4 hour incubation with RNAi agents modified relative to
the parent AD-45165.
[0096] FIG. 9 is a graph depicting improved free uptake silencing
following 24 hour incubation with RNAi agents modified relative to
the parent AD-45165.
[0097] FIG. 10A is a graph depicting silencing of TTR mRNA in
transgenic mice that express hTTR V30M following administration of
a single subcutaneous dose of RNAi agents AD-51544, AD-51545, or
AD-45163.
[0098] FIG. 10B is a graph depicting silencing of TTR mRNA in
transgenic mice that express hTTR V30M following administration of
a single subcutaneous dose of RNAi agents AD-51546, AD-51547, or
AD-45165.
[0099] FIG. 11 is a graph depicting TTR protein suppression in
transgenic mice that express hTTR V30M following administration of
a single subcutaneous dose of 5 mg/kg or 1 mg/kg of RNAi agents
AD-51544, AD-51545, or AD-45163.
[0100] FIG. 12 is a graph depicting TTR protein suppression in
transgenic mice that express hTTR V30M following administration of
a single subcutaneous dose of 5 mg/kg or 1 mg/kg of RNAi agents
AD-51546, AD-51547, or AD-45165.
[0101] FIG. 13 depicts the protocol for post-dose blood draws in
monkeys that received 5.times.5 mg/kg RNAi agent (top line) or
1.times.25 mg/kg RNAi agent (bottom line).
[0102] FIG. 14A is a graph depicting suppression of TTR protein in
non-human primates following subcutaneous administration of five 5
mg/kg doses of AD-45163, AD-51544, AD-51545, AD-51546, or
AD-51547.
[0103] FIG. 14B is a graph depicting suppression of TTR protein in
non-human primates following subcutaneous administration of a
single 25 mg/kg dose of AD-45163, AD-51544, AD-51545, AD-51546, or
AD-51547.
[0104] FIG. 15 is a graph depicting suppression of TTR protein in
non-human primates following subcutaneous administration of
AD-51547 at 2.5 mg/kg (white squares), 5 mg/kg (black squares) or
10 mg/kg (patterned squares) per dose, or administration of PBS as
a negative control (gray squares).
DETAILED DESCRIPTION OF THE INVENTION
[0105] The present invention provides RNAi agents, e.g., double
stranded RNAi agents, and compositions targeting the Transthyretin
(TTR) gene. The present invention also provides methods of
inhibiting expression of TTR and methods of treating or preventing
a TTR-associated disease in a subject using the RNAi agents, e.g.,
double stranded RNAi agents, of the invention. The present
invention is based, at least in part, on the discovery that RNAi
agents that comprise particular chemical modifications show a
superior ability to inhibit expression of TTR. Agents including a
certain pattern of chemical modifications (e.g., an alternating
pattern) and a ligand are shown herein to be effective in silencing
the activity of the TTR gene. Furthermore, agents including one or
more motifs of three identical modifications on three consecutive
nucleotides, including one such motif at or near the cleavage site
of the agents, show surprisingly enhanced TTR gene silencing
activity. When a single such chemical motif is present in the
agent, it is preferred to be at or near the cleavage region for
enhancing of the gene silencing activity. Cleavage region is the
region surrounding the cleavage site, i.e., the site on the target
mRNA at which cleavage occurs.
I. Definitions
[0106] As used herein, each of the following terms has the meaning
associated with it in this section.
[0107] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited
to".
[0108] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0109] As used herein, a "transthyretin" ("TTR") refers to the well
known gene and protein. TTR is also known as prealbumin, HsT2651,
PALB, and TBPA. TTR functions as a transporter of retinol-binding
protein (RBP), thyroxine (T4) and retinol, and it also acts as a
protease. The liver secretes TTR into the blood, and the choroid
plexus secretes TTR into the cerebrospinal fluid. TTR is also
expressed in the pancreas and the retinal pigment epithelium. The
greatest clinical relevance of TTR is that both normal and mutant
TTR protein can form amyloid fibrils that aggregate into
extracellular deposits, causing amyloidosis. See, e.g., Saraiva M.
J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11 for a
review. The molecular cloning and nucleotide sequence of rat
transthyretin, as well as the distribution of mRNA expression, was
described by Dickson, P. W. et al. (1985) J. Biol. Chem.
260(13)8214-8219. The X-ray crystal structure of human TTR was
described in Blake, C C. et al. (1974) J Mol Biol 88, 1-12. The
sequence of a human TTR mRNA transcript can be found at National
Center for Biotechnology Information (NCBI) RefSeq accession number
NM_000371. The sequence of mouse TTR mRNA can be found at RefSeq
accession number NM_013697.2, and the sequence of rat TTR mRNA can
be found at RefSeq accession number NM_012681.1 As used herein,
"target sequence" refers to a contiguous portion of the nucleotide
sequence of an mRNA molecule formed during the transcription of a
TTR gene, including mRNA that is a product of RNA processing of a
primary transcription product.
[0110] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0111] "G," "C," "A" and "U" each generally stand for a nucleotide
that contains guanine, cytosine, adenine, and uracil as a base,
respectively. "T" and "dT" are used interchangeably herein and
refer to a deoxyribonucleotide wherein the nucleobase is thymine,
e.g., deoxyribothymine, 2'-deoxythymidine or thymidine. However, it
will be understood that the term "ribonucleotide" or "nucleotide"
or "deoxyribonucleotide" can also refer to a modified nucleotide,
as further detailed below, or a surrogate replacement moiety. The
skilled person is well aware that guanine, cytosine, adenine, and
uracil may be replaced by other moieties without substantially
altering the base pairing properties of an oligonucleotide
comprising a nucleotide bearing such replacement moiety. For
example, without limitation, a nucleotide comprising inosine as its
base may base pair with nucleotides containing adenine, cytosine,
or uracil. Hence, nucleotides containing uracil, guanine, or
adenine may be replaced in the nucleotide sequences of the
invention by a nucleotide containing, for example, inosine.
Sequences comprising such replacement moieties are embodiments of
the invention.
[0112] A "double stranded RNAi agent," double-stranded RNA (dsRNA)
molecule, also referred to as "dsRNA agent," "dsRNA", "siRNA",
"iRNA agent," as used interchangeably herein, refers to a complex
of ribonucleic acid molecules, having a duplex structure comprising
two anti-parallel and substantially complementary, as defined
below, nucleic acid strands. In general, the majority of
nucleotides of each strand are ribonucleotides, but as described in
detail herein, each or both strands can also include one or more
non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified
nucleotide. In addition, as used in this specification, an "RNAi
agent" may include ribonucleotides with chemical modifications; an
RNAi agent may include substantial modifications at multiple
nucleotides. Such modifications may include all types of
modifications disclosed herein or known in the art. Any such
modifications, as used in a siRNA type molecule, are encompassed by
"RNAi agent" for the purposes of this specification and claims.
[0113] In another embodiment, the RNAi agent may be a
single-stranded siRNA that is introduced into a cell or organism to
inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC
endonuclease Argonaute 2, which then cleaves the target mRNA. The
single-stranded siRNAs are generally 15-30 nucleotides and are
chemically modified. The design and testing of single-stranded
siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al.,
(2012) Cell 150: 883-894, the entire contents of each of which are
hereby incorporated herein by reference. Any of the antisense
nucleotide sequences described herein may be used as a
single-stranded siRNA as described herein or as chemically modified
by the methods described in Lima et al., (2012) Cell
150:883-894.
[0114] The two strands forming the duplex structure may be
different portions of one larger RNA molecule, or they may be
separate RNA molecules. Where the two strands are part of one
larger molecule, and therefore are connected by an uninterrupted
chain of nucleotides between the 3'-end of one strand and the
5'-end of the respective other strand forming the duplex structure,
the connecting RNA chain is referred to as a "hairpin loop." Where
the two strands are connected covalently by means other than an
uninterrupted chain of nucleotides between the 3'-end of one strand
and the 5'-end of the respective other strand forming the duplex
structure, the connecting structure is referred to as a "linker."
The RNA strands may have the same or a different number of
nucleotides. The maximum number of base pairs is the number of
nucleotides in the shortest strand of the dsRNA minus any overhangs
that are present in the duplex. In addition to the duplex
structure, an RNAi agent may comprise one or more nucleotide
overhangs. The term "siRNA" is also used herein to refer to an RNAi
agent as described above.
[0115] In another aspect, the agent is a single-stranded antisense
RNA molecule. An antisense RNA molecule is complementary to a
sequence within the target mRNA. Antisense RNA can inhibit
translation in a stoichiometric manner by base pairing to the mRNA
and physically obstructing the translation machinery, see Dias, N.
et al., (2002) Mol Cancer Ther 1:347-355. The antisense RNA
molecule may have about 15-30 nucleotides that are complementary to
the target mRNA. For example, the antisense RNA molecule may have a
sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous
nucleotides from one of the antisense sequences of Table 1.
[0116] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of an RNAi agent when a 3'-end of one strand of the RNAi
agent extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the double stranded RNAi agent, i.e., no nucleotide
overhang. A "blunt ended" RNAi agent is a dsRNA that is
double-stranded over its entire length, i.e., no nucleotide
overhang at either end of the molecule. The RNAi agents of the
invention include RNAi agents with nucleotide overhangs at one end
(i.e., agents with one overhang and one blunt end) or with
nucleotide overhangs at both ends.
[0117] The term "antisense strand" refers to the strand of a double
stranded RNAi agent which includes a region that is substantially
complementary to a target sequence (e.g., a human TTR mRNA). As
used herein, the term "region complementary to part of an mRNA
encoding transthyretin" refers to a region on the antisense strand
that is substantially complementary to part of a TTR mRNA sequence.
Where the region of complementarity is not fully complementary to
the target sequence, the mismatches are most tolerated in the
terminal regions and, if present, are generally in a terminal
region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the
5' and/or 3' terminus.
[0118] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0119] As used herein, the term "cleavage region" refers to a
region that is located immediately adjacent to the cleavage site.
The cleavage site is the site on the target at which cleavage
occurs. In some embodiments, the cleavage region comprises three
bases on either end of, and immediately adjacent to, the cleavage
site. In some embodiments, the cleavage region comprises two bases
on either end of, and immediately adjacent to, the cleavage site.
In some embodiments, the cleavage site specifically occurs at the
site bound by nucleotides 10 and 11 of the antisense strand, and
the cleavage region comprises nucleotides 11, 12 and 13.
[0120] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0121] Sequences can be "fully complementary" with respect to each
when there is base-pairing of the nucleotides of the first
nucleotide sequence with the nucleotides of the second nucleotide
sequence over the entire length of the first and second nucleotide
sequences. However, where a first sequence is referred to as
"substantially complementary" with respect to a second sequence
herein, the two sequences can be fully complementary, or they may
form one or more, but generally not more than 4, 3 or 2 mismatched
base pairs upon hybridization, while retaining the ability to
hybridize under the conditions most relevant to their ultimate
application. However, where two oligonucleotides are designed to
form, upon hybridization, one or more single stranded overhangs,
such overhangs shall not be regarded as mismatches with regard to
the determination of complementarity. For example, a dsRNA
comprising one oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes described
herein.
[0122] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled. Such non-Watson-Crick base pairs includes,
but not limited to, G:U Wobble or Hoogstein base pairing.
[0123] The terms "complementary," "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0124] As used herein, a polynucleotide that is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide that is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., an mRNA encoding
TTR) including a 5' UTR, an open reading frame (ORF), or a 3' UTR.
For example, a polynucleotide is complementary to at least a part
of a TTR mRNA if the sequence is substantially complementary to a
non-interrupted portion of an mRNA encoding TTR.
[0125] The term "inhibiting," as used herein, is used
interchangeably with "reducing," "silencing," "downregulating,"
"suppressing" and other similar terms, and includes any level of
inhibition.
[0126] The phrase "inhibiting expression of a TTR," as used herein,
includes inhibition of expression of any TTR gene (such as, e.g., a
mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR
gene) as well as variants or mutants of a TTR gene. Thus, the TTR
gene may be a wild-type TTR gene, a mutant TTR gene (such as a
mutant TTR gene giving rise to systemic amyloid deposition), or a
transgenic TTR gene in the context of a genetically manipulated
cell, group of cells, or organism.
[0127] "Inhibiting expression of a TTR gene" includes any level of
inhibition of a TTR gene, e.g., at least partial suppression of the
expression of a TTR gene, such as an inhibition of at least about
5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%. at least about 95%, at least about 96%, at
least about 97%, at least about 98%, or at least about 99%.
[0128] The expression of a TTR gene may be assessed based on the
level of any variable associated with TTR gene expression, e.g.,
TTR mRNA level, TTR protein level, retinol binding protein level,
vitamin A level, or the number or extent of amyloid deposits.
Inhibition may be assessed by a decrease in an absolute or relative
level of one or more of these variables compared with a control
level. The control level may be any type of control level that is
utilized in the art, e.g., a pre-dose baseline level, or a level
determined from a similar subject, cell, or sample that is
untreated or treated with a control (such as, e.g., buffer only
control or inactive agent control).
[0129] The phrase "contacting a cell with an RNAi agent," as used
herein, includes contacting a cell by any possible means.
Contacting a cell with an RNAi agent, e.g., a double stranded RNAi
agent, includes contacting a cell in vitro with the RNAi agent or
contacting a cell in vivo with the RNAi agent. The contacting may
be done directly or indirectly. Thus, for example, the RNAi agent
may be put into physical contact with the cell by the individual
performing the method, or alternatively, the RNAi agent may be put
into a situation that will permit or cause it to subsequently come
into contact with the cell.
[0130] Contacting a cell in vitro may be done, for example, by
incubating the cell with the RNAi agent. Contacting a cell in vivo
may be done, for example, by injecting the RNAi agent into or near
the tissue where the cell is located, or by injecting the RNAi
agent into another area, e.g., the bloodstream or the subcutaneous
space, such that the agent will subsequently reach the tissue where
the cell to be contacted is located. For example, the RNAi agent
may contain and/or be coupled to a ligand, e.g., a GalNAc.sub.3
ligand, that directs the RNAi agent to a site of interest, e.g.,
the liver. Combinations of in vitro and in vivo methods of
contacting are also possible. In connection with the methods of the
invention, a cell might also be contacted in vitro with an RNAi
agent and subsequently transplanted into a subject.
[0131] A "patient" or "subject," as used herein, is intended to
include either a human or non-human animal, preferably a mammal,
e.g., a monkey. Most preferably, the subject or patient is a
human.
[0132] A "TTR-associated disease," as used herein, is intended to
include any disease associated with the TTR gene or protein. Such a
disease may be caused, for example, by excess production of the TTR
protein, by TTR gene mutations, by abnormal cleavage of the TTR
protein, by abnormal interactions between TTR and other proteins or
other endogenous or exogenous substances. A "TTR-associated
disease" includes any type of TTR amyloidosis (ATTR) wherein TTR
plays a role in the formation of abnormal extracellular aggregates
or amyloid deposits. TTR-associated diseases include senile
systemic amyloidosis (SSA), systemic familial amyloidosis, familial
amyloidotic polyneuropathy (FAP), familial amyloidotic
cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS)
amyloidosis, amyloidotic vitreous opacities, carpal tunnel
syndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis
include sensory neuropathy (e.g., paresthesia, hypesthesia in
distal limbs), autonomic neuropathy (e.g., gastrointestinal
dysfunction, such as gastric ulcer, or orthostatic hypotension),
motor neuropathy, seizures, dementia, myelopathy, polyneuropathy,
carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy,
vitreous opacities, renal insufficiency, nephropathy, substantially
reduced mBMI (modified Body Mass Index), cranial nerve dysfunction,
and corneal lattice dystrophy.
[0133] "Therapeutically effective amount," as used herein, is
intended to include the amount of an RNAi agent that, when
administered to a patient for treating a TTR associated disease, is
sufficient to effect treatment of the disease (e.g., by
diminishing, ameliorating or maintaining the existing disease or
one or more symptoms of disease). The "therapeutically effective
amount" may vary depending on the RNAi agent, how the agent is
administered, the disease and its severity and the history, age,
weight, family history, genetic makeup, stage of pathological
processes mediated by TTR expression, the types of preceding or
concomitant treatments, if any, and other individual
characteristics of the patient to be treated.
[0134] "Prophylactically effective amount," as used herein, is
intended to include the amount of an RNAi agent that, when
administered to a subject who does not yet experience or display
symptoms of a TTR-associated disease, but who may be predisposed to
the disease, is sufficient to prevent or ameliorate the disease or
one or more symptoms of the disease. Symptoms that may be
ameliorated include sensory neuropathy (e.g., paresthesia,
hypesthesia in distal limbs), autonomic neuropathy (e.g.,
gastrointestinal dysfunction, such as gastric ulcer, or orthostatic
hypotension), motor neuropathy, seizures, dementia, myelopathy,
polyneuropathy, carpal tunnel syndrome, autonomic insufficiency,
cardiomyopathy, vitreous opacities, renal insufficiency,
nephropathy, substantially reduced mBMI (modified Body Mass Index),
cranial nerve dysfunction, and corneal lattice dystrophy.
Ameliorating the disease includes slowing the course of the disease
or reducing the severity of later-developing disease. The
"prophylactically effective amount" may vary depending on the RNAi
agent, how the agent is administered, the degree of risk of
disease, and the history, age, weight, family history, genetic
makeup, the types of preceding or concomitant treatments, if any,
and other individual characteristics of the patient to be
treated.
[0135] A "therapeutically-effective amount" or "prophylacticaly
effective amount" also includes an amount of an RNAi agent that
produces some desired local or systemic effect at a reasonable
benefit/risk ratio applicable to any treatment. RNAi gents employed
in the methods of the present invention may be administered in a
sufficient amount to produce a reasonable benefit/risk ratio
applicable to such treatment.
[0136] The term "sample," as used herein, includes a collection of
similar fluids, cells, or tissues isolated from a subject, as well
as fluids, cells, or tissues present within a subject. Examples of
biological fluids include blood, serum and serosal fluids, plasma,
cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the
like. Tissue samples may include samples from tissues, organs or
localized regions. For example, samples may be derived from
particular organs, parts of organs, or fluids or cells within those
organs. In certain embodiments, samples may be derived from the
liver (e.g., whole liver or certain segments of liver or certain
types of cells in the liver, such as, e.g., hepatocytes), the
retina or parts of the retina (e.g., retinal pigment epithelium),
the central nervous system or parts of the central nervous system
(e.g., ventricles or choroid plexus), or the pancreas or certain
cells or parts of the pancreas. In some embodiments, a "sample
derived from a subject" refers tocerebrospinal fluid obtained from
the subject. In preferred embodiments, a "sample derived from a
subject" refers to blood or plasma drawn from the subject. In
further embodiments, a "sample derived from a subject" refers to
liver tissue (or subcomponents thereof) or retinal tissue (or
subcomponents thereof) derived from the subject.
II. RNAi Agents
[0137] The present invention provides RNAi agents with superior
gene silencing activity. It is shown herein and in Provisional
Application No. 61/561,710 (to which the present application claims
priority) that a superior result may be obtained by introducing one
or more motifs of three identical modifications on three
consecutive nucleotides into a sense strand and/or antisense strand
of a RNAi agent, particularly at or near the cleavage site. The
sense strand and antisense strand of the RNAi agent may otherwise
be completely modified. The introduction of these motifs interrupts
the modification pattern, if present, of the sense and/or antisense
strand. The RNAi agent also optionally conjugates with a GalNAc
derivative ligand, for instance on the sense strand. The resulting
RNAi agents present superior gene silencing activity.
[0138] The inventors surprisingly discovered that when the sense
strand and antisense strand of the RNAi agent are completely
modified, having one or more motifs of three identical
modifications on three consecutive nucleotides at or near the
cleavage site of at least one strand of a RNAi agent superiorly
enhanced the gene silencing activity of the RNAi agent.
[0139] Accordingly, the invention provides RNAi agents, e.g.,
double stranded RNAi agents, capable of inhibiting the expression
of a target gene (i.e., a TTR gene) in vivo. The RNAi agent
comprises a sense strand and an antisense strand. Each strand of
the RNAi agent can range from 12-30 nucleotides in length. For
example, each strand can be between 14-30 nucleotides in length,
17-30 nucleotides in length, 25-30 nucleotides in length, 27-30
nucleotides in length, 17-23 nucleotides in length, 17-21
nucleotides in length, 17-19 nucleotides in length, 19-25
nucleotides in length, 19-23 nucleotides in length, 19-21
nucleotides in length, 21-25 nucleotides in length, or 21-23
nucleotides in length.
[0140] The sense strand and antisense strand typically form a
duplex double stranded RNA ("dsRNA"), also referred to herein as an
"RNAi agent." The duplex region of an RNAi agent may be 12-30
nucleotide pairs in length. For example, the duplex region can be
between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in
length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in
length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in
length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in
length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in
length, or 21-23 nucleotide pairs in length. In another example,
the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, and 27.
[0141] In one embodiment, the RNAi agent may contain one or more
overhang regions and/or capping groups of RNAi agent at 3'-end, or
5'-end or both ends of a strand. The overhang can be 1-6
nucleotides in length, for instance 2-6 nucleotides in length, 1-5
nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides
in length, 2-4 nucleotides in length, 1-3 nucleotides in length,
2-3 nucleotides in length, or 1-2 nucleotides in length. The
overhangs can be the result of one strand being longer than the
other, or the result of two strands of the same length being
staggered. The overhang can form a mismatch with the target mRNA or
it can be complementary to the gene sequences being targeted or can
be other sequence. The first and second strands can also be joined,
e.g., by additional bases to form a hairpin, or by other non-base
linkers.
[0142] The RNAi agents provided by the present invention include
agents with chemical modifications as disclosed, for example, in
U.S. Provisional Application No. 61/561,710, filed on Nov. 18,
2011, International Application No. PCT/US2011/051597, filed on
Sep. 15, 2010, and PCT Publication WO 2009/073809, the entire
contents of each of which are incorporated herein by reference.
[0143] In one embodiment, the nucleotides in the overhang region of
the RNAi agent can each independently be a modified or unmodified
nucleotide including, but no limited to 2'-sugar modified, such as,
2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine
(Teo), 2'-O-methoxyethyladenosine (Aeo),
2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations
thereof. For example, TT can be an overhang sequence for either end
on either strand. The overhang can form a mismatch with the target
mRNA or it can be complementary to the gene sequences being
targeted or can be other sequence.
[0144] The 5'- or 3'-overhangs at the sense strand, antisense
strand or both strands of the RNAi agent may be phosphorylated. In
some embodiments, the overhang region contains two nucleotides
having a phosphorothioate between the two nucleotides, where the
two nucleotides can be the same or different. In one embodiment,
the overhang is present at the 3'-end of the sense strand,
antisense strand or both strands. In one embodiment, this
3'-overhang is present in the antisense strand. In one embodiment,
this 3'-overhang is present in the sense strand.
[0145] The RNAi agent may contain only a single overhang, which can
strengthen the interference activity of the RNAi, without affecting
its overall stability. For example, the single-stranded overhang is
located at the 3-terminal end of the sense strand or,
alternatively, at the 3-terminal end of the antisense strand. The
RNAi may also have a blunt end, located at the 5'-end of the
antisense strand (or the 3'-end of the sense strand) or vice versa.
Generally, the antisense strand of the RNAi has a nucleotide
overhang at the 3'-end, and the 5'-end is blunt. While the
Applicants are not bound by theory, the theoretical mechanism is
that the asymmetric blunt end at the 5'-end of the antisense strand
and 3'-end overhang of the antisense strand favor the guide strand
loading into RISC process.
[0146] In one embodiment, the RNAi agent is a double ended bluntmer
of 19 nt in length, wherein the sense strand contains at least one
motif of three 2'-F modifications on three consecutive nucleotides
at positions 7, 8, 9 from the 5' end. The antisense strand contains
at least one motif of three 2'-O-methyl modifications on three
consecutive nucleotides at positions 11, 12, 13 from the 5'
end.
[0147] In one embodiment, the RNAi agent is a double ended bluntmer
of 20 nt in length, wherein the sense strand contains at least one
motif of three 2'-F modifications on three consecutive nucleotides
at positions 8, 9, 10 from the 5' end. The antisense strand
contains at least one motif of three 2'-O-methyl modifications on
three consecutive nucleotides at positions 11, 12, 13 from the 5'
end.
[0148] In one embodiment, the RNAi agent is a double ended bluntmer
of 21 nt in length, wherein the sense strand contains at least one
motif of three 2'-F modifications on three consecutive nucleotides
at positions 9, 10, 11 from the 5' end. The antisense strand
contains at least one motif of three 2'-O-methyl modifications on
three consecutive nucleotides at positions 11, 12, 13 from the 5'
end.
[0149] In one embodiment, the RNAi agent comprises a 21 nucleotides
(nt) sense strand and a 23 nucleotides (nt) antisense strand,
wherein the sense strand contains at least one motif of three 2'-F
modifications on three consecutive nucleotides at positions 9, 10,
11 from the 5' end; the antisense strand contains at least one
motif of three 2'-O-methyl modifications on three consecutive
nucleotides at positions 11, 12, 13 from the 5' end, wherein one
end of the RNAi agent is blunt, while the other end comprises a 2
nt overhang. Preferably, the 2 nt overhang is at the 3'-end of the
antisense. Optionally, the RNAi agent further comprises a ligand
(preferably GalNAc.sub.3).
[0150] In one embodiment, the RNAi agent comprises a sense and an
antisense strand, wherein the sense strand is 25-30 nucleotide
residues in length, wherein starting from the 5' terminal
nucleotide (position 1) positions 1 to 23 of the first strand
comprise at least 8 ribonucleotides; antisense strand is 36-66
nucleotide residues in length and, starting from the 3' terminal
nucleotide, comprises at least 8 ribonucleotides in the positions
paired with positions 1-23 of sense strand to form a duplex;
wherein at least the 3' terminal nucleotide of antisense strand is
unpaired with sense strand, and up to 6 consecutive 3' terminal
nucleotides are unpaired with sense strand, thereby forming a 3'
single stranded overhang of 1-6 nucleotides; wherein the 5'
terminus of antisense strand comprises from 10-30 consecutive
nucleotides which are unpaired with sense strand, thereby forming a
10-30 nucleotide single stranded 5' overhang; wherein at least the
sense strand 5' terminal and 3' terminal nucleotides are base
paired with nucleotides of antisense strand when sense and
antisense strands are aligned for maximum complementarity, thereby
forming a substantially duplexed region between sense and antisense
strands; and antisense strand is sufficiently complementary to a
target RNA along at least 19 ribonucleotides of antisense strand
length to reduce target gene expression when the double stranded
nucleic acid is introduced into a mammalian cell; and wherein the
sense strand contains at least one motif of three 2'-F
modifications on three consecutive nucleotides, where at least one
of the motifs occurs at or near the cleavage site. The antisense
strand contains at least one motif of three 2'-O-methyl
modifications on three consecutive nucleotides at or near the
cleavage site.
[0151] In one embodiment, the RNAi agent comprises sense and
antisense strands, wherein the RNAi agent comprises a first strand
having a length which is at least 25 and at most 29 nucleotides and
a second strand having a length which is at most 30 nucleotides
with at least one motif of three 2'-O-methyl modifications on three
consecutive nucleotides at position 11, 12, 13 from the 5' end;
wherein the 3' end of the first strand and the 5' end of the second
strand form a blunt end and the second strand is 1-4 nucleotides
longer at its 3' end than the first strand, wherein the duplex
region which is at least 25 nucleotides in length, and the second
strand is sufficiently complementary to a target mRNA along at
least 19 nt of the second strand length to reduce target gene
expression when the RNAi agent is introduced into a mammalian cell,
and wherein dicer cleavage of the RNAi agent preferentially results
in an siRNA comprising the 3' end of the second strand, thereby
reducing expression of the target gene in the mammal. Optionally,
the RNAi agent further comprises a ligand.
[0152] In one embodiment, the sense strand of the RNAi agent
contains at least one motif of three identical modifications on
three consecutive nucleotides, where one of the motifs occurs at
the cleavage site in the sense strand.
[0153] In one embodiment, the antisense strand of the RNAi agent
can also contain at least one motif of three identical
modifications on three consecutive nucleotides, where one of the
motifs occurs at or near the cleavage site in the antisense
strand
[0154] For RNAi agent having a duplex region of 17-23 nt in length,
the cleavage site of the antisense strand is typically around the
10, 11 and 12 positions from the 5'-end. Thus, the motifs of three
identical modifications may occur at the 9, 10, 11 positions; 10,
11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or
13, 14, 15 positions of the antisense strand, the count starting
from the 1.sup.st nucleotide from the 5'-end of the antisense
strand, or, the count starting from the 1.sup.st paired nucleotide
within the duplex region from the 5'-end of the antisense strand.
The cleavage site in the antisense strand may also change according
to the length of the duplex region of the RNAi from the 5'-end.
[0155] The sense strand of the RNAi agent may contain at least one
motif of three identical modifications on three consecutive
nucleotides at the cleavage site of the strand; and the antisense
strand may have at least one motif of three identical modifications
on three consecutive nucleotides at or near the cleavage site of
the strand. When the sense strand and the antisense strand form a
dsRNA duplex, the sense strand and the antisense strand can be so
aligned that one motif of the three nucleotides on the sense strand
and one motif of the three nucleotides on the antisense strand have
at least one nucleotide overlap, i.e., at least one of the three
nucleotides of the motif in the sense strand forms a base pair with
at least one of the three nucleotides of the motif in the antisense
strand. Alternatively, at least two nucleotides may overlap, or all
three nucleotides may overlap.
[0156] In one embodiment, the sense strand of the RNAi agent may
contain more than one motif of three identical modifications on
three consecutive nucleotides. The first motif should occur at or
near the cleavage site of the strand and the other motifs may be
wing modifications. The term "wing modification" herein refers to a
motif occurring at another portion of the strand that is separated
from the motif at or near the cleavage site of the same strand. The
wing modification is either adjacent to the first motif or is
separated by at least one or more nucleotides. When the motifs are
immediately adjacent to each other than the chemistry of the motifs
are distinct from each other and when the motifs are separated by
one or more nucleotide than the chemistries can be the same or
different. Two or more wing modifications may be present. For
instance, when two wing modifications are present, each wing
modification may occur at one end relative to the first motif which
is at or near cleavage site or on either side of the lead
motif.
[0157] Like the sense strand, the antisense strand of the RNAi
agent may contain at least two motifs of three identical
modifications on three consecutive nucleotides, with at least one
of the motifs occurring at or near the cleavage site of the strand.
This antisense strand may also contain one or more wing
modifications in an alignment similar to the wing modifications
that is present on the sense strand.
[0158] In one embodiment, the wing modification on the sense strand
or antisense strand of the RNAi agent typically does not include
the first one or two terminal nucleotides at the 3'-end, 5'-end or
both ends of the strand.
[0159] In another embodiment, the wing modification on the sense
strand or antisense strand of the RNAi agent typically does not
include the first one or two paired nucleotides within the duplex
region at the 3'-end, 5'-end or both ends of the strand.
[0160] When the sense strand and the antisense strand of the RNAi
agent each contain at least one wing modification, the wing
modifications may fall on the same end of the duplex region, and
have an overlap of one, two or three nucleotides.
[0161] When the sense strand and the antisense strand of the RNAi
agent each contain at least two wing modifications, the sense
strand and the antisense strand can be so aligned that two
modifications each from one strand fall on one end of the duplex
region, having an overlap of one, two or three nucleotides; two
modifications each from one strand fall on the other end of the
duplex region, having an overlap of one, two or three nucleotides;
two modifications one strand fall on each side of the lead motif,
having an overlap of one, two or three nucleotides in the duplex
region.
[0162] In one embodiment, every nucleotide in the sense strand and
antisense strand of the RNAi agent, including the nucleotides that
are part of the motifs, may be modified. Each nucleotide may be
modified with the same or different modification which can include
one or more alteration of one or both of the non-linking phosphate
oxygens and/or of one or more of the linking phosphate oxygens;
alteration of a constituent of the ribose sugar, e.g., of the 2'
hydroxyl on the ribose sugar; wholesale replacement of the
phosphate moiety with "dephospho" linkers; modification or
replacement of a naturally occurring base; and replacement or
modification of the ribose-phosphate backbone.
[0163] As nucleic acids are polymers of subunits, many of the
modifications occur at a position which is repeated within a
nucleic acid, e.g., a modification of a base, or a phosphate
moiety, or a non-linking O of a phosphate moiety. In some cases the
modification will occur at all of the subject positions in the
nucleic acid but in many cases it will not. By way of example, a
modification may only occur at a 3' or 5' terminal position, may
only occur in a terminal region, e.g., at a position on a terminal
nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a
strand. A modification may occur in a double strand region, a
single strand region, or in both. A modification may occur only in
the double strand region of a RNA or may only occur in a single
strand region of a RNA. For example, a phosphorothioate
modification at a non-linking O position may only occur at one or
both termini, may only occur in a terminal region, e.g., at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand, or may occur in double strand and single
strand regions, particularly at termini. The 5' end or ends can be
phosphorylated.
[0164] It may be possible, e.g., to enhance stability, to include
particular bases in overhangs, or to include modified nucleotides
or nucleotide surrogates, in single strand overhangs, e.g., in a 5'
or 3' overhang, or in both. For example, it can be desirable to
include purine nucleotides in overhangs. In some embodiments all or
some of the bases in a 3' or 5' overhang may be modified, e.g.,
with a modification described herein. Modifications can include,
e.g., the use of modifications at the 2' position of the ribose
sugar with modifications that are known in the art, e.g., the use
of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl
modified instead of the ribosugar of the nucleobase, and
modifications in the phosphate group, e.g., phosphorothioate
modifications. Overhangs need not be homologous with the target
sequence.
[0165] In one embodiment, each residue of the sense strand and
antisense strand is independently modified with LNA, HNA, CeNA,
2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy,
2'-hydroxyl, or 2'-fluoro. The strands can contain more than one
modification. In one embodiment, each residue of the sense strand
and antisense strand is independently modified with 2'-O-methyl or
2'-fluoro.
[0166] At least two different modifications are typically present
on the sense strand and antisense strand. Those two modifications
may be the 2'-O-methyl or 2'-fluoro modifications, or others.
[0167] In one embodiment, the N.sub.a and/or N.sub.b comprise
modifications of an alternating pattern. The term "alternating
motif" as used herein refers to a motif having one or more
modifications, each modification occurring on alternating
nucleotides of one strand. The alternating nucleotide may refer to
one per every other nucleotide or one per every three nucleotides,
or a similar pattern. For example, if A, B and C each represent one
type of modification to the nucleotide, the alternating motif can
be "ABABABABABAB . . . ," "AABBAABBAABB . . . ," "AABAABAABAAB . .
. ," "AAABAAABAAAB . . . ," "AAABBBAAABBB . . . ," or "ABCABCABCABC
. . . ," etc.
[0168] The type of modifications contained in the alternating motif
may be the same or different. For example, if A, B, C, D each
represent one type of modification on the nucleotide, the
alternating pattern, i.e., modifications on every other nucleotide,
may be the same, but each of the sense strand or antisense strand
can be selected from several possibilities of modifications within
the alternating motif such as "ABABAB . . . " "ACACAC . . . "
"BDBDBD . . . " or "CDCDCD . . . ," etc.
[0169] In one embodiment, the RNAi agent of the invention comprises
the modification pattern for the alternating motif on the sense
strand relative to the modification pattern for the alternating
motif on the antisense strand is shifted. The shift may be such
that the modified group of nucleotides of the sense strand
corresponds to a differently modified group of nucleotides of the
antisense strand and vice versa. For example, the sense strand when
paired with the antisense strand in the dsRNA duplex, the
alternating motif in the sense strand may start with "ABABAB" from
5'-3' of the strand and the alternating motif in the antisense
strand may start with "BABABA" from 5'-3' of the strand within the
duplex region. As another example, the alternating motif in the
sense strand may start with "AABBAABB" from 5'-3' of the strand and
the alternating motif in the antisenese strand may start with
"BBAABBAA" from 5'-3' of the strand within the duplex region, so
that there is a complete or partial shift of the modification
patterns between the sense strand and the antisense strand.
[0170] In one embodiment, the RNAi agent comprises the pattern of
the alternating motif of 2'-O-methyl modification and 2'-F
modification on the sense strand initially has a shift relative to
the pattern of the alternating motif of 2'-O-methyl modification
and 2'-F modification on the antisense strand initially, i.e., the
2'-O-methyl modified nucleotide on the sense strand base pairs with
a 2'-F modified nucleotide on the antisense strand and vice versa.
The 1 position of the sense strand may start with the 2'-F
modification, and the 1 position of the antisense strand may start
with the 2'-O-methyl modification.
[0171] The introduction of one or more motifs of three identical
modifications on three consecutive nucleotides to the sense strand
and/or antisense strand interrupts the initial modification pattern
present in the sense strand and/or antisense strand. This
interruption of the modification pattern of the sense and/or
antisense strand by introducing one or more motifs of three
identical modifications on three consecutive nucleotides to the
sense and/or antisense strand surprisingly enhances the gene
silencing activity to the target gene.
[0172] In one embodiment, when the motif of three identical
modifications on three consecutive nucleotides is introduced to any
of the strands, the modification of the nucleotide next to the
motif is a different modification than the modification of the
motif. For example, the portion of the sequence containing the
motif is " . . . N.sub.aYYYN.sub.b . . . ," where "Y" represents
the modification of the motif of three identical modifications on
three consecutive nucleotide, and "N.sub.a" and "N.sub.b" represent
a modification to the nucleotide next to the motif "YYY" that is
different than the modification of Y, and where N.sub.a and N.sub.b
can be the same or different modifications. Alternatively, N.sub.a
and/or N.sub.b may be present or absent when there is a wing
modification present.
[0173] The RNAi agent may further comprise at least one
phosphorothioate or methylphosphonate internucleotide linkage. The
phosphorothioate or methylphosphonate internucleotide linkage
modification may occur on any nucleotide of the sense strand or
antisense strand or both in any position of the strand. For
instance, the internucleotide linkage modification may occur on
every nucleotide on the sense strand or antisense strand; each
internucleotide linkage modification may occur in an alternating
pattern on the sense strand or antisense strand; or the sense
strand or antisense strand may contain both internucleotide linkage
modifications in an alternating pattern. The alternating pattern of
the internucleotide linkage modification on the sense strand may be
the same or different from the antisense strand, and the
alternating pattern of the internucleotide linkage modification on
the sense strand may have a shift relative to the alternating
pattern of the internucleotide linkage modification on the
antisense strand.
[0174] In one embodiment, the RNAi comprises the phosphorothioate
or methylphosphonate internucleotide linkage modification in the
overhang region. For example, the overhang region may contain two
nucleotides having a phosphorothioate or methylphosphonate
internucleotide linkage between the two nucleotides.
Internucleotide linkage modifications also may be made to link the
overhang nucleotides with the terminal paired nucleotides within
duplex region. For example, at least 2, 3, 4, or all the overhang
nucleotides may be linked through phosphorothioate or
methylphosphonate internucleotide linkage, and optionally, there
may be additional phosphorothioate or methylphosphonate
internucleotide linkages linking the overhang nucleotide with a
paired nucleotide that is next to the overhang nucleotide. For
instance, there may be at least two phosphorothioate
internucleotide linkages between the terminal three nucleotides, in
which two of the three nucleotides are overhang nucleotides, and
the third is a paired nucleotide next to the overhang nucleotide.
Preferably, these terminal three nucleotides may be at the 3'-end
of the antisense strand.
[0175] In one embodiment, the RNAi agent comprises mismatch(es)
with the target, within the duplex, or combinations thereof. The
mistmatch can occur in the overhang region or the duplex region.
The base pair can be ranked on the basis of their propensity to
promote dissociation or melting (e.g., on the free energy of
association or dissociation of a particular pairing, the simplest
approach is to examine the pairs on an individual pair basis,
though next neighbor or similar analysis can also be used). In
terms of promoting dissociation: A:U is preferred over G:C; G:U is
preferred over G:C; and I:C is preferred over G:C (I=inosine).
Mismatches, e.g., non-canonical or other than canonical pairings
(as described elsewhere herein) are preferred over canonical (A:T,
A:U, G:C) pairings; and pairings which include a universal base are
preferred over canonical pairings.
[0176] In one embodiment, the RNAi agent comprises at least one of
the first 1, 2, 3, 4, or 5 base pairs within the duplex regions
from the 5'-end of the antisense strand can be chosen independently
from the group of: A:U, G:U, I:C, and mismatched pairs, e.g.,
non-canonical or other than canonical pairings or pairings which
include a universal base, to promote the dissociation of the
antisense strand at the 5'-end of the duplex.
[0177] In one embodiment, the nucleotide at the 1 position within
the duplex region from the 5'-end in the antisense strand is
selected from the group consisting of A, dA, dU, U, and dT.
Alternatively, at least one of the first 1, 2 or 3 base pair within
the duplex region from the 5'-end of the antisense strand is an AU
base pair. For example, the first base pair within the duplex
region from the 5'-end of the antisense strand is an AU base
pair.
[0178] In one embodiment, the sense strand sequence may be
represented by formula (I):
5'n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ)j-N.sub.a-n.sub.q-
3' (I)
[0179] wherein:
[0180] i and j are each independently 0 or 1;
[0181] p and q are each independently 0-6;
[0182] each N.sub.a independently represents an oligonucleotide
sequence comprising 0-25 modified nucleotides, each sequence
comprising at least two differently modified nucleotides;
[0183] each N.sub.b independently represents an oligonucleotide
sequence comprising 0-10 modified nucleotides;
[0184] each n.sub.p and n.sub.q independently represent an overhang
nucleotide;
[0185] wherein N.sub.b and Y do not have the same modification;
and
[0186] XXX, YYY and ZZZ each independently represent one motif of
three identical modifications on three consecutive nucleotides.
Preferably YYY is all 2'-F modified nucleotides.
[0187] In one embodiment, the N.sub.a and/or N.sub.b comprise
modifications of alternating pattern.
[0188] In one embodiment, the YYY motif occurs at or near the
cleavage site of the sense strand. For example, when the RNAi agent
has a duplex region of 17-23 nucleotides in length, the YYY motif
can occur at or the vicinity of the cleavage site (e.g.: can occur
at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or
11, 12, 13) of--the sense strand, the count starting from the
1.sup.st nucleotide, from the 5'-end; or optionally, the count
starting at the 1.sup.st paired nucleotide within the duplex
region, from the 5'-end.
[0189] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1,
or both i and j are 1. The sense strand can therefore be
represented by the following formulas:
5'n.sub.p-N.sub.a--YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3' (Ia);
5'n.sub.p-N.sub.a--XXX-N.sub.b-YYY-N.sub.a-n.sub.q3' (Ib); or
5'n.sub.p-N.sub.a--XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3'
(Ic).
[0190] When the sense strand is represented by formula (Ia),
N.sub.b represents an oligonucleotide sequence comprising 0-10,
0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N.sub.a
independently can represent an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0191] When the sense strand is represented as formula (Ib),
N.sub.b represents an oligonucleotide sequence comprising 0-10,
0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each
N.sub.a can independently represent an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0192] When the sense strand is represented as formula (Ic), each
N.sub.b independently represents an oligonucleotide sequence
comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.
Preferably, N.sub.b is 0, 1, 2, 3, 4, 5 or 6 Each N.sub.a can
independently represent an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0193] Each of X, Y and Z may be the same or different from each
other.
[0194] In one embodiment, the antisense strand sequence of the RNAi
may be represented by formula (II):
5'n.sub.q'-N.sub.a'-(Z'Z'Z').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(X'X'X').sub-
.i-N'.sub.a-n.sub.p'3' (II)
[0195] wherein:
[0196] k and l are each independently 0 or 1;
[0197] p' and q' are each independently 0-6;
[0198] each N.sub.a' independently represents an oligonucleotide
sequence comprising 0-25 modified nucleotides, each sequence
comprising at least two differently modified nucleotides;
[0199] each N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10 modified nucleotides;
[0200] each n.sub.p' and n.sub.q' independently represent an
overhang nucleotide;
[0201] wherein N.sub.b' and Y' do not have the same
modification;
[0202] and
[0203] X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one
motif of three identical modifications on three consecutive
nucleotides.
[0204] In one embodiment, the N.sub.a' and/or N.sub.b' comprise
modifications of alternating pattern.
[0205] The Y'Y'Y' motif occurs at or near the cleavage site of the
antisense strand. For example, when the RNAi agent has a duplex
region of 17-23 nt in length, the Y'Y'Y' motif can occur at
positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14,
15 of the antisense strand, with the count starting from the
1.sup.st nucleotide, from the 5'-end; or optionally, the count
starting at the 1.sup.st paired nucleotide within the duplex
region, from the 5'-end. Preferably, the Y'Y'Y' motif occurs at
positions 11, 12, 13.
[0206] In one embodiment, Y'Y'Y' motif is all 2'-OMe modified
nucleotides.
[0207] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1,
or both k and l are 1.
[0208] The antisense strand can therefore be represented by the
following formulas:
5'n.sub.q'-N.sub.a'-Z'Z'Z'-N.sub.b'-Y'Y'Y'-N.sub.a'-n.sub.p'3'
(IIa);
5'n.sub.q'-N.sub.a'-Y'Y'Y'-N.sub.b'-X'X'X'-n.sub.p'3' (Ib); or
5'n.sub.q'-N.sub.a'-Z'Z'Z'-N.sub.b'-Y'Y'Y'-N.sub.b'-X'X'X'-N.sub.a'-n.su-
b.p'3' (IIc).
[0209] When the antisense strand is represented by formula (IIa),
N.sub.b' represents an oligonucleotide sequence comprising 0-10,
0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each
N.sub.a' independently represents an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0210] When the antisense strand is represented as formula (IIb),
N.sub.b' represents an oligonucleotide sequence comprising 0-10,
0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each
N.sub.a' independently represents an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0211] When the antisense strand is represented as formula (IIc),
each N.sub.b' independently represents an oligonucleotide sequence
comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified
nucleotides. Each N.sub.a' independently represents an
oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified
nucleotides. Preferably, N.sub.b is 0, 1, 2, 3, 4, 5 or 6.
[0212] Each of X', Y' and Z' may be the same or different from each
other.
[0213] Each nucleotide of the sense strand and antisense strand may
be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl,
2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, 2'-deoxy or
2'-fluoro. For example, each nucleotide of the sense strand and
antisense strand is independently modified with 2'-O-methyl or
2'-fluoro. Each X, Y, Z, X', Y' and Z', in particular, may
represent a 2'-O-methyl modification or a 2'-fluoro
modification.
[0214] In one embodiment, the sense strand of the RNAi agent may
contain YYY motif occurring at 9, 10 and 11 positions of the strand
when the duplex region is 21 nt, the count starting from the
1.sup.st nucleotide from the 5'-end, or optionally, the count
starting at the 1.sup.st paired nucleotide within the duplex
region, from the 5'-end; and Y represents 2'-F modification. The
sense strand may additionally contain XXX motif or ZZZ motifs as
wing modifications at the opposite end of the duplex region; and
XXX and ZZZ each independently represents a 2'-OMe modification or
2'-F modification.
[0215] In one embodiment the antisense strand may contain Y'Y'Y'
motif occurring at positions 11, 12, 13 of the strand, the count
starting from the 1.sup.st nucleotide from the 5'-end, or
optionally, the count starting at the 1.sup.st paired nucleotide
within the duplex region, from the 5'-end; and Y' represents
2'-O-methyl modification. The antisense strand may additionally
contain X'X'X' motif or Z'Z'Z' motifs as wing modifications at the
opposite end of the duplex region; and X'X'X' and Z'Z'Z' each
independently represents a 2'-OMe modification or 2'-F
modification.
[0216] The sense strand represented by any one of the above
formulas (Ia), (Ib) and (Ic) forms a duplex with a antisense strand
being represented by any one of formulas (IIa), (IIb) and (IIc),
respectively.
[0217] Accordingly, the RNAi agents of the invention may comprise a
sense strand and an antisense strand, each strand having 14 to 30
nucleotides, the RNAi duplex represented by formula (III):
sense:
5'n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ)j-N.sub.a--
n.sub.q3'
antisense:
3'n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub.-
i-N.sub.a'-n.sub.q'5' (III)
[0218] wherein:
[0219] i, j, k, and l are each independently 0 or 1;
[0220] p, p', q, and q' are each independently 0-6;
[0221] each N.sub.a and N.sub.a' independently represents an
oligonucleotide sequence comprising 0-25 modified nucleotides, each
sequence comprising at least two differently modified
nucleotides;
[0222] each N.sub.b and N.sub.b' independently represents an
oligonucleotide sequence comprising 0-10 modified nucleotides;
[0223] wherein
[0224] each n.sub.p', n.sub.p, n.sub.q', and n.sub.q independently
represents an overhang nucleotide; and
[0225] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently
represent one motif of three identical modifications on three
consecutive nucleotides.
[0226] In one embodiment, i is 1 and j is 0; or i is 0 and j is 1;
or both i and j are 1. In another embodiment, k is 1 and l is 0; k
is 0 and l is 1; or both k and l are 1.
[0227] Exemplary combinations of the sense strand and antisense
strand forming a RNAi duplex include the formulas below:
5'n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3'
3'n.sub.p'-N.sub.a'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.a'n.sub.q'5'
(IIIa)
5'n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q3'
3'n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.a'-n.sub.q'5'
(IIIb)
5'n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3'
3'n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.a-n.sub-
.q'5' (IIIc)
[0228] When the RNAi agent is represented by formula (IIIa), each
N.sub.b independently represents an oligonucleotide sequence
comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N.sub.a
independently represents an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0229] When the RNAi agent is represented as formula (IIIb), each
N.sub.b, N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0
modified nucleotides. Each N.sub.a independently represents an
oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified
nucleotides.
[0230] When the RNAi agent is represented as formula (IIIc), each
N.sub.b, N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0
modified nucleotides. Each N.sub.a, N.sub.a' independently
represents an oligonucleotide sequence comprising 2-20, 2-15, or
2-10 modified nucleotides. Each of N.sub.a, N.sub.a', N.sub.b and
N.sub.b' independently comprises modifications of alternating
pattern.
[0231] Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and
(IIIc) may be the same or different from each other.
[0232] When the RNAi agent is represented by formula (III), (IIIa),
(IIIb) or (IIIc), at least one of the Y nucleotides may form a base
pair with one of the Y' nucleotides. Alternatively, at least two of
the Y nucleotides form base pairs with the corresponding Y'
nucleotides; or all three of the Y nucleotides all form base pairs
with the corresponding Y' nucleotides.
[0233] When the RNAi agent is represented by formula (IIIa) or
(IIIc), at least one of the Z nucleotides may form a base pair with
one of the Z' nucleotides. Alternatively, at least two of the Z
nucleotides form base pairs with the corresponding Z' nucleotides;
or all three of the Z nucleotides all form base pairs with the
corresponding Z' nucleotides.
[0234] When the RNAi agent is represented as formula (IIIb) or
(IIIc), at least one of the X nucleotides may form a base pair with
one of the X' nucleotides. Alternatively, at least two of the X
nucleotides form base pairs with the corresponding X' nucleotides;
or all three of the X nucleotides all form base pairs with the
corresponding X' nucleotides.
[0235] In one embodiment, the modification on the Y nucleotide is
different than the modification on the Y' nucleotide, the
modification on the Z nucleotide is different than the modification
on the Z' nucleotide, and/or the modification on the X nucleotide
is different than the modification on the X' nucleotide.
[0236] In one embodiment, the RNAi agent is a multimer containing
at least two duplexes represented by formula (III), (IIIa), (IIIb)
or (IIIc), wherein the duplexes are connected by a linker. The
linker can be cleavable or non-cleavable. Optionally, the multimer
further comprise a ligand. Each of the duplexes can target the same
gene or two different genes; or each of the duplexes can target
same gene at two different target sites.
[0237] In one embodiment, the RNAi agent is a multimer containing
three, four, five, six or more duplexes represented by formula
(III), (IIIa), (IIIb) or (IIIc), wherein the duplexes are connected
by a linker. The linker can be cleavable or non-cleavable.
Optionally, the multimer further comprises a ligand. Each of the
duplexes can target the same gene or two different genes; or each
of the duplexes can target same gene at two different target
sites.
[0238] In one embodiment, two RNAi agents represented by formula
(III), (IIIa), (IIIb) or (IIIc) are linked to each other at the 5'
end, and one or both of the 3' ends of the are optionally
conjugated to to a ligand. Each of the agents can target the same
gene or two different genes; or each of the agents can target same
gene at two different target sites.
[0239] Various publications describe multimeric RNAi agents. Such
publications include WO2007/091269, U.S. Pat. No. 7,858,769,
WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the
entire contents of which are hereby incorporated herein by
reference.
[0240] The RNAi agent that contains conjugations of one or more
carbohydrate moieties to a RNAi agent can optimize one or more
properties of the RNAi agent. In many cases, the carbohydrate
moiety will be attached to a modified subunit of the RNAi agent.
For example, the ribose sugar of one or more ribonucleotide
subunits of a dsRNA agent can be replaced with another moiety,
e.g., a non-carbohydrate (preferably cyclic) carrier to which is
attached a carbohydrate ligand. A ribonucleotide subunit in which
the ribose sugar of the subunit has been so replaced is referred to
herein as a ribose replacement modification subunit (RRMS). A
cyclic carrier may be a carbocyclic ring system, i.e., all ring
atoms are carbon atoms, or a heterocyclic ring system, i.e., one or
more ring atoms may be a heteroatom, e.g., nitrogen, oxygen,
sulfur. The cyclic carrier may be a monocyclic ring system, or may
contain two or more rings, e.g. fused rings. The cyclic carrier may
be a fully saturated ring system, or it may contain one or more
double bonds.
[0241] The ligand may be attached to the polynucleotide via a
carrier. The carriers include (i) at least one "backbone attachment
point," preferably two "backbone attachment points" and (ii) at
least one "tethering attachment point." A "backbone attachment
point" as used herein refers to a functional group, e.g. a hydroxyl
group, or generally, a bond available for, and that is suitable for
incorporation of the carrier into the backbone, e.g., the
phosphate, or modified phosphate, e.g., sulfur containing,
backbone, of a ribonucleic acid. A "tethering attachment point"
(TAP) in some embodiments refers to a constituent ring atom of the
cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from
an atom which provides a backbone attachment point), that connects
a selected moiety. The moiety can be, e.g., a carbohydrate, e.g.
monosaccharide, disaccharide, trisaccharide, tetrasaccharide,
oligosaccharide and polysaccharide. Optionally, the selected moiety
is connected by an intervening tether to the cyclic carrier. Thus,
the cyclic carrier will often include a functional group, e.g., an
amino group, or generally, provide a bond, that is suitable for
incorporation or tethering of another chemical entity, e.g., a
ligand to the constituent ring.
[0242] The RNAi agents may be conjugated to a ligand via a carrier,
wherein the carrier can be cyclic group or acyclic group;
preferably, the cyclic group is selected from pyrrolidinyl,
pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl,
isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,
quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin;
preferably, the acyclic group is selected from serinol backbone or
diethanolamine backbone.
[0243] In certain specific embodiments, the RNAi agent of the
invention is an agent selected from the group of agents listed in
Table 1 and consisting of D1000, D1001, D1002, D1003, D1004, D1005,
D1006, D1007, D1008, D1009, D1010, D1011, D1012, D1013, D1014,
D1015, D1016, D1017, D1018, D1019, D1020, D1021, D1022, D1023,
D1024, D1025, D1026, D1027, D1028, D1029, D1030, D1031, D1032,
D1033, D1034, D1035, D1036, D1037, D1038, D1039, D1040, D1041,
D1042, D1043, D1044, D1045, D1046, D1047, D1048, D1049, D1050,
D1051, D1052, D1053, D1054, D1055, D1056, D1057, D1058, D1059,
D1060, D1061, D1062, D1063, D1064, D1065, D1066, D1067, D1068,
D1069, D1070, D1071, D1072, D1073, D1074, D1075, D1076, D1077,
D1078, D1079, D1080, D1081, D1082, D1083, D1084, D1085, D1086,
D1087, D1088, D1089, D1090, D1091, D1092, D1093, D1094, D1095,
D1096, D1097, D1098, D1099, D1100, D101, D1102, D1103, D1104,
D1105, D1106, D1107, D1108, D1109, D1110, D1111, D112, D1113,
D1114, D1115, D1116, D1117, D1118, D1119, D1120, D1121, D1122,
D1123, D1124, D1125, D1126, D1127, D1128, D1129, D1130, D1131,
D1132, D1133, D1134, D1135, D1136, D1137, D1138, D1139, D1140,
D1141, D1142, D1143, D1144, D1145, D1146, D1147, D1148, D1149,
D1150, D1151, D1152, D1153, D1154, D1155, D1156, D1157, D1158,
D1159, D1160, D1161, D1162, D1163, D1164, D1165, D1166, D1167,
D1168, D1169, D1170, D1171, D1172, D1173, D1174, D1175, D1176,
D1177, D1178, D1179, D1180, D1181, D1182, D1183, D1184, D1185,
D1186, D1187, D1188, D1189, D1190, D1191, D1192, D1193, D1194,
D1195, D1196, D1197, D1198, D1199, D1200, D1201, D1202, D1203,
D1204, D1205, D1206, D1207, D1208, D1209, D1210, D1211, D1212,
D1213, D1214, D1215, D1216, D1217, D1218, D1219, D1220, D1221,
D1222, D1223, D1224, D1225, D1226, D1227, D1228, D1229, D1230,
D1231, D1232, D1233, D1234, D1235, D1236, D1237, D1238, D1239,
D1240, D1241, D1242, D1243, D1244, D1245, D1246, D1247, D1248,
D1249, D1250, D1251, D1252, D1253, D1254, D1255, D1256, D1257,
D1258, D1259, D1260, D1261, D1262, D1263, D1264, D1265, D1266,
D1267, D1268, D1269, D1270, D1271, D1272, D1273, D1274, D1275,
D1276, D1277, D1278, D1279, D1280, D1281, D1282, D1283, D1284,
D1285, D1286, D1287, D1288, D1289, D1290, D1291, D1292, D1293,
D1294, D1295, D1296, D1297, D1298, D1299, D1300, D1301, D1302,
D1303, D1304, D1305, D1306, D1307, D1308, D1309, D1310, D1311,
D1312, D1313, D1314, D1315, D1316, D1317, D1318, D1319, D1320,
D1321, D1322, D1323, D1324, D1325, D1326, D1327, D1328, D1329,
D1330, D1331, D1332, D1333, D1334, D1335, D1336, D1337, D1338,
D1339, D1340, D1341, D1342, D1343, D1344, D1345, D1346, D1347,
D1348, D1349, D1350, D1351, D1352, D1353, D1354, D1355, D1356,
D1357, D1358, D1359, D1360, D1361, D1362, D1363, D1364, D1365,
D1366, D1367, D1368, D1369, D1370, D1371, D1372, D1373, D1374,
D1375, D1376, D1377, D1378, D1379, D1380, D1381, D1382, D1383,
D1384, D1385, D1386, D1387, D1388, D1389, D1390, D1391, D1392,
D1393, D1394, D1395, D1396, D1397, D1398, D1399, D1400, D1401,
D1402, D1403, D1404, D1405, D1406, D1407, D1408, D1409, D1410,
D1411, D1412, D1413, D1414, D1415, D1416, D1417, D1418, D1419,
D1420, D1421, D1422, D1423, D1424, D1425, D1426, D1427, D1428,
D1429, D1430, D1431, D1432, D1433, D1434, D1435, D1436, D1437,
D1438, D1439, D1440, D1441, D1442, D1443, D1444, D1445, D1446,
D1447, D1448, D1449, D1450, D1451, D1452, D1453, D1454, D1455,
D1456, D1457, D1458, D1459, D1460, D1461, D1462, D1463, D1464,
D1465, D1466, D1467, D1468, D1469, D1470, D1471, D1472, D1473,
D1474, D1475, D1476, D1477, D1478, D1479, D1480, D1481, D1482,
D1483, D1484, D1485, D1486, D1487, D1488, D1489, D1490, D1491,
D1492, D1493, D1494, D1495, D1496, D1497, D1498, D1499, D1500,
D1501, D1502, D1503, D1504, D1505, D1506, D1507, D1508, D1509,
D1510, D1511, D1512, D1513, D1514, D1515, D1516, D1517, D1518,
D1519, D1520, D1521, D1522, D1523, D1524, D1525, D1526, D1527,
D1528, D1529, D1530, D1531, D1532, D1533, D1534, D1535, D1536,
D1537, D1538, D1539, D1540, D1541, D1542, D1543, D1544, D1545,
D1546, D1547, D1548, D1549, D1550, D1551, D1552, D1553, D1554,
D1555, D1556, D1557, D1558, D1559, D1560, D1561, D1562, D1563,
D1564, D1565, D1566, D1567, D1568, D1569, D1570, D1571, D1572,
D1573, D1574, D1575, D1576, D1577, D1578, D1579, D1580, D1581,
D1582, D1583, D1584, D1585, D1586, D1587, D1588, D1589, D1590,
D1591, D1592, D1593, D1594, D1595, D1596, D1597, D1598, D1599,
D1600, D1601, D1602, D1603, D1604, D1605, D1606, D1607, D1608,
D1609, D1610, D1611, D1612, D1613, D1614, D1615, D1616, D1617,
D1618, D1619, D1620, D1621, D1622, D1623, D1624, D1625, D1626,
D1627, D1628, D1629, D1630, D1631, D1632, D1633, D1634, D1635,
D1636, D1637, D1638, D1639, D1640, D1641, D1642, D1643, D1644,
D1645, D1646, D1647, D1648, D1649, D1650, D1651, D1652, D1653,
D1654, D1655, D1656, D1657, D1658, D1659, D1660, D1661, D1662,
D1663, D1664, D1665, D1666, D1667, D1668, D1669, D1670, D1671,
D1672, D1673, D1674, D1675, D1676, D1677, D1678, D1679, D1680,
D1681, D1682, D1683, D1684, D1685, D1686, D1687, D1688, D1689,
D1690, D1691, D1692, D1693, D1694, D1695, D1696, D1697, D1698,
D1699, D1700, D1701, D1702, D1703, D1704, D1705, D1706, D1707,
D1708, D1709, D1710, D1711, D1712, D1713, D1714, D1715, D1716,
D1717, D1718, D1719, D1720, D1721, D1722, D1723, D1724, D1725,
D1726, D1727, D1728, D1729, D1730, D1731, D1732, D1733, D1734,
D1735, D1736, D1737, D1738, D1739, D1740, D1741, D1742, D1743,
D1744, D1745, D1746, D1747, D1748, D1749, D1750, D1751, D1752,
D1753, D1754, D1755, D1756, D1757, D1758, D1759, D1760, D1761,
D1762, D1763, D1764, D1765, D1766, D1767, D1768, D1769, D1770,
D1771, D1772, D1773, D1774, D1775, D1776, D1777, D1778, D1779,
D1780, D1781, D1782, D1783, D1784, D1785, D1786, D1787, D1788,
D1789, D1790, D1791, D1792, D1793, D1794, D1795, D1796, D1797,
D1798, D1799, D1800, D1801, D1802, D1803, D1804, D1805, D1806,
D1807, D1808, D1809, D1810, D1811, D1812, D1813, D1814, D1815,
D1816, D1817, D1818, D1819, D1820, D1821, D1822, D1823, D1824,
D1825, D1826, D1827, D1828, D1829, D1830, D1831, D1832, D1833,
D1834, D1835, D1836, D1837, D1838, D1839, D1840, D1841, D1842,
D1843, D1844, D1845, D1846, D1847, D1848, D1849, D1850, D1851,
D1852, D1853, D1854, D1855, D1856, D1857, D1858, D1859, D1860,
D1861, D1862, D1863, D1864, D1865, D1866, D1867, D1868, D1869,
D1870, D1871, D1872, D1873, D1874, D1875, D1876, D1877, D1878,
D1879, D1880, D1881, D1882, D1883, D1884, D1885, D1886, D1887,
D1888, D1889, D1890, D1891, D1892, D1893, D1894, D1895, D1896,
D1897, D1898, D1899, D1900, D1901, D1902, D1903, D1904, D1905,
D1906, D1907, D1908, D1909, D1910, D1911, D1912, D1913, D1914,
D1915, D1916, D1917, D1918, D1919, D1920, D1921, D1922, D1923,
D1924, D1925, D1926, D1927, D1928, D1929, D1930, D1931, D1932,
D1933, D1934, D1935, D1936, D1937, D1938, D1939, D1940, D1941,
D1942, D1943, D1944, D1945, D1946, D1947, D1948, D1949, D1950,
D1951, D1952, D1953, D1954, D1955, D1956, D1957, D1958, D1959,
D1960, D1961, D1962, D1963, D1964, D1965, D1966, D1967, D1968,
D1969, D1970, D1971, D1972, D1973, D1974, D1975, D1976, D1977,
D1978, D1979, D1980, D1981, D1982, D1983, D1984, D1985, D1986,
D1987, D1988, D1989, D1990, D1991, D1992, D1993, D1994, D1995,
D1996, D1997, D1998, D1999, D2000, D2001, D2002, D2003, D2004,
D2005, D2006, D2007, D2008, D2009, D2010, D2011, D2012, D2013,
D2014, D2015, D2016, D2017, D2018, D2019, D2020, D2021, D2022,
D2023, D2024, D2025, D2026, D2027, D2028, D2029, D2030, D2031,
D2032, D2033, D2034, D2035, D2036, D2037, D2038, D2039, D2040,
D2041, D2042, D2043, D2044, D2045, D2046, D2047, D2048, D2049,
D2050, D2051, D2052, D2053, D2054, D2055, D2056, D2057, D2058,
D2059, D2060, D2061, D2062, D2063, D2064, D2065, D2066, D2067,
D2068, D2069, D2070, D2071, D2072, D2073, D2074, D2075, D2076,
D2077, D2078, D2079, D2080, D2081, D2082, D2083, D2084, D2085,
D2086, D2087, D2088, D2089, D2090 and D2091.
[0244] These agents may further comprise a ligand, such as a GalNAc
ligand.
Ligands
[0245] The RNAi agents of the invention, e.g., double stranded RNAi
agents, may optionally be conjugated to one or more ligands. The
ligand can be attached to the sense strand, antisense strand or
both strands, at the 3'-end, 5'-end or both ends. For instance, the
ligand may be conjugated to the sense strand. In preferred
embodiments, the ligand is conjugated to the 3'-end of the sense
strand. In one preferred embodiment, the ligand is a GalNAc ligand.
In particularly preferred embodiments, the ligand is
GalNAc.sub.3:
##STR00004##
[0246] A wide variety of entities can be coupled to the RNAi agents
of the present invention. Preferred moieties are ligands, which are
coupled, preferably covalently, either directly or indirectly via
an intervening tether.
[0247] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of the molecule into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type, compartment, receptor e.g., a cellular or organ
compartment, tissue, organ or region of the body, as, e.g.,
compared to a species absent such a ligand. Ligands providing
enhanced affinity for a selected target are also termed targeting
ligands.
[0248] Some ligands can have endosomolytic properties. The
endosomolytic ligands promote the lysis of the endosome and/or
transport of the composition of the invention, or its components,
from the endosome to the cytoplasm of the cell. The endosomolytic
ligand may be a polyanionic peptide or peptidomimetic which shows
pH-dependent membrane activity and fusogenicity. In one embodiment,
the endosomolytic ligand assumes its active conformation at
endosomal pH. The "active" conformation is that conformation in
which the endosomolytic ligand promotes lysis of the endosome
and/or transport of the composition of the invention, or its
components, from the endosome to the cytoplasm of the cell.
Exemplary endosomolytic ligands include the GALA peptide (Subbarao
et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel
et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their
derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:
56-68). In one embodiment, the endosomolytic component may contain
a chemical group (e.g., an amino acid) which will undergo a change
in charge or protonation in response to a change in pH. The
endosomolytic component may be linear or branched.
[0249] Ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0250] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents; and
nuclease-resistance conferring moieties. General examples include
lipids, steroids, vitamins, sugars, proteins, peptides, polyamines,
and peptide mimics.
[0251] Ligands can include a naturally occurring substance, such as
a protein (e.g., human serum albumin (HSA), low-density lipoprotein
(LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate
(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin
or hyaluronic acid); or a lipid. The ligand may also be a
recombinant or synthetic molecule, such as a synthetic polymer,
e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an
aptamer). Examples of polyamino acids include polyamino acid is a
polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,
styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic lipid,
cationic porphyrin, quaternary salt of a polyamine, or an alpha
helical peptide.
[0252] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an
aptamer.
[0253] Other examples of ligands include dyes, intercalating agents
(e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases or a chelator (e.g., EDTA), lipophilic molecules,
e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene
butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,
geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,
1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2,
polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,
haptens (e.g., biotin), transport/absorption facilitators (e.g.,
aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,
imidazole, bisimidazole, histamine, imidazole clusters,
acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0254] Ligands can be proteins, e.g., glycoproteins, or peptides,
e.g., molecules having a specific affinity for a co-ligand, or
antibodies e.g., an antibody, that binds to a specified cell type
such as a cancer cell, endothelial cell, or bone cell. Ligands may
also include hormones and hormone receptors. They can also include
non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, or aptamers. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-.kappa.B.
[0255] The ligand can be a substance, e.g., a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0256] The ligand can increase the uptake of the oligonucleotide
into the cell by, for example, activating an inflammatory response.
Exemplary ligands that would have such an effect include tumor
necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma
interferon.
[0257] In one aspect, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., a non-kidney target tissue of the body. For example, the
target tissue can be the liver, including parenchymal cells of the
liver. Other molecules that can bind HSA can also be used as
ligands. For example, naproxen or aspirin can be used. A lipid or
lipid-based ligand can (a) increase resistance to degradation of
the conjugate, (b) increase targeting or transport into a target
cell or cell membrane, and/or (c) can be used to adjust binding to
a serum protein, e.g., HSA.
[0258] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0259] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0260] In another preferred embodiment, the lipid based ligand
binds HSA weakly or not at all, such that the conjugate will be
preferably distributed to the kidney. Other moieties that target to
kidney cells can also be used in place of or in addition to the
lipid based ligand.
[0261] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a target cell, e.g., a proliferating cell.
These are particularly useful for treating disorders characterized
by unwanted cell proliferation, e.g., of the malignant or
non-malignant type, e.g., cancer cells. Exemplary vitamins include
vitamin A, E, and K. Other exemplary vitamins include B vitamins,
e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other
vitamins or nutrients taken up by cancer cells. Also included are
HAS, low density lipoprotein (LDL) and high-density lipoprotein
(HDL).
[0262] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0263] The ligand can be a peptide or peptidomimetic. A
peptidomimetic (also referred to herein as an oligopeptidomimetic)
is a molecule capable of folding into a defined three-dimensional
structure similar to a natural peptide. The peptide or
peptidomimetic moiety can be about 5-50 amino acids long, e.g.,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A
peptide or peptidomimetic can be, for example, a cell permeation
peptide, cationic peptide, amphipathic peptide, or hydrophobic
peptide (e.g., consisting primarily of Tyr, Trp or Phe). The
peptide moiety can be a dendrimer peptide, constrained peptide or
crosslinked peptide. In another alternative, the peptide moiety can
include a hydrophobic membrane translocation sequence (MTS). An
exemplary hydrophobic MTS-containing peptide is RFGF having the
amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4). An RFGF
analogue (e.g., amino acid sequence AALLPVLLAAP) (SEQ ID NO:5)
containing a hydrophobic MTS can also be a targeting moiety. The
peptide moiety can be a "delivery" peptide, which can carry large
polar molecules including peptides, oligonucleotides, and protein
across cell membranes. For example, sequences from the HIV Tat
protein (GRKKRRQRRRPPQ) (SEQ ID NO:6) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO:7) have been
found to be capable of functioning as delivery peptides. A peptide
or peptidomimetic can be encoded by a random sequence of DNA, such
as a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et al.,
Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic
tethered to an iRNA agent via an incorporated monomer unit is a
cell targeting peptide such as an arginine-glycine-aspartic acid
(RGD)-peptide, or RGD mimic. A peptide moiety can range in length
from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a structural modification, such as to increase
stability or direct conformational properties. Any of the
structural modifications described below can be utilized. An RGD
peptide moiety can be used to target a tumor cell, such as an
endothelial tumor cell or a breast cancer tumor cell (Zitzmann et
al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate
targeting of an iRNA agent to tumors of a variety of other tissues,
including the lung, kidney, spleen, or liver (Aoki et al., Cancer
Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will
facilitate targeting of an iRNA agent to the kidney. The RGD
peptide can be linear or cyclic, and can be modified, e.g.,
glycosylated or methylated to facilitate targeting to specific
tissues. For example, a glycosylated RGD peptide can deliver an
iRNA agent to a tumor cell expressing .alpha..sub.V.beta..sub.3
(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that
target markers enriched in proliferating cells can be used. For
example, RGD containing peptides and peptidomimetics can target
cancer cells, in particular cells that exhibit an integrin. Thus,
one could use RGD peptides, cyclic peptides containing RGD, RGD
peptides that include D-amino acids, as well as synthetic RGD
mimics. In addition to RGD, one can use other moieties that target
the integrin ligand. Generally, such ligands can be used to control
proliferating cells and angiogeneis. Preferred conjugates of this
type of ligand target PECAM-1, VEGF, or other cancer gene, e.g., a
cancer gene described herein.
[0264] A "cell permeation peptide" is capable of permeating a cell,
e.g., a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). A cell permeation peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation
peptide can be a bipartite amphipathic peptide, such as MPG, which
is derived from the fusion peptide domain of HIV-1 gp41 and the NLS
of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
31:2717-2724, 2003).
[0265] In one embodiment, a targeting peptide can be an amphipathic
.alpha.-helical peptide. Exemplary amphipathic .alpha.-helical
peptides include, but are not limited to, cecropins, lycotoxins,
paradaxins, buforin, CPF, bombinin-like peptide (BLP),
cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal
antimicrobial peptides (HFIAPs), magainines, brevinins-2,
dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus
peptides, esculentinis-1, and caerins. A number of factors will
preferably be considered to maintain the integrity of helix
stability. For example, a maximum number of helix stabilization
residues will be utilized (e.g., leu, ala, or lys), and a minimum
number helix destabilization residues will be utilized (e.g.,
proline, or cyclic monomeric units. The capping residue will be
considered (for example Gly is an exemplary N-capping residue
and/or C-terminal amidation can be used to provide an extra H-bond
to stabilize the helix. Formation of salt bridges between residues
with opposite charges, separated by i.+-.3, or i.+-.4 positions can
provide stability. For example, cationic residues such as lysine,
arginine, homo-arginine, ornithine or histidine can form salt
bridges with the anionic residues glutamate or aspartate.
[0266] Peptide and peptidomimetic ligands include those having
naturally occurring or modified peptides, e.g., D or L peptides;
.alpha., .beta., or .gamma. peptides; N-methyl peptides;
azapeptides; peptides having one or more amide, i.e., peptide,
linkages replaced with one or more urea, thiourea, carbamate, or
sulfonyl urea linkages; or cyclic peptides.
[0267] The targeting ligand can be any ligand that is capable of
targeting a specific receptor. Examples are: folate, GalNAc,
galactose, mannose, mannose-6P, clusters of sugars such as GalNAc
cluster, mannose cluster, galactose cluster, or an apatamer. A
cluster is a combination of two or more sugar units. The targeting
ligands also include integrin receptor ligands, Chemokine receptor
ligands, transferrin, biotin, serotonin receptor ligands, PSMA,
endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands
can also be based on nucleic acid, e.g., an aptamer. The aptamer
can be unmodified or have any combination of modifications
disclosed herein.
[0268] Endosomal release agents include imidazoles, poly or
oligoimidazoles, PEIs, peptides, fusogenic peptides,
polycaboxylates, polyacations, masked oligo or poly cations or
anions, acetals, polyacetals, ketals/polyketyals, orthoesters,
polymers with masked or unmasked cationic or anionic charges,
dendrimers with masked or unmasked cationic or anionic charges.
[0269] PK modulator stands for pharmacokinetic modulator. PK
modulators include lipophiles, bile acids, steroids, phospholipid
analogues, peptides, protein binding agents, PEG, vitamins etc.
Examplary PK modulators include, but are not limited to,
cholesterol, fatty acids, cholic acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,
naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that
comprise a number of phosphorothioate linkages are also known to
bind to serum protein, thus short oligonucleotides, e.g.,
oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,
comprising multiple phosphorothioate linkages in the backbaone are
also amenable to the present invention as ligands (e.g., as PK
modulating ligands).
[0270] In addition, aptamers that bind serum components (e.g.,
serum proteins) are also amenable to the present invention as PK
modulating ligands.
[0271] Other ligand conjugates amenable to the invention are
described in U.S. patent application Ser. No. 10/916,185, filed
Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S.
Ser. No. 10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989
filed Apr. 27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21,
2007, which are incorporated by reference in their entireties for
all purposes.
[0272] When two or more ligands are present, the ligands can all
have same properties, all have different properties or some ligands
have the same properties while others have different properties.
For example, a ligand can have targeting properties, have
endosomolytic activity or have PK modulating properties. In a
preferred embodiment, all the ligands have different
properties.
[0273] Ligands can be coupled to the oligonucleotides at various
places, for example, 3'-end, 5'-end, and/or at an internal
position. In preferred embodiments, the ligand is attached to the
oligonucleotides via an intervening tether, e.g., a carrier
described herein. The ligand or tethered ligand may be present on a
monomer when the monomer is incorporated into the growing strand.
In some embodiments, the ligand may be incorporated via coupling to
a "precursor" monomer after the "precursor" monomer has been
incorporated into the growing strand. For example, a monomer
having, e.g., an amino-terminated tether (i.e., having no
associated ligand), e.g., TAP-(CH.sub.2).sub.nNH.sub.2 may be
incorporated into a growing oligonucleotide strand. In a subsequent
operation, i.e., after incorporation of the precursor monomer into
the strand, a ligand having an electrophilic group, e.g., a
pentafluorophenyl ester or aldehyde group, can subsequently be
attached to the precursor monomer by coupling the electrophilic
group of the ligand with the terminal nucleophilic group of the
precursor monomer's tether.
[0274] In another example, a monomer having a chemical group
suitable for taking part in Click Chemistry reaction may be
incorporated, e.g., an azide or alkyne terminated tether/linker. In
a subsequent operation, i.e., after incorporation of the precursor
monomer into the strand, a ligand having complementary chemical
group, e.g. an alkyne or azide can be attached to the precursor
monomer by coupling the alkyne and the azide together.
[0275] For double-stranded oligonucleotides, ligands can be
attached to one or both strands. In some embodiments, a
double-stranded iRNA agent contains a ligand conjugated to the
sense strand. In other embodiments, a double-stranded iRNA agent
contains a ligand conjugated to the antisense strand.
[0276] In some embodiments, ligand can be conjugated to
nucleobases, sugar moieties, or internucleosidic linkages of
nucleic acid molecules. Conjugation to purine nucleobases or
derivatives thereof can occur at any position including, endocyclic
and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or
8-positions of a purine nucleobase are attached to a conjugate
moiety. Conjugation to pyrimidine nucleobases or derivatives
thereof can also occur at any position. In some embodiments, the
2-, 5-, and 6-positions of a pyrimidine nucleobase can be
substituted with a conjugate moiety. Conjugation to sugar moieties
of nucleosides can occur at any carbon atom. Example carbon atoms
of a sugar moiety that can be attached to a conjugate moiety
include the 2', 3', and 5' carbon atoms. The 1' position can also
be attached to a conjugate moiety, such as in an abasic residue.
Internucleosidic linkages can also bear conjugate moieties. For
phosphorus-containing linkages (e.g., phosphodiester,
phosphorothioate, phosphorodithiotate, phosphoroamidate, and the
like), the conjugate moiety can be attached directly to the
phosphorus atom or to an O, N, or S atom bound to the phosphorus
atom. For amine- or amide-containing internucleosidic linkages
(e.g., PNA), the conjugate moiety can be attached to the nitrogen
atom of the amine or amide or to an adjacent carbon atom.
[0277] Any suitable ligand in the field of RNA interference may be
used, although the ligand is typically a carbohydrate e.g.
monosaccharide (such as GalNAc), disaccharide, trisaccharide,
tetrasaccharide, polysaccharide.
[0278] Linkers that conjugate the ligand to the nucleic acid
include those discussed above. For example, the ligand can be one
or more GalNAc (N-acetylglucosamine) derivatives attached through a
bivalent or trivalent branched linker.
[0279] In one embodiment, the dsRNA of the invention is conjugated
to a bivalent and trivalent branched linkers include the structures
shown in any of formula (IV)-(VII):
##STR00005##
wherein.
[0280] q.sup.2A, q.sup.2B, q.sup.3A, q.sup.3B q4.sup.A, q.sup.4B,
q.sup.5A, q.sup.5B and q.sup.5C represent independently for each
occurrence 0-20 and wherein the repeating unit can be the same or
different; P.sup.2A, P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A,
P.sup.4B, P.sup.5A, P.sup.5B, P.sup.5C, T.sup.2A, T.sup.2B,
T.sup.3A, T.sup.3B, T.sup.4A, T.sup.4B, T.sup.4A, T.sup.5B,
T.sup.5C are each independently for each occurrence absent, CO, NH,
O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH or CH.sub.2O;
[0281] Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B, Q.sup.4A, Q.sup.4B,
Q.sup.5A, Q.sup.5B, Q.sup.5C are independently for each occurrence
absent, alkylene, substituted alkylene wherin one or more
methylenes can be interrupted or terminated by one or more of O, S,
S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''), C.ident.C or C(O);
R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B, R.sup.4A, R.sup.4B,
R.sup.5A, R.sup.5B, R.sup.5C are each independently for each
occurrence absent, NH, O, S, CH.sub.2, C(O)O, C(O)NH,
NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO, CH.dbd.N--O,
##STR00006##
or heterocyclyl;
[0282] L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A, L.sup.4B,
L.sup.5A, L.sup.5B, and L.sup.5C, represent the ligand; i.e. each
independently for each occurrence a monosaccharide (such as
GalNAc), disaccharide, trisaccharide, tetrasaccharide,
oligosaccharide, or polysaccharide; and
[0283] R.sup.a is H or amino acid side chain.
[0284] Trivalent conjugating GalNAc derivatives are particularly
useful for use with RNAi agents for inhibiting the expression of a
target gene, such as those of formula (VII):
##STR00007##
[0285] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a
monosaccharide, such as GalNAc derivative. Examples of suitable
bivalent and trivalent branched linker groups conjugating GalNAc
derivatives include, but are not limited to, the following
compounds:
##STR00008## ##STR00009## ##STR00010##
[0286] In other embodiments, the RNAi agent of the invention is an
agent selected from the group consisting of AD-45163, AD-45165,
AD-51544, AD-51545, AD-51546, and AD-51547.
III. Pharmaceutical Compositions
[0287] The RNAi agents of the invention may be formulated for
administration in any convenient way for use in human or veterinary
medicine, by analogy with other pharmaceuticals. The pharmaceutical
compositions comprising RNAi agents of the invention may be, for
example, solutions with or without a buffer, or compositions
containing pharmaceutically acceptable carriers. Such compositions
include, for example, aqueous or crystalline compositions,
liposomal formulations, micellar formulations, emulsions, and gene
therapy vectors.
[0288] In the methods of the invention, the RNAi agent may be
administered in a solution. A free RNAi agent may be administered
in an unbuffered solution, e.g., in saline or in water.
Alternatively, the free siRNA may also be administered in a
suitable buffer solution. The buffer solution may comprise acetate,
citrate, prolamine, carbonate, or phosphate, or any combination
thereof. In a preferred embodiment, the buffer solution is
phosphate buffered saline (PBS). The pH and osmolarity of the
buffer solution containing the RNAi agent can be adjusted such that
it is suitable for administering to a subject.
[0289] In some embodiments, the buffer solution further comprises
an agent for controlling the osmolarity of the solution, such that
the osmolarity is kept at a desired value, e.g., at the physiologic
values of the human plasma. Solutes which can be added to the
buffer solution to control the osmolarity include, but are not
limited to, proteins, peptides, amino acids, non-metabolized
polymers, vitamins, ions, sugars, metabolites, organic acids,
lipids, or salts. In some embodiments, the agent for controlling
the osmolarity of the solution is a salt. In certain embodiments,
the agent for controlling the osmolarity of the solution is sodium
chloride or potassium chloride.
[0290] In other embodiments, the RNAi agent is formulated as a
composition that includes one or more RNAi agents and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0291] In one embodiment, the RNAi agent preparation includes at
least a second therapeutic agent (e.g., an agent other than an RNA
or a DNA). For example, an RNAi agent composition for the treatment
of a TTR-associated disease, e.g., a transthyretin-related
hereditary amyloidosis (familial amyloid polyneuropathy, FAP), may
include a known drug for the amelioration of FAP, e.g., Tafamidis
(INN, or Fx-1006A or Vyndagel).
[0292] A formulated RNAi agent composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., it
contains less than 80, 50, 30, 20, or 10% of water). In another
example, the RNAi agent is in an aqueous phase, e.g., in a solution
that includes water.
[0293] The aqueous phase or the crystalline compositions can be
incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase) or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the RNAi agent composition is formulated in a manner
that is compatible with the intended method of administration, as
described herein. For example, in particular embodiments the
composition is prepared by at least one of the following methods:
spray drying, lyophilization, vacuum drying, evaporation, fluid bed
drying, or a combination of these techniques; or sonication with a
lipid, freeze-drying, condensation and other self-assembly.
[0294] An RNAi agent preparation can be formulated in combination
with another agent, e.g., another therapeutic agent or an agent
that stabilizes RNAi agent, e.g., a protein that complexes with the
RNAi agent to form an iRNP. Still other agents include chelators,
e.g., EDTA (e.g., to remove divalent cations such as Mg.sup.2+),
salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor
such as RNAsin) and so forth.
[0295] In one embodiment, the RNAi agent preparation includes
another siRNA compound, e.g., a second RNAi agent that can mediate
RNAi with respect to a second gene, or with respect to the same
gene. Still other preparation can include at least 3, 5, ten,
twenty, fifty, or a hundred or more different RNAi agent species.
Such RNAi agents can mediate RNAi with respect to a similar number
of different genes.
[0296] The iRNA agents of the invention may be formulated for
pharmaceutical use. Pharmaceutically acceptable compositions
comprise a therapeutically- or prophylactically effective amount of
one or more of the the dsRNA agents in any of the preceding
embodiments, taken alone or formulated together with one or more
pharmaceutically acceptable carriers (additives), excipient and/or
diluents.
[0297] Methods of preparing pharmaceutical compositions of the
invention include the step of bringing into association an RNAi
agent of the present invention with the carrier and, optionally,
one or more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing into association an
RNAi agent of the present invention with liquid carriers, or finely
divided solid carriers, or both, and then, if necessary, shaping
the product.
[0298] The pharmaceutical compositions may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, e.g.,
those targeted for buccal, sublingual, and systemic absorption,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural injection as, for example, a
sterile solution or suspension, or sustained-release formulation;
(3) topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)
nasally. Delivery using subcutaneous or intravenous methods can be
particularly advantageous.
[0299] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0300] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the composition and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, ethyl cellulose and cellulose
acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)
lubricating agents, such as magnesium state, sodium lauryl sulfate
and talc; (8) excipients, such as cocoa butter and suppository
waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such
as propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; (22) bulking agents, such as
polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; and (22) other non-toxic compatible
substances employed in pharmaceutical compositions.
[0301] The compositions may conveniently be presented in unit
dosage form and may be prepared by any methods well known in the
art of pharmacy. The amount of RNAi agent which can be combined
with a carrier material to produce a single dosage form will vary
depending upon the host being treated, and the particular mode of
administration. The RNAi agent which can be combined with a carrier
material to produce a single dosage form will generally be that
amount of the RNAi agent which produces a desired effect, e.g.,
therapeutic or prophylactic effect. Generally, out of one hundred
percent, this amount will range from about 0.1 percent to about
ninety-nine percent of RNAi agent, preferably from about 5 percent
to about 70 percent, most preferably from about 10 percent to about
30 percent.
[0302] In certain embodiments, a composition of the present
invention comprises an excipient selected from the group consisting
of cyclodextrins, celluloses, liposomes, micelle forming agents,
e.g., bile acids, and polymeric carriers, e.g., polyesters and
polyanhydrides; and an RNAi agent of the present invention. In
certain embodiments, an aforementioned composition renders orally
bioavailable an RNAi agent of the present invention.
[0303] In some cases, in order to prolong the effect of an RNAi
agent, it is desirable to slow the absorption of the agent from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material having poor water solubility. The rate of absorption of
the RNAi agent then depends upon its rate of dissolution which, in
turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-administered
RNAi agent may be accomplished by dissolving or suspending the
agent in an oil vehicle.
[0304] Liposomes
[0305] An RNAi agent of the invention can be formulated for
delivery in a membranous molecular assembly, e.g., a liposome or a
micelle. As used herein, the term "liposome" refers to a vesicle
composed of amphiphilic lipids arranged in at least one bilayer,
e.g., one bilayer or a plurality of bilayers. Liposomes include
unilamellar and multilamellar vesicles that have a membrane formed
from a lipophilic material and an aqueous interior. The aqueous
portion contains the RNAi agent composition. The lipophilic
material isolates the aqueous interior from an aqueous exterior,
which typically does not include the RNAi agent composition,
although in some examples, it may. Liposomes are useful for the
transfer and delivery of active ingredients to the site of action.
Because the liposomal membrane is structurally similar to
biological membranes, when liposomes are applied to a tissue, the
liposomal bilayer fuses with bilayer of the cellular membranes. As
the merging of the liposome and cell progresses, the internal
aqueous contents that include the RNAi agent are delivered into the
cell where the RNAi agent can specifically bind to a target RNA and
can mediate RNAi. In some cases the liposomes are also specifically
targeted, e.g., to direct the RNAi agent to particular cell
types.
[0306] A liposome containing an RNAi agent can be prepared by a
variety of methods. In one example, the lipid component of a
liposome is dissolved in a detergent so that micelles are formed
with the lipid component. For example, the lipid component can be
an amphipathic cationic lipid or lipid conjugate. The detergent can
have a high critical micelle concentration and may be nonionic.
Exemplary detergents include cholate, CHAPS, octylglucoside,
deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is
then added to the micelles that include the lipid component. The
cationic groups on the lipid interact with the RNAi agent and
condense around the RNAi agent to form a liposome. After
condensation, the detergent is removed, e.g., by dialysis, to yield
a liposomal preparation of RNAi agent.
[0307] If necessary a carrier compound that assists in condensation
can be added during the condensation reaction, e.g., by controlled
addition. For example, the carrier compound can be a polymer other
than a nucleic acid (e.g., spermine or spermidine). pH can also be
adjusted to favor condensation.
[0308] Methods for producing stable polynucleotide delivery
vehicles, which incorporate a polynucleotide/cationic lipid complex
as structural components of the delivery vehicle, are further
described in, e.g., WO 96/37194, the entire contents of which are
incorporated herein by reference. Liposome formation can also
include one or more aspects of exemplary methods described in
Felgner, P. L. et al., Proc. Nat. Acad. Sci., USA 8:7413-7417,
1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol.
Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9,
1979; Szoka, et al. Proc. Nat. Acad. Sci. 75: 4194, 1978; Mayhew,
et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim.
Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol.
115:757, 1984. Commonly used techniques for preparing lipid
aggregates of appropriate size for use as delivery vehicles include
sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.
Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be
used when consistently small (50 to 200 nm) and relatively uniform
aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta
775:169, 1984). These methods are readily adapted to packaging RNAi
agent preparations into liposomes.
[0309] Liposomes that are pH-sensitive or negatively-charged entrap
nucleic acid molecules rather than complex with them. Since both
the nucleic acid molecules and the lipid are similarly charged,
repulsion rather than complex formation occurs. Nevertheless, some
nucleic acid molecules are entrapped within the aqueous interior of
these liposomes. pH-sensitive liposomes have been used to deliver
DNA encoding the thymidine kinase gene to cell monolayers in
culture. Expression of the exogenous gene was detected in the
target cells (Zhou et al., Journal of Controlled Release, 19,
(1992) 269-274).
[0310] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0311] Examples of other methods to introduce liposomes into cells
in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678;
WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem.
269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993;
Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143,
1993; and Strauss EMBO J. 11:417, 1992.
[0312] In one embodiment, cationic liposomes are used. Cationic
liposomes possess the advantage of being able to fuse to the cell
membrane. Non-cationic liposomes, although not able to fuse as
efficiently with the plasma membrane, are taken up by macrophages
in vivo and can be used to deliver RNAi agents to macrophages.
[0313] Further advantages of liposomes include: liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated RNAi agents in their
internal compartments from metabolism and degradation (Rosoff, in
"Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.),
1988, volume 1, p. 245). Important considerations in the
preparation of liposome formulations are the lipid surface charge,
vesicle size and the aqueous volume of the liposomes.
[0314] A positively charged synthetic cationic lipid,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA) can be used to form small liposomes that interact
spontaneously with nucleic acid to form lipid-nucleic acid
complexes which are capable of fusing with the negatively charged
lipids of the cell membranes of tissue culture cells, resulting in
delivery of RNAi agent (see, e.g., Felgner, P. L. et al., Proc.
Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355
for a description of DOTMA and its use with DNA).
[0315] A DOTMA analogue,
1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used
in combination with a phospholipid to form DNA-complexing vesicles.
Lipofectin.TM. Bethesda Research Laboratories, Gaithersburg, Md.)
is an effective agent for the delivery of highly anionic nucleic
acids into living tissue culture cells that comprise positively
charged DOTMA liposomes which interact spontaneously with
negatively charged polynucleotides to form complexes. When enough
positively charged liposomes are used, the net charge on the
resulting complexes is also positive. Positively charged complexes
prepared in this way spontaneously attach to negatively charged
cell surfaces, fuse with the plasma membrane, and efficiently
deliver functional nucleic acids into, for example, tissue culture
cells. Another commercially available cationic lipid,
1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP")
(Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in
that the oleoyl moieties are linked by ester, rather than ether
linkages.
[0316] Other reported cationic lipid compounds include those that
have been conjugated to a variety of moieties including, for
example, carboxyspermine which has been conjugated to one of two
types of lipids and includes compounds such as
5-carboxyspermylglycine dioctaoleoylamide ("DOGS")
(Transfectam.TM., Promega, Madison, Wis.) and
dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).
[0317] Another cationic lipid conjugate includes derivatization of
the lipid with cholesterol ("DC-Chol") which has been formulated
into liposomes in combination with DOPE (See, Gao, X. and Huang,
L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine,
made by conjugating polylysine to DOPE, has been reported to be
effective for transfection in the presence of serum (Zhou, X. et
al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines,
these liposomes containing conjugated cationic lipids, are said to
exhibit lower toxicity and provide more efficient transfection than
the DOTMA-containing compositions. Other commercially available
cationic lipid products include DMRIE and DMRIE-HP (Vical, La
Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc.,
Gaithersburg, Md.). Other cationic lipids suitable for the delivery
of oligonucleotides are described in WO 98/39359 and WO
96/37194.
[0318] Liposomal formulations are particularly suited for topical
administration, liposomes present several advantages over other
formulations. Such advantages include reduced side effects related
to high systemic absorption of the administered drug, increased
accumulation of the administered drug at the desired target, and
the ability to administer RNAi agent into the skin. In some
implementations, liposomes are used for delivering RNAi agent to
epidermal cells and also to enhance the penetration of RNAi agent
into dermal tissues, e.g., into skin. For example, the liposomes
can be applied topically. Topical delivery of drugs formulated as
liposomes to the skin has been documented (see, e.g., Weiner et
al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis
et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and
Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al.
Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,
1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz.
101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.
Sci. USA 84:7851-7855, 1987).
[0319] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome I (glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and
Novasome II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver a drug into the dermis of mouse skin. Such formulations
with RNAi agent are useful for treating a dermatological
disorder.
[0320] Liposomes that include RNAi agent can be made highly
deformable. Such deformability can enable the liposomes to
penetrate through pore that are smaller than the average radius of
the liposome. For example, transfersomes are a type of deformable
liposomes. Transferosomes can be made by adding surface edge
activators, usually surfactants, to a standard liposomal
composition. Transfersomes that include RNAi agent can be
delivered, for example, subcutaneously by infection in order to
deliver RNAi agent to keratinocytes in the skin. In order to cross
intact mammalian skin, lipid vesicles must pass through a series of
fine pores, each with a diameter less than 50 nm, under the
influence of a suitable transdermal gradient. In addition, due to
the lipid properties, these transferosomes can be self-optimizing
(adaptive to the shape of pores, e.g., in the skin),
self-repairing, and can frequently reach their targets without
fragmenting, and often self-loading.
[0321] Other formulations amenable to the present invention are
described in U.S. provisional application Ser. No. 61/018,616,
filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748,
filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and
61/051,528, filed May 8, 2008. PCT application no
PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations
that are amenable to the present invention.
[0322] Surfactants
[0323] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes (see above).
RNAi agent (or a precursor, e.g., a larger dsiRNA which can be
processed into a siRNA, or a DNA which encodes a siRNA or
precursor) compositions can include a surfactant. In one
embodiment, the siRNA is formulated as an emulsion that includes a
surfactant. The most common way of classifying and ranking the
properties of the many different types of surfactants, both natural
and synthetic, is by the use of the hydrophile/lipophile balance
(HLB). The nature of the hydrophilic group provides the most useful
means for categorizing the different surfactants used in
formulations (Rieger, in "Pharmaceutical Dosage Forms," Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0324] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical products and are usable over a wide
range of pH values. In general their HLB values range from 2 to
about 18 depending on their structure. Nonionic surfactants include
nonionic esters such as ethylene glycol esters, propylene glycol
esters, glyceryl esters, polyglyceryl esters, sorbitan esters,
sucrose esters, and ethoxylated esters. Nonionic alkanolamides and
ethers such as fatty alcohol ethoxylates, propoxylated alcohols,
and ethoxylated/propoxylated block polymers are also included in
this class. The polyoxyethylene surfactants are the most popular
members of the nonionic surfactant class.
[0325] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0326] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0327] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0328] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage
Forms," Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0329] Micelles and other Membranous Formulations
[0330] The RNAi agents of the invention can also be provided as
micellar formulations. "Micelles" are defined herein as a
particular type of molecular assembly in which amphipathic
molecules are arranged in a spherical structure such that all the
hydrophobic portions of the molecules are directed inward, leaving
the hydrophilic portions in contact with the surrounding aqueous
phase. The converse arrangement exists if the environment is
hydrophobic.
[0331] A mixed micellar formulation suitable for delivery through
transdermal membranes may be prepared by mixing an aqueous solution
of the siRNA composition, an alkali metal C.sub.8 to C.sub.22 alkyl
sulphate, and a micelle forming compound. Exemplary micelle forming
compounds include lecithin, hyaluronic acid, pharmaceutically
acceptable salts of hyaluronic acid, glycolic acid, lactic acid,
chamomile extract, cucumber extract, oleic acid, linoleic acid,
linolenic acid, monoolein, monooleates, monolaurates, borage oil,
evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine
and pharmaceutically acceptable salts thereof, glycerin,
polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers
and analogues thereof, polidocanol alkyl ethers and analogues
thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The
micelle forming compounds may be added at the same time or after
addition of the alkali metal alkyl sulphate. Mixed micelles will
form with substantially any kind of mixing of the ingredients but
vigorous mixing in order to provide smaller size micelles.
[0332] In one method a first micellar composition is prepared which
contains the siRNA composition and at least the alkali metal alkyl
sulphate. The first micellar composition is then mixed with at
least three micelle forming compounds to form a mixed micellar
composition. In another method, the micellar composition is
prepared by mixing the siRNA composition, the alkali metal alkyl
sulphate and at least one of the micelle forming compounds,
followed by addition of the remaining micelle forming compounds,
with vigorous mixing.
[0333] Phenol and/or m-cresol may be added to the mixed micellar
composition to stabilize the formulation and protect against
bacterial growth. Alternatively, phenol and/or m-cresol may be
added with the micelle forming ingredients. An isotonic agent such
as glycerin may also be added after formation of the mixed micellar
composition.
[0334] For delivery of the micellar formulation as a spray, the
formulation can be put into an aerosol dispenser and the dispenser
is charged with a propellant. The propellant, which is under
pressure, is in liquid form in the dispenser. The ratios of the
ingredients are adjusted so that the aqueous and propellant phases
become one, i.e., there is one phase. If there are two phases, it
is necessary to shake the dispenser prior to dispensing a portion
of the contents, e.g., through a metered valve. The dispensed dose
of pharmaceutical agent is propelled from the metered valve in a
fine spray.
[0335] Propellants may include hydrogen-containing
chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl
ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2
tetrafluoroethane) may be used.
[0336] The specific concentrations of the essential ingredients can
be determined by relatively straightforward experimentation. For
absorption through the oral cavities, it is often desirable to
increase, e.g., at least double or triple, the dosage for through
injection or administration through the gastrointestinal tract.
[0337] Particles
[0338] In another embodiment, an RNAi agent of the invention may be
incorporated into a particle, e.g., a microparticle. Microparticles
can be produced by spray-drying, but may also be produced by other
methods including lyophilization, evaporation, fluid bed drying,
vacuum drying, or a combination of these techniques.
IV. Methods for Inhibiting TTR Expression
[0339] The present invention also provides methods of inhibiting
expression of a transthyretin (TTR) in a cell. The methods include
contacting a cell with an RNAi agent, e.g., double stranded RNAi
agent, in an amount effective to inhibit expression of TTR in the
cell, thereby inhibiting expression of TTR in the cell.
[0340] Contacting of a cell with an RNAi agent, e.g., a double
stranded RNAi agent, may be done in vitro or in vivo. Contacting a
cell in vivo with the RNAi agent includes contacting a cell or
group of cells within a subject, e.g., a human subject, with the
RNAi agent. Combinations of in vitro and in vivo methods of
contacting a cell are also possible. Contacting a cell may be
direct or indirect, as discussed above. Furthermore, contacting a
cell may be accomplished via a targeting ligand, including any
ligand described herein or known in the art. In preferred
embodiments, the targeting ligand is a carbohydrate moiety, e.g., a
GalNAc.sub.3 ligand, or any other ligand that directs the RNAi
agent to a site of interest, e.g., the liver of a subject.
[0341] The term "inhibiting," as used herein, is used
interchangeably with "reducing," "silencing," "downregulating",
"suppressing", and other similar terms, and includes any level of
inhibition.
[0342] The phrase "inhibiting expression of a TTR" is intended to
refer to inhibition of expression of any TTR gene (such as, e.g., a
mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR
gene) as well as variants or mutants of a TTR gene. Thus, the TTR
gene may be a wild-type TTR gene, a mutant TTR gene (such as a
mutant TTR gene giving rise to amyloid deposition), or a transgenic
TTR gene in the context of a genetically manipulated cell, group of
cells, or organism.
[0343] "Inhibiting expression of a TTR gene" includes any level of
inhibition of a TTR gene, e.g., at least partial suppression of the
expression of a TTR gene. The expression of the TTR gene may be
assessed based on the level, or the change in the level, of any
variable associated with TTR gene expression, e.g., TTR mRNA level,
TTR protein level, or the number or extent of amyloid deposits.
This level may be assessed in an individual cell or in a group of
cells, including, for example, a sample derived from a subject.
[0344] Inhibition may be assessed by a decrease in an absolute or
relative level of one or more variables that are associated with
TTR expression compared with a control level. The control level may
be any type of control level that is utilized in the art, e.g., a
pre-dose baseline level, or a level determined from a similar
subject, cell, or sample that is untreated or treated with a
control (such as, e.g., buffer only control or inactive agent
control).
[0345] In some embodiments of the methods of the invention,
expression of a TTR gene is inhibited by at least about 5%, at
least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%. at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99%.
[0346] Inhibition of the expression of a TTR gene may be manifested
by a reduction of the amount of mRNA expressed by a first cell or
group of cells (such cells may be present, for example, in a sample
derived from a subject) in which a TTR gene is transcribed and
which has or have been treated (e.g., by contacting the cell or
cells with an RNAi agent of the invention, or by administering an
RNAi agent of the invention to a subject in which the cells are or
were present) such that the expression of a TTR gene is inhibited,
as compared to a second cell or group of cells substantially
identical to the first cell or group of cells but which has not or
have not been so treated (control cell(s)). In preferred
embodiments, the inhibition is assessed by expressing the level of
mRNA in treated cells as a percentage of the level of mRNA in
control cells, using the following formula:
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) .cndot.100 % ##EQU00001##
[0347] Alternatively, inhibition of the expression of a TTR gene
may be assessed in terms of a reduction of a parameter that is
functionally linked to TTR gene expression, e.g., TTR protein
expression, retinol binding protein level, vitamin A level, or
presence of amyloid deposits comprising TTR. TTR gene silencing may
be determined in any cell expressing TTR, either constitutively or
by genomic engineering, and by any assay known in the art. The
liver is the major site of TTR expression. Other significant sites
of expression include the choroid plexus, retina and pancreas.
[0348] Inhibition of the expression of a TTR protein may be
manifested by a reduction in the level of the TTR protein that is
expressed by a cell or group of cells (e.g., the level of protein
expressed in a sample derived from a subject). As explained above
for the assessment of mRNA suppression, the inhibition of protein
expression levels in a treated cell or group of cells may similarly
be expressed as a percentage of the level of protein in a control
cell or group of cells.
[0349] A control cell or group of cells that may be used to assess
the inhibition of the expression of a TTR gene includes a cell or
group of cells that has not yet been contacted with an RNAi agent
of the invention. For example, the control cell or group of cells
may be derived from an individual subject (e.g., a human or animal
subject) prior to treatment of the subject with an RNAi agent.
[0350] The level of TTR mRNA that is expressed by a cell or group
of cells, or the level of circulating TTR mRNA, may be determined
using any method known in the art for assessing mRNA expression. In
one embodiment, the level of expression of TTR in a sample is
determined by detecting a transcribed polynucleotide, or portion
thereof, e.g., mRNA of the TTR gene. RNA may be extracted from
cells using RNA extraction techniques including, for example, using
acid phenol/guanidine isothiocyanate extraction (RNAzol B;
Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene
(PreAnalytix, Switzerland). Typical assay formats utilizing
ribonucleic acid hybridization include nuclear run-on assays,
RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res.
12:7035), Northern blotting, in situ hybridization, and microarray
analysis. Circulating TTR mRNA may be detected using methods the
described in PCT/US2012/043584, the entire contents of which are
hereby incorporated herein by reference.
[0351] In one embodiment, the level of expression of TTR is
determined using a nucleic acid probe. The term "probe", as used
herein, refers to any molecule that is capable of selectively
binding to a specific TTR. Probes can be synthesized by one of
skill in the art, or derived from appropriate biological
preparations. Probes may be specifically designed to be labeled.
Examples of molecules that can be utilized as probes include, but
are not limited to, RNA, DNA, proteins, antibodies, and organic
molecules.
[0352] Isolated mRNA can be used in hybridization or amplification
assays that include, but are not limited to, Southern or Northern
analyses, polymerase chain reaction (PCR) analyses and probe
arrays. One method for the determination of mRNA levels involves
contacting the isolated mRNA with a nucleic acid molecule (probe)
that can hybridize to TTR mRNA. In one embodiment, the mRNA is
immobilized on a solid surface and contacted with a probe, for
example by running the isolated mRNA on an agarose gel and
transferring the mRNA from the gel to a membrane, such as
nitrocellulose. In an alternative embodiment, the probe(s) are
immobilized on a solid surface and the mRNA is contacted with the
probe(s), for example, in an Affymetrix gene chip array. A skilled
artisan can readily adapt known mRNA detection methods for use in
determining the level of TTR mRNA.
[0353] An alternative method for determining the level of
expression of TTR in a sample involves the process of nucleic acid
amplification and/or reverse transcriptase (to prepare cDNA) of for
example mRNA in the sample, e.g., by RT-PCR (the experimental
embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202),
ligase chain reaction (Barany (1991) Proc. Nat. Acad. Sci. USA
88:189-193), self sustained sequence replication (Guatelli et al.
(1990) Proc. Nat. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh et al. (1989) Proc. Nat. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988)
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers. In
particular aspects of the invention, the level of expression of TTR
is determined by quantitative fluorogenic RT-PCR (i.e., the
TaqMan.TM. System).
[0354] The expression levels of TTR mRNA may be monitored using a
membrane blot (such as used in hybridization analysis such as
Northern, Southern, dot, and the like), or microwells, sample
tubes, gels, beads or fibers (or any solid support comprising bound
nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305,
5,677,195 and 5,445,934, which are incorporated herein by
reference. The determination of TTR expression level may also
comprise using nucleic acid probes in solution.
[0355] In preferred embodiments, the level of mRNA expression is
assessed using branched DNA (bDNA) assays or real time PCR (qPCR).
The use of these methods is described and exemplified in the
Examples presented herein.
[0356] The level of TTR protein expression may be determined using
any method known in the art for the measurement of protein levels.
Such methods include, for example, electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography,
fluid or gel precipitin reactions, absorption spectroscopy, a
colorimetric assays, spectrophotometric assays, flow cytometry,
immunodiffusion (single or double), immunoelectrophoresis, Western
blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent
assays (ELISAs), immunofluorescent assays, electrochemiluminescence
assays, and the like.
[0357] In some embodiments, the efficacy of the methods of the
invention can be monitored by detecting or monitoring a reduction
in an amyloid TTR deposit. Reducing an amyloid TTR deposit, as used
herein, includes any decrease in the size, number, or severity of
TTR deposits, or to a prevention or reduction in the formation of
TTR deposits, within an organ or area of a subject, as may be
assessed in vitro or in vivo using any method known in the art. For
example, some methods of assessing amyloid deposits are described
in Gertz, M. A. & Rajukumar, S. V. (Editors) (2010),
Amyloidosis: Diagnosis and Treatment, New York: Humana Press.
Methods of assessing amyloid deposits may include biochemical
analyses, as well as visual or computerized assessment of amyloid
deposits, as made visible, e.g., using immunohistochemical
staining, fluorescent labeling, light microscopy, electron
microscopy, fluorescence microscopy, or other types of microscopy.
Invasive or noninvasive imaging modalities, including, e.g., CT,
PET, or NMR/MRI imaging may be employed to assess amyloid
deposits.
[0358] The methods of the invention may reduce TTR deposits in any
number of tissues or regions of the body including but not limited
to the heart, liver, spleen, esophagus, stomach, intestine (ileum,
duodenum and colon), brain, sciatic nerve, dorsal root ganglion,
kidney and retina.
[0359] The term "sample" as used herein refers to a collection of
similar fluids, cells, or tissues isolated from a subject, as well
as fluids, cells, or tissues present within a subject. Examples of
biological fluids include blood, serum and serosal fluids, plasma,
lymph, urine, cerebrospinal fluid, saliva, ocular fluids, and the
like. Tissue samples may include samples from tissues, organs or
localized regions. For example, samples may be derived from
particular organs, parts of organs, or fluids or cells within those
organism. In certain embodiments, samples may be derived from the
liver (e.g., whole liver or certain segments of liver or certain
types of cells in the liver, such as, e.g., hepatocytes), the
retina or parts of the retina (e.g., retinal pigment epithelium),
the central nervous system or parts of the central nervous system
(e.g., ventricles or choroid plexus), or the pancreas or certain
cells or parts of the pancreas. In preferred embodiments, a "sample
derived from a subject" refers to blood or plasma drawn from the
subject. In further embodiments, a "sample derived from a subject"
refers to liver tissue or retinal tissue derived from the
subject.
[0360] In some embodiments of the methods of the invention, the
RNAi agent is administered to a subject such that the RNAi agent is
delivered to a specific site within the subject. The inhibition of
expression of TTR may be assessed using measurements of the level
or change in the level of TTR mRNA or TTR protein in a sample
derived from fluid or tissue from the specific site within the
subject. In preferred embodiments, the site is selected from the
group consisting of liver, choroid plexus, retina, and pancreas.
The site may also be a subsection or subgroup of cells from any one
of the aforementioned sites (e.g., hepatocytes or retinal pigment
epithelium). The site may also include cells that express a
particular type of receptor (e.g., hepatocytes that express the
asialogycloprotein receptor).
V. Methods for Treating or Preventing a TTR-Associated Disease
[0361] The present invention also provides methods for treating or
preventing a TTR-associated disease in a subject. The methods
include administering to the subject a therapeutically effective
amount or prophylactically effective amount of an RNAi agent of the
invention.
[0362] As used herein, a "subject" includes either a human or a
non-human animal, preferably a vertebrate, and more preferably a
mammal. A subject may include a transgenic organism. Most
preferably, the subject is a human, such as a human suffering from
or predisposed to developing a TTR-associated disease.
[0363] In some embodiments, the subject is suffering from a
TTR-associated disease. In other embodiments, the subject is a
subject at risk for developing a TTR-associated disease, e.g., a
subject with a TTR gene mutation that is associated with the
development of a TTR associated disease, a subject with a family
history of TTR-associated disease, or a subject who has signs or
symptoms suggesting the development of TTR amyloidosis.
[0364] A "TTR-associated disease," as used herein, includes any
disease caused by or associated with the formation of amyloid
deposits in which the fibril precurosors consist of variant or
wild-type TTR protein. Mutant and wild-type TTR give rise to
various forms of amyloid deposition (amyloidosis). Amyloidosis
involves the formation and aggregation of misfolded proteins,
resulting in extracellular deposits that impair organ function.
Clinical syndromes associated with TTR aggregation include, for
example, senile systemic amyloidosis (SSA); systemic familial
amyloidosis; familial amyloidotic polyneuropathy (FAP); familial
amyloidotic cardiomyopathy (FAC); and leptomeningeal amyloidosis,
also known as leptomeningeal or meningocerebrovascular amyloidosis,
central nervous system (CNS) amyloidosis, or amyloidosis VII
form.
[0365] In some embodiments of the methods of the invention, RNAi
agents of the invention are administered to subjects suffering from
familial amyloidotic cardiomyopathy (FAC) and senile systemic
amyloidosis (SSA). Normal-sequence TTR causes cardiac amyloidosis
in people who are elderly and is termed senile systemic amyloidosis
(SSA) (also called senile cardiac amyloidosis (SCA) or cardiac
amyloidosis). SSA often is accompanied by microscopic deposits in
many other organs. TTR mutations accelerate the process of TTR
amyloid formation and are the most important risk factor for the
development of clinically significant TTR amyloidosis (also called
ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic
TTR variants are known to cause systemic familial amyloidosis.
[0366] In some embodiments of the methods of the invention, RNAi
agents of the invention are administered to subjects suffering from
transthyretin (TTR)-related familial amyloidotic polyneuropathy
(FAP). Such subjects may suffer from ocular manifestations, such as
vitreous opacity and glaucoma. It is known to one of skill in the
art that amyloidogenic transthyretin (ATTR) synthesized by retinal
pigment epithelium (RPE) plays important roles in the progression
of ocular amyloidosis. Previous studies have shown that panretinal
laser photocoagulation, which reduced the RPE cells, prevented the
progression of amyloid deposition in the vitreous, indicating that
the effective suppression of ATTR expression in RPE may become a
novel therapy for ocular amyloidosis (see, e.g., Kawaji, T., et
al., Ophthalmology. (2010) 117: 552-555). The methods of the
invention are useful for treatment of ocular manifestations of TTR
related FAP, e.g., ocular amyloidosis. The RNAi agent can be
delivered in a manner suitable for targeting a particular tissue,
such as the eye. Modes of ocular delivery include retrobulbar,
subcutaneous eyelid, subconjunctival, subtenon, anterior chamber or
intravitreous injection (or internal injection or infusion).
Specific formulations for ocular delivery include eye drops or
ointments.
[0367] Another TTR-associated disease is hyperthyroxinemia, also
known as "dystransthyretinemic hyperthyroxinemia" or
"dysprealbuminemic hyperthyroxinemia". This type of
hyperthyroxinemia may be secondary to an increased association of
thyroxine with TTR due to a mutant TTR molecule with increased
affinity for thyroxine. See, e.g., Moses et al. (1982) J. Cin.
Invest., 86, 2025-2033.
[0368] The RNAi agents of the invention may be administered to a
subject using any mode of administration known in the art,
including, but not limited to subcutaneous, intravenous,
intramuscular, intraocular, intrabronchial, intrapleural,
intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any
combinations thereof. In preferred embodiments, the agents are
administered subcutaneously.
[0369] In some embodiments, the administration is via a depot
injection. A depot injection may release the RNAi agent in a
consistent way over a prolonged time period. Thus, a depot
injection may reduce the frequency of dosing needed to obtain a
desired effect, e.g., a desired inhibition of TTR, or a therapeutic
or prophylactic effect. A depot injection may also provide more
consistent serum concentrations. Depot injections may include
subcutaneous injections or intramuscular injections. In preferred
embodiments, the depot injection is a subcutaneous injection.
[0370] In some embodiments, the administration is via a pump. The
pump may be an external pump or a surgically implanted pump. In
certain embodiments, the pump is a subcutaneously implanted osmotic
pump. In other embodiments, the pump is an infusion pump. An
infusion pump may be used for intravenous, subcutaneous, arterial,
or epidural infusions. In preferred embodiments, the infusion pump
is a subcutaneous infusion pump. In other embodiments, the pump is
a surgically implanted pump that delivers the RNAi agent to the
liver.
[0371] Other modes of administration include epidural,
intracerebral, intracerebroventricular, nasal administration,
intraarterial, intracardiac, intraosseous infusion, intrathecal,
and intravitreal, and pulmonary. The mode of administration may be
chosen based upon whether local or systemic treatment is desired
and based upon the area to be treated. The route and site of
administration may be chosen to enhance targeting. In some
embodiments, the RNAi agent is administered to a subject in an
amount effective to inhibit TTR expression in a cell within the
subject. The amount effective to inhibit TTR expression in a cell
within a subject may be assessed using methods discussed above,
including methods that involve assessment of the inhibition of TTR
mRNA, TTR protein, or related variables, such as amyloid
deposits.
[0372] In some embodiments, the RNAi agent is administered to a
subject in a therapeutically or prophylactically effective
amount.
[0373] "Therapeutically effective amount," as used herein, is
intended to include the amount of an RNAi agent that, when
administered to a patient for treating a TTR associated disease, is
sufficient to effect treatment of the disease (e.g., by
diminishing, ameliorating or maintaining the existing disease or
one or more symptoms of disease). The "therapeutically effective
amount" may vary depending on the RNAi agent, how the agent is
administered, the disease and its severity and the history, age,
weight, family history, genetic makeup, stage of pathological
processes mediated by TTR expression, the types of preceding or
concomitant treatments, if any, and other individual
characteristics of the patient to be treated.
[0374] "Prophylactically effective amount," as used herein, is
intended to include the amount of an RNAi agent that, when
administered to a subject who does not yet experience or display
symptoms of a TTR-associated disease, but who may be predisposed to
the disease, is sufficient to prevent or ameliorate the disease or
one or more symptoms of the disease. Symptoms that may be
ameliorated include sensory neuropathy (e.g., paresthesia,
hypesthesia in distal limbs), autonomic neuropathy (e.g.,
gastrointestinal dysfunction, such as gastric ulcer, or orthostatic
hypotension), motor neuropathy, seizures, dementia, myelopathy,
polyneuropathy, carpal tunnel syndrome, autonomic insufficiency,
cardiomyopathy, vitreous opacities, renal insufficiency,
nephropathy, substantially reduced mBMI (modified Body Mass Index),
cranial nerve dysfunction, and corneal lattice dystrophy.
Ameliorating the disease includes slowing the course of the disease
or reducing the severity of later-developing disease. The
"prophylactically effective amount" may vary depending on the RNAi
agent, how the agent is administered, the degree of risk of
disease, and the history, age, weight, family history, genetic
makeup, the types of preceding or concomitant treatments, if any,
and other individual characteristics of the patient to be
treated.
[0375] A "therapeutically-effective amount" or "prophylacticaly
effective amount" also includes an amount of an RNAi agent that
produces some desired local or systemic effect at a reasonable
benefit/risk ratio applicable to any treatment. RNAi agents
employed in the methods of the present invention may be
administered in a sufficient amount to produce a reasonable
benefit/risk ratio applicable to such treatment.
[0376] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" also include an
amount that provides a benefit in the treatment, prevention, or
management of pathological processes or symptom(s) of pathological
processes mediated by TTR expression. Symptoms of TTR amyloidosis
include sensory neuropathy (e.g. paresthesia, hypesthesia in distal
limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction,
such as gastric ulcer, or orthostatic hypotension), motor
neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal
tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous
opacities, renal insufficiency, nephropathy, substantially reduced
mBMI (modified Body Mass Index), cranial nerve dysfunction, and
corneal lattice dystrophy.
[0377] The dose of an RNAi agent that is administered to a subject
may be tailored to balance the risks and benefits of a particular
dose, for example, to achieve a desired level of TTR gene
suppression (as assessed, e.g., based on TTR mRNA suppression, TTR
protein expression, or a reduction in an amyloid deposit, as
defined above) or a desired therapeutic or prophylactic effect,
while at the same time avoiding undesirable side effects.
[0378] In one embodiment, the RNAi agent is administered at a dose
of between about 0.25 mg/kg to about 50 mg/kg, e.g., between about
0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to about 1
mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between about
0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about 10
mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10
mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg,
between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to
about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
[0379] In some embodiments, the RNAi agent is administered at a
dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2
mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,
about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about
11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15
mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19
mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23
mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27
mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg,
about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg,
about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,
about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg,
about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg,
about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
[0380] In some embodiments, the RNAi agent is administered in two
or more doses. If desired to facilitate repeated or frequent
infusions, implantation of a delivery device, e.g., a pump,
semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular), or reservoir may be advisable. In
some embodiments, the number or amount of subsequent doses is
dependent on the achievement of a desired effect, e.g., the
suppression of a TTR gene, or the achievement of a therapeutic or
prophylactic effect, e.g., reducing an amyloid deposit or reducing
a symptom of a TTR-associated disease. In some embodiments, the
RNAi agent is administered according to a schedule. For example,
the RNAi agent may be administered twice per week, three times per
week, four times per week, or five times per week. In some
embodiments, the schedule involves regularly spaced
administrations, e.g., hourly, every four hours, every six hours,
every eight hours, every twelve hours, daily, every 2 days, every 3
days, every 4 days, every 5 days, weekly, biweekly, or monthly. In
other embodiments, the schedule involves closely spaced
administrations followed by a longer period of time during which
the agent is not administered. For example, the schedule may
involve an initial set of doses that are administered in a
relatively short period of time (e.g., about every 6 hours, about
every 12 hours, about every 24 hours, about every 48 hours, or
about every 72 hours) followed by a longer time period (e.g., about
1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, or about 8 weeks) during which the
RNAi agent is not administered. In one embodiment, the RNAi agent
is initially administered hourly and is later administered at a
longer interval (e.g., daily, weekly, biweekly, or monthly). In
another embodiment, the RNAi agent is initially administered daily
and is later administered at a longer interval (e.g., weekly,
biweekly, or monthly). In certain embodiments, the longer interval
increases over time or is determined based on the achievement of a
desired effect. In a specific embodiment, the RNAi agent is
administered once daily during a first week, followed by weekly
dosing starting on the eighth day of administration. In another
specific embodiment, the RNAi agent is administered every other day
during a first week followed by weekly dosing starting on the
eighth day of administration.
[0381] Any of these schedules may optionally be repeated for one or
more iterations. The number of iterations may depend on the
achievement of a desired effect, e.g., the suppression of a TTR
gene, retinol binding protein level, vitamin A level, and/or the
achievement of a therapeutic or prophylactic effect, e.g., reducing
an amyloid deposit or reducing a symptom of a TTR-associated
disease.
[0382] In some embodiments, the RNAi agent is administered with
other therapeutic agents or other therapeutic regimens. For
example, other agents or other therapeutic regimens suitable for
treating a TTR-associated disease may include a liver transplant,
which can reduce mutant TTR levels in the body; Tafamidis
(Vyndagel), which kinetically stabilizes the TTR tetramer
preventing tetramer dissociation required for TTR amyloidogenesis;
and diuretics, which may be employed, for example, to reduce edema
in TTR amyloidosis with cardiac involvement.
[0383] In one embodiment, a subject is administered an initial dose
and one or more maintenance doses of an RNAi agent. The maintenance
dose or doses can be the same or lower than the initial dose, e.g.,
one-half of the initial dose. A maintenance regimen can include
treating the subject with a dose or doses ranging from 0.01 g to 15
mg/kg of body weight per day, e.g., 10 mg/kg, 1 mg/kg, 0.1 mg/kg,
0.01 mg/kg, 0.001 mg/kg, or 0.00001 mg/kg of bodyweight per day.
The maintenance doses are, for example, administered no more than
once every 2 days, once every 5 days, once every 7 days, once every
10 days, once every 14 days, once every 21 days, or once every 30
days. Further, the treatment regimen may last for a period of time
which will vary depending upon the nature of the particular
disease, its severity and the overall condition of the patient. In
certain embodiments the dosage may be delivered no more than once
per day, e.g., no more than once per 24, 36, 48, or more hours,
e.g., no more than once every 5 or 8 days. Following treatment, the
patient can be monitored for changes in his/her condition. The
dosage of the RNAi agent may either be increased in the event the
patient does not respond significantly to current dosage levels, or
the dose may be decreased if an alleviation of the symptoms of the
disease state is observed, if the disease state has been ablated,
or if undesired side-effects are observed.
VI. Kits
[0384] The present invention also provides kits for performing any
of the methods of the invention. Such kits include one or more RNAi
agent(s) and instructions for use, e.g., instructions for
inhibiting expression of a TTR in a cell by contacting the cell
with the RNAi agent(s) in an amount effective to inhibit expression
of the TTR. The kits may optionally further comprise means for
contacting the cell with the RNAi agent (e.g., an injection
device), or means for measuring the inhibition of TTR (e.g., means
for measuring the inhibition of TTR mRNA or TTR protein). Such
means for measuring the inhibition of TTR may comprise a means for
obtaining a sample from a subject, such as, e.g., a plasma sample.
The kits of the invention may optionally further comprise means for
administering the RNAi agent(s) to a subject or means for
determining the therapeutically effective or prophylactically
effective amount.
[0385] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated herein by
reference.
Examples
Example 1: Inhibition of TTR with TTR-GalNAc Conjugates
[0386] A single dose of the TTR RNAi agent AD-43527 was
administered to mice subcutaneously and TTR mRNA levels were
determined 72 hours post administration.
[0387] The mouse/rat cross-reactive GalNAc-conjugate, AD-43527, was
chosen for in vivo evaluation in WT C57BL/6 mice for silencing of
TTR mRNA in liver. The sequence of each strand of AD-43527 is shown
below.
[0388] Strand: s=sense; as=antisense
TABLE-US-00002 Oligo Duplex # Strand # Sequence 5' to 3' AD-43527 s
A- AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfL96 (SEQ ID 89592 NO: 8) as A-
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu (SEQ 83989 ID NO: 9 L96 =
GalNAc3; lowercase nts (a, u, g, c) are 2'-O-methyl nucleotides, Nf
(i.e., Af) is a 2'-fluoro nucleotide
The ligand used was GalNAc.sub.3:
##STR00011##
This GalNAc3 ligand was conjugated to the 3'-end of the sense
strand using the linker and tether as shown below:
##STR00012##
The structure of the resulting GalNAc.sub.3 conjugated sense strand
is shown in the following schematic:
##STR00013##
Additional RNAi agents that target TTR and have the following
sequences and modifications were synthesized and assayed.
Mouse/Rat Cross Reactive TTR RNAi Agents
TABLE-US-00003 [0389] Duplex Sense strand 5'-3' Antisense strand
5'-3' AD- AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfQ11L96
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 43528 (SEQ ID NO: 10) (SEQ ID
NO: 11)
Human/cyno cross reactive TTR RNAi agents; parent duplex is
AD-18328 [having a sense strand 5'-3' sequence of
GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 12) and antisense strand 5' to
3' sequence of AUGGAAuACUCUUGGUuACdTdT (SEQ ID NO: 13) with the
following modifications: alternating 2'F/2'OMe w/2 PS on AS.
TABLE-US-00004 Duplex Sense strand 5'-3' Antisense strand 5'-3' AD-
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfaUfL96
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 45163 (SEQ ID NO: 14) (SEQ ID
NO: 16) AD- AfuGfuAfaCfcAfaGfaGfuAfuUfcCfaUfQ11L96
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 45164 (SEQ ID NO: 15) (SEQ ID
NO: 17)
L96=GalNAc.sub.3; lowercase nts (a,u,g,c) are 2'-O-methyl
nucleotides, Nf (i.e., Af) is a 2'-fluoro nucleotide; Q11 is
cholesterol; s is phosphorothioate.
[0390] AD-43527 was administered to female C57BL/6 mice (6-10
weeks, 5 per group) via subcutaneous injection at a dose volume of
10 .mu.l/g at a dose of 30, 15, 7.5, 3.5, 1.75 or 0.5 mg/kg of
AD-43527. Control animals received PBS by subcutaneous injection at
the same dose volume.
[0391] After approximately seventy two hours, mice were
anesthetized with 200 .mu.l of ketamine, and then exsanguinated by
severing the right caudal artery. Liver tissue was collected,
flash-frozen and stored at -80.degree. C. until processing.
[0392] Efficacy of treatment was evaluated by measurement of TTR
mRNA in the liver at 72 hours post-dose. TTR liver mRNA levels were
assayed utilizing the Branched DNA assays--QuantiGene 1.0
(Panomics). Briefly, mouse liver samples were ground and tissue
lysates were prepared. Liver lysis mixture (a mixture of 1 volume
of lysis mixture, 2 volume of nuclease-free water and 10 .mu.l of
Proteinase-K/ml for a final concentration of 20 mg/ml) was
incubated at 65.degree. C. for 35 minutes. 5 .mu.l of liver lysate
and 95 .mu.l of working probe set (TTR probe for gene target and
GAPDH for endogenous control) were added into the Capture Plate.
Capture Plates were incubated at 53.degree. C. 1.degree. C. (aprx.
16-20 hrs). The next day, the Capture Plates were washed 3 times
with 1.times. Wash Buffer (nuclease-free water, Buffer Component 1
and Wash Buffer Component 2), then dried by centrifuging for 1
minute at 240 g. 100 .mu.l of Amplifier Probe mix per well was
added into the Capture Plate, which was sealed with aluminum foil
and incubated for 1 hour at 46.degree. C. 1.degree. C. Following a
1 hour incubation, the wash step was repeated, then 100p of Label
Probe mix per well was added. Capture plates were incubated at
46.degree. C. 1.degree. C. for 1 hour. The plates were then washed
with 1.times. Wash Buffer, dried and 100 .mu.l substrate per well
was added into the Capture Plates. Capture Plates were incubated
for 30 minutes at 46.degree. C. followed by incubation for 30
minutes at room temperature. Plates were read using the SpectraMax
Luminometer following incubation. bDNA data were analyzed by
subtracting the average background from each duplicate sample,
averaging the resultant duplicate GAPDH (control probe) and TTR
(experimental probe) values, and then computing the ratio:
(experimental probe-background)/(control probe-background). The
average TTR mRNA level was calculated for each group and normalized
to the PBS group average to give relative TTR mRNA as a % of the
PBS control group.
[0393] The results are shown in FIG. 1. The GalNAc conjugated RNAi
agent targeting TTR had an ED.sub.50 of approximately 5 mg/kg for
TTR mRNA knockdown. These results demonstrate that GalNAc
conjugated RNAi agents that target TTR are effective at inhibiting
expression of TTR mRNA.
Example 2: Inhibition of TTR with TTR-GalNAc Conjugates is
Durable
[0394] Mice were administered a subcutaneous dose (either 7.5 or
30.0 mg/kg) of AD-43527, a GalNAc conjugated RNAi agent that
targets TTR. The TTR mRNA levels in the liver were evaluated at 1,
3, 5, 7, 10, 13, 15, 19, 26, 33, and 41 days post treatment using
the method described in Example 1.
[0395] The results are shown in FIG. 2. At day 19, administration
of 30.0 mg/kg GalNAc conjugated RNAi agents still showed about 50%
silencing. Full recovery of expression occurred at day 41.
[0396] These results demonstrated that the inhibition provided by
GalNAc conjugated siRNA targeting TTR is durable, lasting up to 3,
5, 7, 10, 13, 15, 19, 26 or 33 days post treatment.
Example 3. RNA Synthesis and Duplex Annealing
1. Oligonucleotide Synthesis
[0397] Oligonucleotides were synthesized on an AKTAoligopilot
synthesizer or an ABI 394 synthsizer. Commercially available
controlled pore glass solid support (dT-CPG, 500 .ANG., Prime
Synthesis) and RNA phosphoramidites with standard protecting
groups, 5'-O-dimethoxytrityl
N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--N,N'-diisopropyl-2-cya-
noethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies)
were used for the oligonucleotide synthesis unless otherwise
specified. The 2'-F phosphoramidites,
5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-O--N,N'-diisopropyl-2-
-cyanoethyl-phosphoramidite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O--N,N'-diisopropyl-2-cyanoethyl-
-phosphoramidite were purchased from (Promega). All
phosphoramidites were used at a concentration of 0.2M in
acetonitrile (CH.sub.3CN) except for guanosine which was used at
0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of
16 minutes was used. The activator was 5-ethyl thiotetrazole
(0.75M, American International Chemicals), for the PO-oxidation
Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in
2,6-lutidine/ACN (1:1 v/v) was used.
[0398] Ligand conjugated strands were synthesized using a solid
support containing the corresponding ligand. For example, the
introduction of a carbohydrate moiety/ligand (for e.g., GalNAc) at
the 3'-end of a sequence was achieved by starting the synthesis
with the corresponding carbohydrate solid support. Similarly a
cholesterol moiety at the 3'-end was introduced by starting the
synthesis on the cholesterol support. In general, the ligand moiety
was tethered to trans-4-hydroxyprolinol via a tether of choice as
described in the previous examples to obtain a
hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety
was then coupled to a solid support via a succinate linker or was
converted to phosphoramidite via standard phosphitylation
conditions to obtain the desired carbohydrate conjugate building
blocks. Fluorophore labeled siRNAs were synthesized from the
corresponding phosphoramidite or solid support, purchased from
Biosearch Technologies. The oleyl lithocholic (GalNAc).sub.3
polymer support made in house at a loading of 38.6 .mu.mol/gram.
The Mannose (Man).sub.3 polymer support was also made in house at a
loading of 42.0 .mu.mol/gram.
[0399] Conjugation of the ligand of choice at the desired position,
for example at the 5'-end of the sequence, was achieved by coupling
of the corresponding phosphoramidite to the growing chain under
standard phosphoramidite coupling conditions unless otherwise
specified. An extended 15 minute coupling of 0.1M solution of
phosphoramidite in anhydrous CH.sub.3CN in the presence of
5-(ethylthio)-1H-tetrazole activator to a solid bound
oligonucleotide. Oxidation of the internucleotide phosphite to the
phosphate was carried out using standard iodine-water as reported
in Beaucage, S. L. (2008) Solid-phase synthesis of siRNA
oligonucleotides. Curr. Opin. Drug Discov. Devel., 11, 203-216;
Mueller, S., Wolf, J. and Ivanov, S. A. (2004) Current Strategies
for the Synthesis of RNA. Curr. Org. Synth., 1, 293-307; Xia, J.,
Noronha, A., Toudjarska, I., Li, F., Akinc, A., Braich, R.,
Frank-Kamenetsky, M., Rajeev, K. G., Egli, M. and Manoharan, M.
(2006) Gene Silencing Activity of siRNAs with a Ribo-difluorotoluyl
Nucleotide. ACS Chem. Biol., 1, 176-183 or by treatment with
tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with a 10
minute oxidation wait time conjugated oligonucleotide.
Phosphorothioate was introduced by the oxidation of phosphite to
phosphorothioate by using a sulfur transfer reagent such as DDTT
(purchased from AM Chemicals), PADS and or Beaucage reagent The
cholesterol phosphoramidite was synthesized in house, and used at a
concentration of 0.1 M in dichloromethane. Coupling time for the
cholesterol phosphoramidite was 16 minutes.
2. Deprotection-I (Nucleobase Deprotection)
[0400] After completion of synthesis, the support was transferred
to a 100 ml glass bottle (VWR). The oligonucleotide was cleaved
from the support with simultaneous deprotection of base and
phosphate groups with 80 mL of a mixture of ethanolic ammonia
[ammonia:ethanol (3:1)] for 6.5 h at 55.degree. C. The bottle was
cooled briefly on ice and then the ethanolic ammonia mixture was
filtered into a new 250 ml bottle. The CPG was washed with
2.times.40 mL portions of ethanol/water (1:1 v/v). The volume of
the mixture was then reduced to 30 ml by roto-vap. The mixture was
then frozen on dry ice and dried under vacuum on a speed vac.
3. Deprotection-I (Removal of 2' TBDMS Group)
[0401] The dried residue was resuspended in 26 ml of triethylamine,
triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO
(3:4:6) and heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction was then quenched with 50 ml of 20 mM sodium acetate and
pH adjusted to 6.5, and stored in freezer until purification.
4. Analysis
[0402] The oligonucleotides were analyzed by high-performance
liquid chromatography (HPLC) prior to purification and selection of
buffer and column depends on nature of the sequence and or
conjugated ligand.
5. HPLC Purification
[0403] The ligand conjugated oligonucleotides were purified by
reverse phase preparative HPLC. The unconjugated oligonucleotides
were purified by anion-exchange HPLC on a TSK gel column packed in
house. The buffers were 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN, 1M NaBr (buffer B). Fractions containing full-length
oligonucleotides were pooled, desalted, and lyophilized.
Approximately 0.15 OD of desalted oligonucleotidess were diluted in
water to 150 .mu.l and then pipetted in special vials for CGE and
LC/MS analysis. Compounds were finally analyzed by LC-ESMS and
CGE.
6. RNAi Agent Preparation
[0404] For the preparation of an RNAi agent, equimolar amounts of
sense and antisense strand were heated in 1.times.PBS at 95.degree.
C. for 5 minutes and slowly cooled to room temperature. The
integrity of the duplex was confirmed by HPLC analysis. Table 1
below reflects the RNAi agents which target human or rodent TTR
mRNA.
TABLE-US-00005 TABLE 1 RNAi Agents and Results of In Vitro
Screening % of mRNA remained SEQ SEQ conc. of siRNA Duplex ID ID
0.1 0.01 IC50 ID S ID NO: Sense strand (S) AS ID NO: Antisense
strand (AS) 1 nM nM nM (nM) D1000 S1000 18
AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasu AS1000 1110
AfUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.1 0.47 0.006 D1001
S1001 19 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1001 1111
aUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.10 0.49 0.0065 D1002
S1002 20 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1002 1112
aUfgGfAfAfuAfcUfcuuGfgsUfuAfcAfusGfsa 0.04 0.10 0.46 0.0068 D1003
S1003 21 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1003 1113
aUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa 0.05 0.12 0.56 0.0073 D1004
S1004 22 aUGuaACccAGagUAuuCCasu AS1004 1114
AUggAAuaCUcuUGguUAcaUsGsa 0.07 0.13 0.44 0.008 D1005 S1005 23
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1005 1115
aUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa 0.06 0.11 0.53 0.0093 D1006
S1006 24 AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1006 1116
aUfgGfaAfuAfcUfcuuGfGfuuAfcAfusGfsa 0.05 0.16 0.55 0.0095 D1007
S1007 25 AfuGfuAfAfCfcAfAfGfaGfuAfuUfcCfasUf AS1007 1117
aUfgGfaAfuAfcUfcuuGfguuAfcAfusGfsa 0.05 0.14 0.48 0.0098 D1008
S1008 26 auguaaccaadGadGudAudAcdGasu AS1008 1118
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.07 0.11 0.33 0.010 D1009
S1009 27 UfgGfGfAfuUfuCfAfUfgUfaAfcCfAfAfgsAf AS1009 1119
uCfuugGfuUfaCfaugAfaAfuccCfasUfsc 0.03 0.14 0.56 0.0101 D1010 S1010
28 UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf AS1010 1120
uCfuUfgGfuUfaCfaugAfaAfUfCfcCfasUfsc 0.03 0.14 0.65 0.0101 D1011
S1011 29 aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1011 1121
aUfgGfaAfuAfcUfcuuGfGfuuAfcaUfsgsa 0.06 0.10 0.55 0.011 D1012 S1012
30 UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAf AS1012 1122
uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.13 0.54 0.0114 D1013
S1013 31 auguaaccaadGadGudAudAcdGasu AS1013 1123
aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa 0.11 0.19 0.49 0.011 D1014
S1014 32 AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf AS1014 1124
aUfgGfaAfuAfcUfcuuGfgUfUfAfcAfusGfsa 0.04 0.16 0.59 0.013 D1015
S1015 33 AfuguAfaccAfaGfdAGfdTAdTudCcdAsu AS1015 1125
dAUdGgdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa 0.07 0.15 0.51 0.013 D1016
S1016 34 auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1016 1126
aUfgGfaAfuAfcUfcuuGfgUfuAfcAfUfsGfsa 0.05 0.14 0.64 0.013 D1017
S1017 35 UfGfggAfuUfuCfAfUfgUfAfAfcCfaAfgsAf AS1017 1127
uCfuUfgGfuuaCfaugAfaAfuCfCfcasUfsc 0.09 0.41 0.74 0.0133 D1018
S1018 36 AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1018 1128
aUfgGfaAfuAfcUfcuuGfgUfuAfCfAfusGfsa 0.03 0.14 0.61 0.014 D1019
S1019 37 AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf AS1019 1129
aUfgGfaAfuAfcUfcuuGfGfUfuAfcAfusGfsa 0.02 0.2 0.7 0.014 D1020 S1020
38 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1020 1130
asUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.04 0.16 0.67 0.0156 D1021
S1021 39 aUfguAfAfccAfAfgagUfaUfuCfcasUf AS1021 1131
aUfGfgAfaUfaCfUfCfuuGfGfuuAfCfaUfsgsa 0.11 0.24 0.64 0.016 D1022
S1022 40 dTdGggdAdTuudCdAugdTdAacdCdAagsdA AS1022 1132
udCdTugdGdTuadCdAugdAdAaudCdCcasdTsc 0.08 0.27 0.64 0.0161 D1023
S1023 41 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1023 1133
aUfgsGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.19 0.63 0.0163 D1024
S1024 42 UfgGfgAfuUfuCfAfUfguaAfcCfaAfgsAf AS1024 1134
uCfuUfgGfuUfAfCfaugAfaAfuCfcCfasUfsc 0.05 0.25 0.69 0.0164 D1025
S1025 43 UfgGfgAfuUfuCfAfUfgUfAfAfcCfaAfgsAf AS1025 1135
uCfuUfgGfuuaCfaugAfaAfuCfcCfasUfsc 0.04 0.18 0.75 0.0166 D1026
S1026 44 UfgGfgAfuUfuCfAfUfgUfaAfcCfaAfgsAf AS1026 1136
uCfuUfgGfuUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.19 0.66 0.0178 D1027
S1027 45 UfgGfgAfuUfuCfAfUfgUfaAfccaAfgsAf AS1027 1137
uCfuUfGfGfuUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.19 0.69 0.018 D1028
S1028 46 dAdTgudAdAccdAdAgadGdTaudTdCcasdT AS1028 1138
adTdGgadAdTacdTdCuudGdGuudAdCausdGsa 0.15 0.29 0.72 0.018 D1029
S1029 47 AdTGdTAdACdCAdAGdAGdTAdTUdCCdAsU AS1029 1139
dAUdGGdAAdTAdCUdCUdTGdGUdTAdCAdTsGsdA 0.1 0.27 0.61 0.018 D1030
S1030 48 UfgGfGfAfuuuCfAfUfgUfaAfcCfaAfgsAf AS1030 1140
uCfuUfgGfuUfaCfaugAfAfAfuccCfasUfsc 0.04 0.21 0.64 0.0187 D1031
S1031 49 AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu AS1031 1141
AfUfGfGfAfAfuAfCfUfCfUfuGfGfuuAfcAfusGfsa 0.06 0.15 0.62 0.019
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UfgUfcUfuGfcAfAfGfcAfaagCfaCfgUfL96 AS1986 2096
aCfgUfgCfUfUfuGfcuuGfcAfaGfaCfasAfsu D1987 S1987 1005
UfgUfcUfuGfcAfAfGfcAfaAfGfCfaCfgUfL96 AS1987 2097
aCfgUfgcuUfuGfcuuGfcAfaGfaCfasAfsu D1988 S1988 1006
GfuCfuUfgCfaAfGfCfaAfagcAfcGfuAfL96 AS1988 2098
uAfcGfuGfCfUfuUfgcuUfgCfaAfgAfcsAfsa D1989 S1989 1007
GfuCfuUfgCfaAfGfCfaAfaGfCfAfcGfuAfL96 AS1989 2099
uAfcGfugcUfuUfgcuUfgCfaAfgAfcsAfsa D1990 S1990 1008
UfcUfuGfcAfaGfCfAfaAfgcaCfgUfaUfL96 AS1990 2100
aUfaCfgUfGfCfuUfugcUfuGfcAfaGfasCfsa D1991 S1991 1009
UfcUfuGfcAfaGfCfAfaAfgCfAfCfgUfaUfL96 AS1991 2101
aUfaCfgugCfuUfugcUfuGfcAfaGfasCfsa D1992 S1992 1010
CfuUfgCfaAfgCfAfAfaGfcacGfuAfuUfL96 AS1992 2102
aAfuAfcGfUfGfcUfuugCfuUfgCfaAfgsAfsc D1993 S1993 1011
CfuUfgCfaAfgCfAfAfaGfcAfCfGfuAfuUfL96 AS1993 2103
aAfuAfcguGfcUfuugCfuUfgCfaAfgsAfsc D1994 S1994 1012
UfuGfcAfaGfcAfAfAfgCfacgUfaUfuAfL96 AS1994 2104
uAfaUfaCfGfUfgCfuuuGfcUfuGfcAfasGfsa D1995 S1995 1013
UfuGfcAfaGfcAfAfAfgCfaCfGfUfaUfuAfL96 AS1995 2105
uAfaUfacgUfgCfuuuGfcUfuGfcAfasGfsa D1996 S1996 1014
UfgCfaAfgCfaAfAfGfcAfcguAfuUfaAfL96 AS1996 2106
uUfaAfuAfCfGfuGfcuuUfgCfuUfgCfasAfsg D1997 S1997 1015
UfgCfaAfgCfaAfAfGfcAfcGfUfAfuUfaAfL96 AS1997 2107
uUfaAfuacGfuGfcuuUfgCfuUfgCfasAfsg D1998 S1998 1016
GfcAfaGfcAfaAfGfCfaCfguaUfuAfaAfL96 AS1998 2108
uUfuAfaUfAfCfgUfgcuUfuGfcUfuGfcsAfsa D1999 S1999 1017
GfcAfaGfcAfaAfGfCfaCfgUfAfUfuAfaAfL96 AS1999 2109
uUfuAfauaCfgUfgcuUfuGfcUfuGfcsAfsa D2000 S2000 1018
CfaAfgCfaAfaGfCfAfcGfuauUfaAfaUfL96 AS2000 2110
aUfuUfaAfUfAfcGfugcUfuUfgCfuUfgsCfsa
D2001 S2001 1019 CfaAfgCfaAfaGfCfAfcGfuAfUfUfaAfaUfL96 AS2001 2111
aUfuUfaauAfcGfugcUfuUfgCfuUfgsCfsa D2002 S2002 1020
AfaGfcAfaAfgCfAfCfgUfauuAfaAfuAfL96 AS2002 2112
uAfuUfuAfAfUfaCfgugCfuUfuGfcUfusGfsc D2003 S2003 1021
AfaGfcAfaAfgCfAfCfgUfaUfUfAfaAfuAfL96 AS2003 2113
uAfuUfuaaUfaCfgugCfuUfuGfcUfusGfsc D2004 S2004 1022
AfgCfaAfaGfcAfCfGfuAfuuaAfaUfaUfL96 AS2004 2114
aUfaUfuUfAfAfuAfcguGfcUfuUfgCfusUfsg D2005 S2005 1023
AfgCfaAfaGfcAfCfGfuAfuUfAfAfaUfaUfL96 AS2005 2115
aUfaUfuuaAfuAfcguGfcUfuUfgCfusUfsg D2006 S2006 1024
GfcAfaAfgCfaCfGfUfaUfuaaAfuAfuGfL96 AS2006 2116
cAfuAfuUfUfAfaUfacgUfgCfuUfuGfcsUfsu D2007 S2007 1025
GfcAfaAfgCfaCfGfUfaUfuAfAfAfuAfuGfL96 AS2007 2117
cAfuAfuuuAfaUfacgUfgCfuUfuGfcsUfsu D2008 S2008 1026
CfaAfaGfcAfcGfUfAfuUfaaaUfaUfgAfL96 AS2008 2118
uCfaUfaUfUfUfaAfuacGfuGfcUfuUfgsCfsu D2009 S2009 1027
CfaAfaGfcAfcGfUfAfuUfaAfAfUfaUfgAfL96 AS2009 2119
uCfaUfauuUfaAfuacGfuGfcUfuUfgsCfsu D2010 S2010 1028
AfaAfgCfaCfgUfAfUfuAfaauAfuGfaUfL96 AS2010 2120
aUfcAfuAfUfUfuAfauaCfgUfgCfuUfusGfsc D2011 S2011 1029
AfaAfgCfaCfgUfAfUfuAfaAfUfAfuGfaUfL96 AS2011 2121
aUfcAfuauUfuAfauaCfgUfgCfuUfusGfsc D2012 S2012 1030
AfaGfcAfcGfuAfUfUfaAfauaUfgAfuCfL96 AS2012 2122
gAfuCfaUfAfUfuUfaauAfcGfuGfcUfusUfsg D2013 S2013 1031
AfaGfcAfcGfuAfUfUfaAfaUfAfUfgAfuCfL96 AS2013 2123
gAfuCfauaUfuUfaauAfcGfuGfcUfusUfsg D2014 S2014 1032
AfgCfaCfgUfaUfUfAfaAfuauGfaUfcUfL96 AS2014 2124
aGfaUfcAfUfAfuUfuaaUfaCfgUfgCfusUfsu D2015 S2015 1033
AfgCfaCfgUfaUfUfAfaAfuAfUfGfaUfcUfL96 AS2015 2125
aGfaUfcauAfuUfuaaUfaCfgUfgCfusUfsu D2016 S2016 1034
GfcAfcGfuAfuUfAfAfaUfaugAfuCfuGfL96 AS2016 2126
cAfgAfuCfAfUfaUfuuaAfuAfcGfuGfcsUfsu D2017 S2017 1035
GfcAfcGfuAfuUfAfAfaUfaUfGfAfuCfuGfL96 AS2017 2127
cAfgAfucaUfaUfuuaAfuAfcGfuGfcsUfsu D2018 S2018 1036
CfaCfgUfaUfuAfAfAfuAfugaUfcUfgCfL96 AS2018 2128
gCfaGfaUfCfAfuAfuuuAfaUfaCfgUfgsCfsu D2019 S2019 1037
CfaCfgUfaUfuAfAfAfuAfuGfAfUfcUfgCfL96 AS2019 2129
gCfaGfaucAfuAfuuuAfaUfaCfgUfgsCfsu D2020 S2020 1038
AfcGfuAfuUfaAfAfUfaUfgauCfuGfcAfL96 AS2020 2130
uGfcAfgAfUfCfaUfauuUfaAfuAfcGfusGfsc D2021 S2021 1039
AfcGfuAfuUfaAfAfUfaUfgAfUfCfuGfcAfL96 AS2021 2131
uGfcAfgauCfaUfauuUfaAfuAfcGfusGfsc D2022 S2022 1040
CfgUfaUfuAfaAfUfAfuGfaucUfgCfaGfL96 AS2022 2132
cUfgCfaGfAfUfcAfuauUfuAfaUfaCfgsUfsg D2023 S2023 1041
CfgUfaUfuAfaAfUfAfuGfaUfCfUfgCfaGfL96 AS2023 2133
cUfgCfagaUfcAfuauUfuAfaUfaCfgsUfsg D2024 S2024 1042
GfuAfuUfaAfaUfAfUfgAfucuGfcAfgCfL96 AS2024 2134
gCfuGfcAfGfAfuCfauaUfuUfaAfuAfcsGfsu D2025 S2025 1043
GfuAfuUfaAfaUfAfUfgAfuCfUfGfcAfgCfL96 AS2025 2135
gCfuGfcagAfuCfauaUfuUfaAfuAfcsGfsu D2026 S2026 1044
UfaUfuAfaAfuAfUfGfaUfcugCfaGfcCfL96 AS2026 2136
gGfcUfgCfAfGfaUfcauAfuUfuAfaUfasCfsg D2027 S2027 1045
UfaUfuAfaAfuAfUfGfaUfcUfGfCfaGfcCfL96 AS2027 2137
gGfcUfgcaGfaUfcauAfuUfuAfaUfasCfsg D2028 S2028 1046
AfuUfaAfaUfaUfGfAfuCfugcAfgCfcAfL96 AS2028 2138
uGfgCfuGfCfAfgAfucaUfaUfuUfaAfusAfsc D2029 S2029 1047
AfuUfaAfaUfaUfGfAfuCfuGfCfAfgCfcAfL96 AS2029 2139
uGfgCfugcAfgAfucaUfaUfuUfaAfusAfsc D2030 S2030 1048
UfuAfaAfuAfuGfAfUfcUfgcaGfcCfaUfL96 AS2030 2140
aUfgGfcUfGfCfaGfaucAfuAfuUfuAfasUfsa D2031 S2031 1049
UfuAfaAfuAfuGfAfUfcUfgCfAfGfcCfaUfL96 AS2031 2141
aUfgGfcugCfaGfaucAfuAfuUfuAfasUfsa D2032 S2032 1050
UfaAfaUfaUfgAfUfCfuGfcagCfcAfuUfL96 AS2032 2142
aAfuGfgCfUfGfcAfgauCfaUfaUfuUfasAfsu D2033 S2033 1051
UfaAfaUfaUfgAfUfCfuGfcAfGfCfcAfuUfL96 AS2033 2143
aAfuGfgcuGfcAfgauCfaUfaUfuUfasAfsu D2034 S2034 1052
AfaAfuAfuGfaUfCfUfgCfagcCfaUfuAfL96 AS2034 2144
uAfaUfgGfCfUfgCfagaUfcAfuAfuUfusAfsa D2035 S2035 1053
AfaAfuAfuGfaUfCfUfgCfaGfCfCfaUfuAfL96 AS2035 2145
uAfaUfggcUfgCfagaUfcAfuAfuUfusAfsa D2036 S2036 1054
AfaUfaUfgAfuCfUfGfcAfgccAfuUfaAfL96 AS2036 2146
uUfaAfuGfGfCfuGfcagAfuCfaUfaUfusUfsa D2037 S2037 1055
AfaUfaUfgAfuCfUfGfcAfgCfCfAfuUfaAfL96 AS2037 2147
uUfaAfuggCfuGfcagAfuCfaUfaUfusUfsa D2038 S2038 1056
AfuAfuGfaUfcUfGfCfaGfccaUfuAfaAfL96 AS2038 2148
uUfuAfaUfGfGfcUfgcaGfaUfcAfuAfusUfsu D2039 S2039 1057
AfuAfuGfaUfcUfGfCfaGfcCfAfUfuAfaAfL96 AS2039 2149
uUfuAfaugGfcUfgcaGfaUfcAfuAfusUfsu D2040 S2040 1058
UfaUfgAfuCfuGfCfAfgCfcauUfaAfaAfL96 AS2040 2150
uUfuUfaAfUfGfgCfugcAfgAfuCfaUfasUfsu D2041 S2041 1059
UfaUfgAfuCfuGfCfAfgCfcAfUfUfaAfaAfL96 AS2041 2151
uUfuUfaauGfgCfugcAfgAfuCfaUfasUfsu D2042 S2042 1060
AfuGfaUfcUfgCfAfGfcCfauuAfaAfaAfL96 AS2042 2152
uUfuUfuAfAfUfgGfcugCfaGfaUfcAfusAfsu D2043 S2043 1061
AfuGfaUfcUfgCfAfGfcCfaUfUfAfaAfaAfL96 AS2043 2153
uUfuUfuaaUfgGfcugCfaGfaUfcAfusAfsu D2044 S2044 1062
UfgAfuCfuGfcAfGfCfcAfuuaAfaAfaGfL96 AS2044 2154
cUfuUfuUfAfAfuGfgcuGfcAfgAfuCfasUfsa D2045 S2045 1063
UfgAfuCfuGfcAfGfCfcAfuUfAfAfaAfaGfL96 AS2045 2155
cUfuUfuuaAfuGfgcuGfcAfgAfuCfasUfsa D2046 S2046 1064
GfaUfcUfgCfaGfCfCfaUfuaaAfaAfgAfL96 AS2046 2156
uCfuUfuUfUfAfaUfggcUfgCfaGfaUfcsAfsu D2047 S2047 1065
GfaUfcUfgCfaGfCfCfaUfuAfAfAfaAfgAfL96 AS2047 2157
uCfuUfuuuAfaUfggcUfgCfaGfaUfcsAfsu D2048 S2048 1066
AfuCfuGfcAfgCfCfAfuUfaaaAfaGfaCfL96 AS2048 2158
gUfcUfuUfUfUfaAfuggCfuGfcAfgAfusCfsa D2049 S2049 1067
AfuCfuGfcAfgCfCfAfuUfaAfAfAfaGfaCfL96 AS2049 2159
gUfcUfuuuUfaAfuggCfuGfcAfgAfus Cfsa D2050 S2050 1068
UfcUfgCfaGfcCfAfUfuAfaaaAfgAfcAfL96 AS2050 2160
uGfuCfuUfUfUfuAfaugGfcUfgCfaGfasUfsc D2051 S2051 1069
UfcUfgCfaGfcCfAfUfuAfaAfAfAfgAfcAfL96 AS2051 2161
uGfuCfuuuUfuAfaugGfcUfgCfaGfasUfsc D2052 S2052 1070
CfuGfcAfgCfcAfUfUfaAfaaaGfaCfaCfL96 AS2052 2162
gUfgUfcUfUfUfuUfaauGfgCfuGfcAfgsAfsu D2053 S2053 1071
CfuGfcAfgCfcAfUfUfaAfaAfAfGfaCfaCfL96 AS2053 2163
gUfgUfcuuUfuUfaauGfgCfuGfcAfgsAfsu D2054 S2054 1072
UfgCfaGfcCfaUfUfAfaAfaagAfcAfcAfL96 AS2054 2164
uGfuGfuCfUfUfuUfuaaUfgGfcUfgCfasGfsa D2055 S2055 1073
UfgCfaGfcCfaUfUfAfaAfaAfGfAfcAfcAfL96 AS2055 2165
uGfuGfucuUfuUfuaaUfgGfcUfgCfasGfsa D2056 S2056 1074
GfcAfgCfcAfuUfAfAfaAfagaCfaCfaUfL96 AS2056 2166
aUfgUfgUfCfUfuUfuuaAfuGfgCfuGfcsAfsg D2057 S2057 1075
GfcAfgCfcAfuUfAfAfaAfaGfAfCfaCfaUfL96 AS2057 2167
aUfgUfgucUfuUfuuaAfuGfgCfuGfcsAfsg D2058 S2058 1076
CfaGfcCfaUfuAfAfAfaAfgacAfcAfuUfL96 AS2058 2168
aAfuGfuGfUfCfuUfuuuAfaUfgGfcUfgsCfsa D2059 S2059 1077
CfaGfcCfaUfuAfAfAfaAfgAfCfAfcAfuUfL96 AS2059 2169
aAfuGfuguCfuUfuuuAfaUfgGfcUfgsCfsa D2060 S2060 1078
AfgCfcAfuUfaAfAfAfaGfacaCfaUfuCfL96 AS2060 2170
gAfaUfgUfGfUfcUfuuuUfaAfuGfgCfusGfsc D2061 S2061 1079
AfgCfcAfuUfaAfAfAfaGfaCfAfCfaUfuCfL96 AS2061 2171
gAfaUfgugUfcUfuuuUfaAfuGfgCfusGfsc D2062 S2062 1080
GfcCfaUfuAfaAfAfAfgAfcacAfuUfcUfL96 AS2062 2172
aGfaAfuGfUfGfuCfuuuUfuAfaUfgGfcsUfsg D2063 S2063 1081
GfcCfaUfuAfaAfAfAfgAfcAfCfAfuUfcUfL96 AS2063 2173
aGfaAfuguGfuCfuuuUfuAfaUfgGfcsUfsg D2064 S2064 1082
CfcAfuUfaAfaAfAfGfaCfacaUfuCfuGfL96 AS2064 2174
cAfgAfaUfGfUfgUfcuuUfuUfaAfuGfgsCfsu D2065 S2065 1083
CfcAfuUfaAfaAfAfGfaCfaCfAfUfuCfuGfL96 AS2065 2175
cAfgAfaugUfgUfcuuUfuUfaAfuGfgsCfsu D2066 S2066 1084
CfaUfuAfaAfaAfGfAfcAfcauUfcUfgUfL96 AS2066 2176
aCfaGfaAfUfGfuGfucuUfuUfuAfaUfgsGfsc D2067 S2067 1085
CfaUfuAfaAfaAfGfAfcAfcAfUfUfcUfgUfL96 AS2067 2177
aCfaGfaauGfuGfucuUfuUfuAfaUfgsGfsc D2068 S2068 1086
AfuUfaAfaAfaGfAfCfaCfauuCfuGfuAfL96 AS2068 2178
uAfcAfgAfAfUfgUfgucUfuUfuUfaAfusGfsg D2069 S2069 1087
AfuUfaAfaAfaGfAfCfaCfaUfUfCfuGfuAfL96 AS2069 2179
uAfcAfgaaUfgUfgucUfuUfuUfaAfusGfsg D2070 S2070 1088
UfuAfaAfaAfgAfCfAfcAfuucUfgUfaAfL96 AS2070 2180
uUfaCfaGfAfAfuGfuguCfuUfuUfuAfasUfsg D2071 S2071 1089
UfuAfaAfaAfgAfCfAfcAfuUfCfUfgUfaAfL96 AS2071 2181
uUfaCfagaAfuGfuguCfuUfuUfuAfasUfsg D2072 S2072 1090
UfaAfaAfaGfaCfAfCfaUfucuGfuAfaAfL96 AS2072 2182
uUfuAfcAfGfAfaUfgugUfcUfuUfuUfasAfsu D2073 S2073 1091
UfaAfaAfaGfaCfAfCfaUfuCfUfGfuAfaAfL96 AS2073 2183
uUfuAfcagAfaUfgugUfcUfuUfuUfasAfsu D2074 S2074 1092
AfaAfaAfgAfcAfCfAfuUfcugUfaAfaAfL96 AS2074 2184
uUfuUfaCfAfGfaAfuguGfuCfuUfuUfusAfsa D2075 S2075 1093
AfaAfaAfgAfcAfCfAfuUfcUfGfUfaAfaAfL96 AS2075 2185
uUfuUfacaGfaAfuguGfuCfuUfuUfusAfsa D2076 S2076 1094
AfaAfaGfaCfaCfAfUfuCfuguAfaAfaAfL96 AS2076 2186
uUfuUfuAfCfAfgAfaugUfgUfcUfuUfusUfsa D2077 S2077 1095
AfaAfaGfaCfaCfAfUfuCfuGfUfAfaAfaAfL96 AS2077 2187
uUfuUfuacAfgAfaugUfgUfcUfuUfusUfsa D2078 S2078 1096
AfaAfgAfcAfcAfUfUfcUfguaAfaAfaAfL96 AS2078 2188
uUfuUfuUfAfCfaGfaauGfuGfuCfuUfusUfsu D2079 S2079 1097
AfaAfgAfcAfcAfUfUfcUfgUfAfAfaAfaAfL96 AS2079 2189
uUfuUfuuaCfaGfaauGfuGfuCfuUfusUfsu D2080 S2080 1098
AfaGfaCfaCfaUfUfCfuGfuaaAfaAfaAfL96 AS2080 2190
uUfuUfuUfUfAfcAfgaaUfgUfgUfcUfusUfsu D2081 S2081 1099
AfaGfaCfaCfaUfUfCfuGfuAfAfAfaAfaAfL96 AS2081 2191
uUfuUfuuuAfcAfgaaUfgUfgUfcUfusUfsu D2082 S2082 1100
AfgAfcAfcAfuUfCfUfgUfaaaAfaAfaAfL96 AS2082 2192
uUfuUfuUfUfUfaCfagaAfuGfuGfuCfusUfsu D2083 S2083 1101
AfgAfcAfcAfuUfCfUfgUfaAfAfAfaAfaAfL96 AS2083 2193
uUfuUfuuuUfaCfagaAfuGfuGfuCfusUfsu D2084 S2084 1102
GfaCfaCfaUfuCfUfGfuAfaaaAfaAfaAfL96 AS2084 2194
uUfuUfuUfUfUfuAfcagAfaUfgUfgUfcsUfsu D2085 S2085 1103
GfaCfaCfaUfuCfUfGfuAfaAfAfAfaAfaAfL96 AS2085 2195
uUfuUfuuuUfuAfcagAfaUfgUfgUfcsUfsu D2086 S2086 1104
AfcAfcAfuUfcUfGfUfaAfaaaAfaAfaAfL96 AS2086 2196
uUfuUfuUfUfUfuUfacaGfaAfuGfuGfusCfsu D2087 S2087 1105
AfcAfcAfuUfcUfGfUfaAfaAfAfAfaAfaAfL96 AS2087 2197
uUfuUfuuuUfuUfacaGfaAfuGfuGfusCfsu D2088 S2088 1106
CfaCfaUfuCfuGfUfAfaAfaaaAfaAfaAfL96 AS2088 2198
uUfuUfuUfUfUfuUfuacAfgAfaUfgUfgsUfsc D2089 S2089 1107
CfaCfaUfuCfuGfUfAfaAfaAfAfAfaAfaAfL96 AS2089 2199
uUfuUfuuuUfuUfuacAfgAfaUfgUfgsUfsc D2090 S2090 1108
AfcAfuUfcUfgUfAfAfaAfaaaAfaAfaAfL96 AS2090 2200
uUfuUfuUfUfUfuUfuuaCfaGfaAfuGfusGfsu D2091 S2091 1109
AfcAfuUfcUfgUfAfAfaAfaAfAfAfaAfaAfL96 AS2091 2201
uUfuUfuuuUfuUfuuaCfaGfaAfuGfusGfsu Lowercase nucleotides (a, u, g,
c) are 2'-O-methyl nucleotides; Nf (e.g., Af) is a 2'-fluoro
nucleotide; s is aphosphothiorate linkage; L96 indicates a
GalNAc.sub.3 ligand.
Example 4: In Vitro Screening of RNAi Agents
Cell Culture and Transfections
[0405] Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas,
Va.) were grown to near confluence at 37.degree. C. in an
atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS,
streptomycin, and glutamine (ATCC) before being released from the
plate by trypsinization. Transfection was carried out by adding
14.8 .mu.l of Opti-MEM plus 0.2 .mu.l of Lipofectamine RNAiMax per
well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 .mu.l of
siRNA duplexes per well into a 96-well plate and incubated at room
temperature for 15 minutes. 80 .mu.l of complete growth media
without antibiotic containing .about.2.times.104 Hep3B cells were
then added to the siRNA mixture. Cells were incubated for either 24
or 120 hours prior to RNA purification. Single dose experiments
were performed at 10 nM and 0.1 nM final duplex concentration and
dose response experiments were done using 8, 4 fold serial
dilutions with a maximum dose of 10 nM final duplex
concentration.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen,
Part #: 610-12)
[0406] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding
Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf
Thermomixer (the mixing speed was the same throughout the process).
Ten microliters of magnetic beads and 80 .mu.l Lysis/Binding Buffer
mixture were added to a round bottom plate and mixed for 1 minute.
Magnetic beads were captured using magnetic stand and the
supernatant was removed without disturbing the beads. After
removing the supernatant, the lysed cells were added to the
remaining beads and mixed for 5 minutes. After removing the
supernatant, magnetic beads were washed 2 times with 150 .mu.l Wash
Buffer A and mixed for 1 minute. Beads were capture again and
supernatant removed. Beads were then washed with 150 .mu.l Wash
Buffer B, captured and supernatant was removed. Beads were next
washed with 150 .mu.l Elution Buffer, captured and supernatant
removed. Beads were allowed to dry for 2 minutes. After drying, 50
.mu.l of Elution Buffer was added and mixed for 5 minutes at
70.degree. C. Beads were captured on magnet for 5 minutes. 40 .mu.l
of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription
Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
[0407] A master mix of 1 .mu.l 10.times. Buffer, 0.4 .mu.l
25.times.dNTPs, 1 .mu.l Random primers, 0.5 .mu.l Reverse
Transcriptase, 0.5 .mu.l RNase inhibitor and 1.6 .mu.l of H.sub.2O
per reaction were added into 5 .mu.l total RNA. cDNA was generated
using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.)
through the following steps: 25.degree. C. 10 min, 37.degree. C.
120 min, 85.degree. C. 5 sec, 4.degree. C. hold.
Real Time PCR
[0408] 2 .mu.l of cDNA were added to a master mix containing 0.5l
GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E (human) Cat
#4308313 (rodent)), 0.5p TTR TaqMan probe (Applied Biosystems cat #
HS00174914 ml (human) cat # Rn00562124_ml (rat)) and 5 .mu.l
Lightcycler 480 probe master mix (Roche Cat #04887301001) per well
in a 384 well plate (Roche cat #04887301001). Real time PCR was
done in a Roche LC 480 Real Time PCR machine (Roche). Each duplex
was tested in at least two independent transfections and each
transfection was assayed in duplicate, unless otherwise noted.
[0409] To calculate relative fold change, real time data were
analyzed using the .DELTA..DELTA.Ct method and normalized to assays
performed with cells transfected with 10 nM AD-1955, or mock
transfected cells. IC.sub.50s were calculated using a 4 parameter
fit model using XLFit and normalized to cells transfected with
AD-1955 (sense sequence: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO:
2202); antisense sequence: UCGAAGuCUcAGCGuAAGdTsdT (SEQ ID NO:
2203)) or naive cells over the same dose range, or to its own
lowest dose. IC.sub.50s were calculated for each individual
transfection as well as in combination, where a single IC.sub.50
was fit to the data from both transfections.
[0410] The results of gene silencing of the exemplary siRNA duplex
with various motif modifications of the invention are shown in
Table 1 above.
Example 5: In Vitro Silencing Activity of Chemically Modified RNAi
Agents that Target TTR
[0411] The following experiments demonstrated the beneficial
effects of chemical modifications, including the introduction of
triplet repeat motifs, together with a GalNAc.sub.3 ligand, on the
silencing activity of RNAi agents that target TTR. The sequences of
the agents investigated are provided in Table 2 below. The regions
of complementarity to the TTR mRNA are as follows: the region of
complementarity of RNAi agents AD-45165, AD-51546 and AD-51547 is
GGATGGGATTTCATGTAACCAAGA (SEQ ID NO: 2204) and the region or
complemetarity of RNAi agents AD-45163, AD-51544, and AD-51545 is
TTCATGTAACCAAGAGTATTCCAT (SEQ ID NO: 2205).
Protocol for Assessment of IC.sub.50 in Hep3B Cells
[0412] The IC.sub.50 for each modified siRNA was determined in
Hep3B cells (a human hepatoma cell line) by standard reverse
transfection using Lipofectamine RNAiMAX. In brief, reverse
transfection was carried out by adding 5 .mu.L of Opti-MEM to 5
.mu.L of siRNA duplex per well into a 96-well plate along with 10
.mu.L of Opti-MEM plus 0.5 .mu.L of Lipofectamine RNAiMax per well
(Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at room
temperature for 15-20 minutes. Following incubation, 100 .mu.L of
complete growth media without antibiotic containing 12,000-15,000
Hep3B cells was then added to each well. Cells were incubated for
24 hours at 37.degree. C. in an atmosphere of 5% CO.sub.2 prior to
lysis and analysis of TTR and GAPDH mRNA by bDNA (Quantigene).
Seven different siRNA concentrations ranging from 10 nM to 0.6
.mu.M were assessed for IC.sub.50 determination and TTR/GAPDH for
siRNA transfected cells was normalized to cells transfected with 10
nM Luc siRNA. The results are shown in Table 2.
Protocol for Assessment of Free-Uptake IC.sub.50
[0413] Free uptake silencing in primary cynomolgus hepatocytes was
assessed following incubation with TTR siRNA for either 4 hours or
24 hours. Silencing was measured at 24 hours from the initial
exposure. In brief, 96-well culture plates were coated with
0.05%-0.1% collagen (Sigma C3867-1VL) at room temperature, 24 hours
prior to the start of the experiment. On the day of assay, siRNAs
were diluted in pre-warmed Plating Media consisting of DMEM
supplemented with GIBCO's Maintenance Media Kit (Serum-Free, Life
Technologies CM4000), and added to the collagen-coated 96-well
culture plates. Cryopreserved primary cynomolgus hepatocytes were
rapidly thawed in a 37.degree. C. water bath, and immediately
diluted in Plating Media to a concentration of 360,000 cells/mL. A
volume of cell suspension was gently pipetted on top of the
pre-plated siRNAs such that the final cell count was 18,000
cells/well. The plate was lightly swirled to mix and spread cells
evenly across the wells and placed in a 37.degree. C., 5% CO.sub.2
incubator for 24 hours prior to lysis and analysis of TTR and GAPDH
mRNA by bDNA (Quantigene, Affymetrix). In the case of the 4 h
incubation with siRNA, the media was decanted after 4 hours of
exposure to the cells, and replaced with fresh Plating Media for
the remaining 20 hours of incubation. Downstream analysis for TTR
and GAPDH mRNA was the same as described above. For a typical dose
response curve, siRNAs were titrated from 1 uM to 0.24 nM by 4 fold
serial dilution.
TABLE-US-00006 TABLE 2 In vitro Activity Summary for Alternating
TTR-GalNAc and Variants with Triplet Motifs Free-Uptake Hep3B
Duplex IC50 (.mu.M) IC50 ID S (5'-3') AS (5'-3'') 4 h 24 h (nM)
AD-45163 AfuGfuAfaCfcAfaGfaGfu aUfgGfaAfuAfcUfcUfuGfg 0.04101
0.00820 0.0115 AfuUfcCfaUfL96 UfuAfcAfusGfsa (SEQ ID NO: 2206) (SEQ
ID NO: 2212) AD-51544 AfuGfuAfaCfcAfAfGfaGf aUfgGfAfAfuAfcUfcuuGfg
0.00346 0.00374 0.0014 uAfuucCfaUfL96 UfuAfcAfusGfsa (SEQ ID NO:
2207) (SEQ ID NO: 2213) AD-51545 AfuGfuAfAfCfcAfAfGfaG
aUfgGfaAfuAfcUfcuuGfgu 0.00395 0.00389 0.0018 fuAfuUfcCfaUfL96
uAfcAfusGfsa (SEQ ID NO: 2208) (SEQ ID NO: 2214) AD-45165
UfgGfgAfuUfuCfaUfgUfa uCfuUfgGfuUfaCfaUfgAfa 0.02407 0.00869 0.0112
AfcCfaAfgAfL96 AfuCfcCfasUfsc (SEQ ID NO: 2209) (SEQ ID NO: 2215)
AD-51546 UfgGfGfAfuUfuCfAfUfgU uCfuugGfuUfaCfaugAfaAf 0.00317
0.00263 0.0017 faAfcCfAfAfgAfL96 uccCfasUfsc (SEQ ID NO: 2210) (SEQ
ID NO: 2216) AD-51547 UfgGfgAfuUfuCfAfUfgUf uCfuUfgGfUfUfaCfaugAfa
0.00460 0.00374 0.0028 aacCfaAfgAfL96 AfuCfcCfasUfsc (SEQ ID NO:
2211) (SEQ ID NO: 2217) Lowercase nucleotides (a, u, g, c) indicate
2'-O-methyl nucleotides; Nf (e.g., Af) indicates a 2'-fluoro
nucleotide; s indicates a phosphothiorate linkage; L96 indicates a
GalNAc.sub.3 ligand; bold nucleotides indicate changes relative to
the corresponding parent agent. Each bold nucleotide is at the
center of a triplet motif.
[0414] The results are provided in Table 2 and demonstrate that
modified RNAi agents that target TTR provide enhanced silencing
activity.
Results: Improved Activity of Modified RNAi Agents
[0415] Parent RNAi agents with alternating chemical modifications
and a GalNAc3 ligand provided an IC.sub.50 in Hep3B cells of about
0.01 nM. As shown in FIGS. 4-5 and in Table 2, agents modified
relative to the parent agents, for example, by the addition of one
or more repeating triplets of 2'-fluoro and 2'-O-methyl
modifications, showed unexpectedly enhanced silencing activity,
achieving IC.sub.50 values in Hep3B cells that were 5-8 fold better
than the corresponding parent agent.
Results: Free Uptake IC.sub.50s in Hep3B Cells
[0416] As shown in Table 2 and FIGS. 6-7, RNAi agents modified
relative to the parent AD-45163 also showed enhanced free uptake
silencing. The modified agents showed more than double the
silencing activity of the parent after a 24 hour incubation period
and nearly 10 times the silencing activity of the parent after a 4
hour incubation period.
[0417] As shown in Table 2 and FIGS. 8-9, RNAi agents modified
relative to the parent AD-45165 also showed enhanced free uptake
silencing. The modified agents showed 2-3 times the silencing
activity of the parent after a 24 hour incubation period and 5-8
times the silencing activity of the parent after a 4 hour
incubation period.
[0418] Taken collectively, these results demonstrate that the
modified RNAi agents presented herein, e.g., AD-51544, AD-51545,
AD-51546, and AD-51547, all showed unexpectedly good inhibition of
TTR mRNA in in vitro silencing experiments.
Example 6: TTR mRNA Silencing and TTR Protein Suppression in
Transgenic Mice
[0419] To assess the efficacy of the RNAi agents AD-45163,
AD-51544, AD-51545, AD45165, AD-51546, and AD-51547, these agents
were administered to transgenic mice that express human
transthyretin with the V30M mutation (see Santos, S D., Fernaandes,
R., and Saraiva, M J. (2010) Neurobiology of Aging, 31, 280-289).
The V30M mutation is known to cause familial amyloid polyneuropathy
type I in humans. See, e.g., Lobato, L. (2003) JNephrol.,
16(3):438-42.
[0420] The RNAi agents (in PBS buffer) or PBS control were
administered to mice (2 male and 2 female) of 18-24 months of age
in a single subcutaneous dose of 5 mg/kg or 1 mg/kg. After
approximately 48 hours, mice were anesthetized with 200 .mu.l of
ketamine, and then exsanguinated by severing the right caudal
artery. Whole blood was isolated and plasma was isolated and stored
at -80.degree. C. until assaying. Liver tissue was collected,
flash-frozen and stored at -80.degree. C. until processing.
[0421] Efficacy of treatment was evaluated by (i) measurement of
TTR mRNA in liver at 48 hours post-dose, and (ii) measurement of
TTR protein in plasma at pre-bleed and at 48 hours post-dose. TTR
liver mRNA levels were assayed utilizing the Branched DNA
assays--QuantiGene 2.0 (Panomics cat #: QS0011). Briefly, mouse
liver samples were ground and tissue lysates were prepared. Liver
lysis mixture (a mixture of 1 volume of lysis mixture, 2 volume of
nuclease-free water and 10 ul of Proteinase-K/ml for a final
concentration of 20 mg/ml) was incubated at 65.degree. C. for 35
minutes. 20 .mu.l of Working Probe Set (TTR probe for gene target
and GAPDH for endogenous control) and 80 ul of tissue-lysate were
then added into the Capture Plate. Capture Plates were incubated at
55.degree. C. 1.degree. C. (aprx. 16-20 hrs). The next day, the
Capture Plates were washed 3 times with 1.times. Wash Buffer
(nuclease-free water, Buffer Component 1 and Wash Buffer Component
2), then dried by centrifuging for 1 minute at 240 g. 100 .mu.l of
pre-Amplifier Working Reagent was added into the Capture Plate,
which was sealed with aluminum foil and incubated for 1 hour at
55.degree. C. 1.degree. C. Following 1 hour incubation, the wash
step was repeated, then 100 .mu.l of Amplifier Working Reagent was
added. After 1 hour, the wash and dry steps were repeated, and 100
.mu.l of Label Probe was added. Capture plates were incubated
50.degree. C. 1.degree. C. for 1 hour. The plate was then washed
with 1.times. Wash Buffer, dried and 1001 Substrate was added into
the Capture Plate. Capture Plates were read using the SpectraMax
Luminometer following a 5 to 15 minute incubation. bDNA data were
analyzed by subtracting the average background from each triplicate
sample, averaging the resultant triplicate GAPDH (control probe)
and TTR (experimental probe) values, and then computing the ratio:
(experimental probe-background)/(control probe-background).
[0422] Plasma TTR levels were assayed utilizing the commercially
available kit "AssayMax Human Prealbumin ELISA Kit" (AssayPro, St.
Charles, Mo., Catalog # EP3010-1) according to manufacturer's
guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1.times.
mix diluents and added to pre-coated plates along with kit
standards, and incubated for 2 hours at room temperature followed
by 5.times. washes with kit wash buffer. Fifty microliters of
biotinylated prealbumin antibody was added to each well and
incubated for 1 hr at room temperature, followed by 5.times. washes
with wash buffer. Fifty microliters of streptavidin-peroxidase
conjugate was added to each well and plates were incubated for 30
minutes at room temperature followed by washing as previously
described. The reaction was developed by the addition of 50
.mu.l/well of chromogen substrate and incubation for 10 minutes at
room temperature with stopping of reaction by the addition of 50
.mu.l/well of stop solution. Absorbance at 450 nm was read on a
Versamax microplate reader (Molecular Devices, Sunnyvale, Calif.)
and data were analyzed utilizing the Softmax 4.6 software package
(Molecular Devices).
[0423] The results are shown in FIGS. 10-12. FIGS. 10A and 10B show
that the RNAi agents modified relative to the parent agents
AD-45163 and AD-45165 showed RNA silencing activity that was
similar or more potent compared with that of the parent agents.
FIG. 11 shows that the agents AD-51544 and AD-51545 showed dose
dependent silencing activity and that the silencing activity of
these agents at a dose of 5 mg/kg was similar to that of the
corresponding parent AD-45163. FIG. 12 shows that the agents
AD-51546 and AD-51547 also showed dose-dependent silencing
activity. Furthermore, the silencing activity of AD-51546 and
AD-51547 at a dose of 5 mg/kg was superior to that of the
corresponding parent AD-45165.
Example 7: Serum and Liver Pharmacokinetic Profiles of RNAi Agents
that Target TTR in Mice
[0424] To assess the pharmacokinetic profiles of the RNAi agents
AD-45163, AD-51544, AD-51545, AD-51546, and AD-51547, these agents,
in PBS buffer, were administered to C57BL/6 mice using a single IV
bolus or subcutaneous (SC) administration. The plasma
concentrations and liver concentrations of the agents were assessed
at various timepoints after the administration.
[0425] The plasma pharmacokinetic parameters are presented in
Tables 3 and 4 below. The mean resident time (MRT) in plasma was
about 0.2 hours after IV dosing and about 1 hour after SC dosing.
At a dose of 25 mg/kg, the agents AD-51544, AD-51545, AD-51546, and
AD-51547 showed similar plasma pharmacokinetic properties. Each of
these agents had more than 75% bioavailability from the
subcutaneous space. Their bioavailability was superior to that of
the parent agent AD-45163 that was administered at a higher dose of
30 mg/kg. The subcutaneous bioavailability of AD-51544 and AD-51547
was about 100%, whereas that of AD-51545 was 90% and that of and
AD-51546 was 76%.
TABLE-US-00007 TABLE 3 Summary of Plasma PK Parameter Estimates
After SC Administration of TTR-GalNAc siRNAs in Mice 30 mpk 25 mpk
25 mpk 25 mpk 25 mpk AD- AD- AD- AD- AD- 45163 51544 51545 51546
51547 (h/c (h/c (h/c (h/c (h/c TTR- TTR- TTR- TTR- TTR- Parameter
GalNAc) GalNAc) GalNAc) GalNAc) GalNAc) Plasma 0.25 1 0.5 1 0.5
Tmax (h) Plasma 9.6 11.7 10.9 11.7 12.1 Cmax (.mu.g/mL) Plasma 12.4
21.9 19.9 20.9 25.3 AUC (h*.mu.g/mL) F.sub.sc(%) 79 100 90.1 76.0
99.2
TABLE-US-00008 TABLE 4 Plasma siRNA PK Parameters in Mice after an
IV Bolus or SC Dose of AD-51544, 51545, 51546 or 51547 at 25 mg/kg
Test Article AD-51544 AD-51545 AD-51546 AD-51547 siRNA Dose (mg/kg)
25 25 25 25 Route of Administration IV SC IV SC IV SC IV SC
t.sub.max (h) 0.083 1 0.083 0.5 0.083 1 0.083 0.5 C.sub.max
(.mu.g/mL) 96.5.sup.a 11.7 108.sup.a 10.9 128.sup.a 10.9 123.sup.a
12.1 AUC.sub.0-last (h .mu.g/mL) 21.6 21.9 22.1 19.9 27.5 20.9 25.5
25.3 MRT.sub.0-last (h) 0.17 1.2 0.16 1.1 0.22 1.4 0.19 1.3
Apparent t.sub.1/2 .beta. (h).sup.b ND ND ND 0.49 ND 1.2 ND 0.56
F.sub.SC (%).sup.c -- 102 -- 90.1 -- 76.0 -- 99.2
.sup.aConcentration at the 1.sup.st sampling time (5 min) after IV
dosing .sup.bApparent elimination half-life (t.sub.1/2 .beta.)
could not be determined (ND) for all 4 test articles after IV
dosing as the terminal phase of the concentration-time profiles was
not well defined, as a result, the t.sub.1/2 .beta.-associated PK
parameters (eg, AUC.sub.0-.infin., CL and Vss) were not reported.
.sup.cSC bioavailability, calculated as percentage ratio of
AUC.sub.0-last after SC and IV dosing at 25 mg/kg
[0426] The results also indicated that the RNAi agents AD-45163,
AD-51544, AD-51545, AD-51546, and AD-51547 achieved similar or
higher concentrations in the liver when administered subcutaneously
than when administered by IV bolus. The liver pharmacokinetic
parameters are presented in Tables 5 and 6 below. The peak
concentration (C.sub.max) and area under the curve (AUC.sub.0-last)
in the liver were two to three times higher after subcutaneous
administration as compared with IV administration of the same agent
at the same dose. Liver exposures were highest for AD-51547 and
lowest for AD-51545. The mean resident time (MRT) and elimination
half-life were longer for AD-51546 and AD-51547 compared with
AD-51544 and AD-51545. Following subcutaneous administration, the
approximate MRTs were 40 hours for AD-51546 and 25 hours for
AD-51547, whereas the MRTs for AD-51544 and AD-51545 were lower
(about 6-9 hours). The elimination half life of AD-51546 and
AD-51547 was also higher (41-53 hours) than was the elimination
half life of AD-51544 and AD-51545 (6-10 hours).
TABLE-US-00009 TABLE 5 Summary of Liver PK Parameter Estimates
After SC Administration of TTR-GalNAc siRNAs in Mice 30 mpk 25 mpk
25 mpk 25 mpk 25 mpk AD- AD- AD- AD- AD- 45163 51544 51545 51546
51547 (h/c (h/c (h/c (h/c (h/c TTR- TTR- TTR- TTR- TTR- Parameter
GalNAc) GalNAc) GalNAc) GalNAc) GalNAc) Liver Tmax 8 4 4 2 8 (h)
Liver Cmax 313 126 80 117 174 (.mu.g/g) Liver AUC 4519 1092 763
2131 4583 (h*.mu.g/g)
TABLE-US-00010 TABLE 6 Liver siRNA PK Parameters in Mice after an
IV Bolus or SC Dose of AD-51544, 51545, 51546 or 51547 at 25 mg/kg
Test Article AD-51544 AD-51545 AD-51546 AD-51547 siRNA Dose (mg/kg)
25 25 25 25 Route of Administration IV SC IV SC IV SC IV SC
t.sub.max (h) 1 4 1 4 4 2 2 8 C.sub.max (.mu.g/g) 67.9 126 37.0
80.5 35.3 117 73.8 174 AUC.sub.0-last (h .mu.g/g) 632 1092 324 763
984 2131 1429 4583 MRT.sub.0-last (h) 8.7 6.5 5.9 8.5 45.7 40.2
29.4 25.3 Apparent t.sub.1/2.beta. (h) 8.1 8.2 5.7 10.0 51.1 45.3
41.1 52.7
Example 8: In Vitro Stability of RNAi Agents in Monkey Serum
[0427] The serum stability of RNAi agents AD-51544, AD-51545,
AD-51546, and AD-51547 was also assessed in monkeys. The results
demonstrated that the antisense and sense strands of AD-51544,
AD-51545, and AD-51547 showed serum stability over a period of
about 24 hours (data not shown).
Example 9: RNAi Agents Produce Lasting Suppression of TTR Protein
in Non-Human Primates
[0428] The RNA silencing activity of RNAi agents AD-45163,
AD-51544, AD-51545, AD-51546, and AD-51547 was assessed by
measuring suppression of TTR protein in serum of cynomologous
monkeys following subcutaneous administration of five 5 mg/kg doses
(one dose each day for 5 days) or a single 25 mg/kg dose. Pre-dose
TTR protein levels in serum were assessed by averaging the levels
at 11 days prior to the first dose, 7 days prior to the first dose,
and 1 day prior to the first dose. Post-dose serum levels of TTR
protein were assessed by determining the level in serum beginning
at 1 day after the final dose (i.e., study day 5 in the 5.times.5
mg/kg group and study day 1 in the 1.times.25 mg/kg group) until 49
days after the last dose (i.e., study day 53 in the 5.times.5 mg/kg
group and study day 49 in the 1.times.25 mg/kg group). See FIG.
13.
[0429] TTR protein levels were assessed as described in Example 6.
The results are shown in FIGS. 14A and 14B and in Tables 7 and
8.
[0430] A maximal suppression of TTR protein of up to about 50% was
achieved in the groups that received 25 mg/kg of AD-45163,
AD-51544, AD-51546, and AD-51547 (see Table 8). A greater maximal
suppression of TTR protein of about 70% was achieved in the groups
that received 5.times.5 mg/kg of AD-45163, AD-51544, AD-51546, and
AD-51547 (see Table 7). The agent AD-51545 produced a lesser degree
of suppression in both administration protocols. Significant
suppression of about 20% or more persisted for up to 49 days after
the last dose of AD-51546 and AD-51547 in both the 1.times.25 mg/kg
and 5.times.5 mg/kg protocols. Generally, better suppression was
achieved in the 5.times.5 mg/kg protocol than in the 1.times.25
mg/kg protocol.
TABLE-US-00011 TABLE 7 Fraction Serum Transthyretin Relative to
Pre-dose in Cynomolgus Monkeys (5 mg/kg daily for 5 days) D-11 D-7
D-1 D5 D7 D9 D11 D14 D18 D22 D26 D32 D39 D46 D53 AD-45163 0.98 0.99
1.03 0.71 0.52 0.40 0.34 0.27 0.31 0.39 0.48 0.64 0.68 0.81 0.88
AD-51544 1.02 0.99 0.99 0.60 0.47 0.37 0.35 0.39 0.48 0.58 0.66
0.74 0.83 0.91 0.92 AD-51545 1.03 0.97 1.00 0.73 0.65 0.63 0.69
0.68 0.78 0.87 0.97 1.00 1.03 1.06 1.09 AD-51546 1.01 0.97 1.02
0.59 0.42 0.35 0.30 0.32 0.43 0.58 0.66 0.77 0.92 0.93 0.97
AD-51547 0.99 0.99 1.02 0.74 0.54 0.41 0.34 0.34 0.39 0.49 0.51
0.53 0.65 0.70 0.77
TABLE-US-00012 TABLE 8 Fraction Serum Transthyretin Relative to
Pre-dose in Cynomolgus Monkeys (25 mg/kg) D-11 D-7 D-1 D1 D3 D5 D7
D10 D14 D18 D22 D28 D35 D42 D49 AD-45163 1.04 1.01 0.95 0.99 0.84
0.67 0.57 0.44 0.45 0.51 0.58 0.66 0.72 0.78 0.85 AD-51544 1.01
1.04 0.95 0.92 0.69 0.57 0.49 0.48 0.56 0.65 0.69 0.77 0.83 0.87
0.94 AD-51545 0.98 1.02 0.99 0.87 0.77 0.69 0.71 0.72 0.84 0.90
0.92 0.99 1.00 1.00 1.00 AD-51546 1.04 1.03 0.93 0.89 0.71 0.62
0.53 0.50 0.55 0.70 0.70 0.69 0.72 0.79 0.84 AD-51547 0.96 1.03
1.01 1.19 0.90 0.70 0.54 0.48 0.50 0.50 0.52 0.58 0.62 0.70
0.72
Example 10: Tolerability of RNAi Agents that Target TTR
In Cytokine Evaluation in Whole Blood Assay
[0431] To assess the tolerability of RNAi agents that target TTR
(including AD-45163, AD-51544, AD-51545, AD-51546, and AD-51547),
each agent was tested in a whole blood assay using blood from three
human donors. The agents were either 300 nM DOTAP transfected or 1
.mu.M without transfection reagent (free siRNA). There was less
than a two fold change for the following cytokines/chemokines:
G-CSF, IFN-.gamma., IL-10, IL-12 (p70), IL1.beta., IL-1ra, IL-6,
IL-8, IP-10, MCP-1, MIP-1.alpha., MIP-1.beta., TNF.alpha.. (Results
not shown).
In Vivo Evaluation
[0432] To assess in vivo tolerability, RNAi agents were injected
subcutaneously in CD1 mice at a dose of 125 mg/kg. No cytokine
induction was observed at 2, 4, 6, 24, or 48 hours after
subcutaneous injection of AD-45163. No significant cytokine
induction was observed at 6 or 24 hours after subcutaneous
injection of AD-51544, AD-51545, AD-51546, or AD-51547.
[0433] To further assess in vivo tolerability, multiple RNAi agents
(including AD-45163, AD-51544, AD-51545, AD-51546, and AD-51547)
were tested by subcutaneous injection of 5 and 25 mg in non-human
primates (cynomologous monkeys) with dose volumes between 1-2 ml
per site. No erythema or edema was observed at injection sites.
Single SC Dose Rat Tolerability Study
[0434] To assess toxicity, rats were injected with a single
subcutaneous dose of 100, 250, 500, or 750 mg/kg of AD-45163 (see
Table 9). The following assessments were made: clinical signs of
toxicity, body weight, hematology, clinical chemistry and
coagulation, organ weights (liver & spleen); gross and
microscopic evaluation (kidney, liver, lung, lymph node, spleen,
testes, thymus, aorta, heart, intestine (small and large).
TABLE-US-00013 TABLE 9 Single SC Dose Rat Tolerability Study: 100,
250, 500 & 750 mg/kg of AD-45163 in Sprague Dawley Rats Dose
Dose No. Male Day of Level Volume Route & Sprague Necropsy
Group (mg/kg) (ml/kg) Regimen Dawley Rats PBS 0 10 SC Injection
7/group Day 4 AD-45163 100 Day 1 (5 Tox Parent (2 sites) animals, 2
TK animals)
[0435] The results showed no test article-related clinical signs of
toxicity, effects on body weight, organ weights, or clinical
chemistry. No histopathology was observed in heart, kidneys,
testes, spleen, liver, and thymus. There was a non-adverse, slight
test article-related increase in WBC (68%, primarily attributed to
increase in NEUT and MONO) at 750 mg/kg. These results indicate
that a single-dose of up to 750 mg/kg is well tolerated in
rats.
Tolerability of Repeated Subcutaneous Administrations in Rats
[0436] To assess the tolerability of repeated subcutaneous
administrations of AD-45163, daily subcutaneous injections of 300
mg/kg were given for 5 days, and a necropsy was performed on day 6.
The study design is shown in Table 10.
TABLE-US-00014 TABLE 10 Five Day Repeat Dose Tolerability Study in
Rat Dose Level Cone No of Tox Group (kmg/kg (mg/mL) Animals Nx Day
6 PBS 0 0 2M, 2F 2M, 2F AD-45163 300 150 2M, 2F 2M, 2F
[0437] The following outcome variables were assessed: clinical
signs, body weights, hematology, clinical chemistry and
coagulation, organ weights, gross and microscopic evaluation
(liver, spleen, kidney, heart, GI tract and first and last
injection site). The results showed no test article-related
clinical signs, body weight or organ weight effects, and also no
test article-related findings in clinical hematology or chemistry.
There was a possible slight prolongation of activated partial
thromboplastin time (APTT) on day 6 (20.4 vs. 17.4 sec).
Histopathology revealed no test article-related findings in the
liver, spleen, heart, and GI tract. In the kidney, minimal to
slight hypertrophy of the tubular epithelium (not adverse) was
observed. At the last injection site, there was minimal multifocal
mononuclear infiltration, not adverse. These results indicate that
five daily 300 mg/kg doses of the parent RNAi agent AD-45163 are
well tolerated in rats.
Example 11: RNAi Agents Produce Lasting Suppression of TTR Protein
in Non-Human Primates
[0438] The RNA silencing activity of RNAi agent AD-51547 was
assessed by measuring suppression of TTR protein in the serum of
cynomologous monkeys following subcutaneous administration of a
"loading phase" of the RNAi agent: five daily doses of either 2.5
mg/kg, 5 mg/kg or 10 mg/kg (one dose each day for 5 days) followed
by a "maintenance phase" of the RNAi agent: weekly dosing of either
2.5 mg/kg, 5 mg/kg or 10 mg/kg for 4 weeks. Pre-dose TTR protein
levels in serum were assessed by averaging the levels at 11 days
prior to the first dose, 7 days prior to the first dose, and 1 day
prior to the first dose. Post-dose serum levels of TTR protein were
assessed by determining the level in serum relative to pre-dose
beginning at 1 day after the loading phase was completed until 40
days after the last dose of the maintenance phase (i.e., study day
70).
[0439] TTR protein levels were assessed as described in Example 6.
The results are shown in FIG. 15.
[0440] A maximal suppression of TTR protein of up to about 80% was
achieved in all of the groups that received either 2.5 mg/kg, 5
mg/kg or 10 mg/kg of AD-51547. Nadir knockdown was achieved in all
of the groups by about day 14, the suppression sustained at nadir
knockdown levels with a weekly maintenance dose of either 2.5
mg/kg, 5 mg/kg or 10 mg/kg of AD-51547. The levels of TTR had not
returned to baseline more than 40 days after the day of
administration of the last maintenance dose for the 5 and 2.5 mg/kg
dose levels.
EQUIVALENTS
[0441] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200318111A1).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200318111A1).
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