U.S. patent application number 13/936732 was filed with the patent office on 2014-02-13 for folate conjugates.
This patent application is currently assigned to Alnylam Pharmaceuticals, Inc.. The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to Muthusamy Jayaraman, Muthiah Manoharan, Jayaprakash Nair, Kallanthottathil G. Rajeev.
Application Number | 20140045919 13/936732 |
Document ID | / |
Family ID | 40626650 |
Filed Date | 2014-02-13 |
United States Patent
Application |
20140045919 |
Kind Code |
A1 |
Manoharan; Muthiah ; et
al. |
February 13, 2014 |
Folate Conjugates
Abstract
The present invention provides iRNA agent including at least one
monomer having the structure shown in formula (I') ##STR00001##
wherein: A and B are each independently for each occurrence O,
N(R.sup.N) or S; X is H, a protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide; Y is H, a protecting group, a
phosphate group, a phosphodiester group, an activated phosphate
group, an activated phosphite group, a phosphoramidite, a solid
support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a
lipid, a PEG, a steroid, a lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide; R is folate, a folate analog a
folate mimic or a folate receptor binding ligand; L.sup.6 and
L.sup.7 are each independently for each occurrence
--(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--; Q' is NH, O, S,
CH.sub.2, C(O)O, C(O)NH, --NH--CH(R.sup.a)--C(O)--,
--C(O)--CH(R.sup.a)--NH--, CO, ##STR00002## where R.sup.a is H or
amino acid side; chain. R' and R'' are each independently H,
CH.sub.3, OH, SH, NH.sub.2, NH(Alkyl=Me, Et, Pr, isoPr, Bu, Bn) or
N(diAlkyl=Me.sub.2, Et.sub.2, Bn.sub.2); Z', Z'', Z''' and Z''''
are independently O or S; n represent independently for each
occurrence 1-20; and m represent independently for each occurrence
0-50.
Inventors: |
Manoharan; Muthiah;
(Cambridge, MA) ; Rajeev; Kallanthottathil G.;
(Cambridge, MA) ; Jayaraman; Muthusamy;
(Cambridge, MA) ; Nair; Jayaprakash; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Alnylam Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
40626650 |
Appl. No.: |
13/936732 |
Filed: |
July 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12328537 |
Dec 4, 2008 |
8507455 |
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13936732 |
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60992309 |
Dec 4, 2007 |
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61013597 |
Dec 13, 2007 |
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Current U.S.
Class: |
514/44A ;
435/375; 536/24.5 |
Current CPC
Class: |
A61K 47/55 20170801;
C12N 15/113 20130101 |
Class at
Publication: |
514/44.A ;
536/24.5; 435/375 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The work described herein was carried out, at least in part,
using funds from the United States government under contract number
HHSN266200600012C from the Department of Health and Human Services
(DHHS) and contract number HDTRA-1-07-C-0082, from the Department
of Defense and Defense Threat Reduction Agency (DOD/DTRA). The
government may therefore have certain rights in the invention.
Claims
1. An iRNA agent comprising at least one monomer having the
structure shown in formula (I') ##STR00078## wherein: A and B are
each independently for each occurrence O, N(R.sup.N) or S; X is H,
a protecting group, a phosphate group, a phosphodiester group, an
activated phosphate group, an activated phosphite group, a
phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside,
--P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a
polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-P(Z''')(Z'''')O-oligonucleotide, a
nucleotide, or an oligonucleotide; Y is H, a protecting group, a
phosphate group, a phosphodiester group, an activated phosphate
group, an activated phosphite group, a phosphoramidite, a solid
support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a
lipid, a PEG, a steroid, a lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide; R is folate, a folate analog a
folate mimic or a folate receptor binding ligand; L.sup.6 and
L.sup.7 are each independently for each occurrence
--(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--; Q' is NH, O, S,
CH.sub.2, C(O)O, C(O)NH, --NH--CH(R.sup.a)--C(O)--,
--C(O)--CH(R.sup.a)--NH--, CO, ##STR00079## R.sup.a is H or amino
acid side chain; R' and R'' are each independently H, CH.sub.3, OH,
SH, NH.sub.2, NH(Alkyl) or N(diAlkyl); Z', Z'', Z''' and Z'''' are
independently O or S; n represent independently for each occurrence
1-20; and m represent independently for each occurrence 0-50.
2. An iRNA agent comprising at least one monomer having the
structure shown in formula (I) ##STR00080## wherein: X is H, a
hydroxyl protecting group, a phosphate group, a phosphodiester
group, an activated phosphate group, an activated phosphite group,
a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside,
--P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a
polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide; Y is H, a hydroxyl protecting
group, a phosphate group, a phosphodiester group, an activated
phosphate group, an activated phosphite group, a phosphoramidite, a
solid support, --P(Z')(Z'')O-nucleoside,
--P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a
lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide; Q is a tether; R is folate, a
folate analog a folate mimic or a folate receptor binding ligand;
L.sup.6 and L.sup.7 are each independently for each occurrence
--(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--; Q' is NH, O, S,
CH.sub.2, C(O)O, C(O)NH, --NH--CH(R.sup.a)--C(O)--,
--C(O)--CH(R.sup.a)--NH--, CO, ##STR00081## R.sup.a is H or amino
acid side chain; R' and R'' are each independently H, CH3, OH, SH,
NH2, NH(Alkyl) or N(diAlkyl); Z', Z'', Z''' and Z'''' are
independently O or S; n represent independently for each occurrence
1-20; and m represent independently for each occurrence 0-50.
3. The iRNA agent of claim 1, wherein said iRNA agent is double
stranded.
4. The iRNA agent of claim 3, wherein said monomer is at the 3'-end
of one of the strands.
5. The iRNA agent of claim 4, wherein said monomer is at the 3'-end
of sense strand.
6. The iRNA agent of claim 1, wherein the iRNA agent further
comprises at least one monomer of formula (LI) ##STR00082## wherein
X.sup.6 and Y.sup.6 are each independently H, a hydroxyl protecting
group, a phosphate group, a phosphodiester group, an activated
phosphate group, an activated phosphite group, a phosphoramidite, a
solid support, --P(Z')(Z'')O-nucleoside,
--P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a
polymer,
--P(Z')(Z'')O--R.sup.1-Q'-R.sup.2--OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide, --P(Z')(Z'')-formula (I) or
--P(Z')(Z'')--; Q.sup.6 is absent or
--(P.sup.6-Q.sup.6-R.sup.6).sub.v-T.sup.6-; P.sup.6 and T.sup.6 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; Q.sup.6 is independently
for each occurrence absent, substituted alkylene wherein 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.6 is 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, ##STR00083## or
heterocyclyl; R' and R'' are each independently H, C.sub.1-C.sub.6
alkyl OH, SH, N(R.sup.N).sub.2; R.sup.N is independently for each
occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl;
R.sup.a is H or amino acid side chain; Z', Z'', Z''' and Z'''' are
each independently for each occurrence O or S; v represent
independently for each occurrence 0-20; R.sup.L is a lipophile or a
cationic lipid.
7. The RNAi agent of claim 6, wherein R.sup.L is a lipophile.
8. The RNAi agent of claim 7, wherein R.sup.L is cholesterol.
9. The RNAi agent of claim 1, wherein R is chosen from a group
consisting of ##STR00084##
10. The RNAi agent of claim 1, wherein said monomer is chosen from
a group consisting of ##STR00085##
11. The RNAi agent of claim 10, wherein R is ##STR00086##
12. A method of modulating the expression of a target gene in a
cell, comprising providing to said cell an iRNA agent of claim
1.
13. The method of claim 18, wherein the target gene is selected
from the group consisting of CD45, GFP, Factor VII, Eg5, PCSK9,
TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene,
CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene,
Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2
gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E
gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB
gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I
gene, topoisomerase II alpha gene, mutations in the p73 gene,
mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1)
gene, mutations in the PPM1D gene, mutations in the RAS gene,
mutations in the caveolin I gene, mutations in the MIB I gene,
mutations in the MTAI gene, mutations in the M68 gene, mutations in
tumor suppressor genes, and mutations in the p53 tumor suppressor
gene.
14. A pharmaceutical composition comprising an iRNA agent of claim
1 alone or in combination with a pharmaceutically acceptable
carrier or excipient.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/328,537, filed Dec. 4, 2008, which claims
the benefit of priority to U.S. Provisional Patent Application Ser.
No. 60/992,309, filed Dec. 4, 2007; and U.S. Provisional Patent
Application Ser. No. 61/013,597 filed Dec. 13, 2007, all of which
are incorporated herein by reference in their entirety.
BACKGROUND
[0003] Oligonucleotide compounds have important therapeutic
applications in medicine. Oligonucleotides can be used to silence
genes that are responsible for a particular disease. Gene-silencing
prevents formation of a protein by inhibiting translation.
Importantly, gene-silencing agents are a promising alternative to
traditional small, organic compounds that inhibit the function of
the protein linked to the disease. siRNA, antisense RNA, and
micro-RNA are oligonucleotides that prevent the formation of
proteins by gene-silencing.
[0004] RNA interference or "RNAi" is a term initially coined by
Fire and co-workers to describe the observation that
double-stranded RNA (dsRNA) can block gene expression when it is
introduced into worms (Fire et al. (1998) Nature 391, 806-811).
Short dsRNA directs gene-specific, post-transcriptional silencing
in many organisms, including vertebrates, and has provided a new
tool for studying gene function. RNAi is mediated by RNA-induced
silencing complex (RISC), a sequence-specific, multi-component
nuclease that destroys messenger RNAs homologous to the silencing
trigger. RISC is known to contain short RNAs (approximately 22
nucleotides) derived from the double-stranded RNA trigger, but the
protein components of this activity remained unknown.
[0005] siRNA compounds are promising agents for a variety of
diagnostic and therapeutic purposes. siRNA compounds can be used to
identify the function of a gene. In addition, siRNA compounds offer
enormous potential as a new type of pharmaceutical agent which acts
by silencing disease-causing genes. Research is currently underway
to develop interference RNA therapeutic agents for the treatment of
many diseases including central-nervous-system diseases,
inflammatory diseases, metabolic disorders, oncology, infectious
diseases, and ocular disease.
[0006] siRNA has been shown to be extremely effective as a
potential anti-viral therapeutic with numerous published examples
appearing recently. siRNA molecules directed against targets in the
viral genome dramatically reduce viral titers by orders of
magnitude in animal models of influenza (Ge et. al., Proc. Natl.
Acd. Sci. USA, 101:8676-8681 (2004); Tompkins et. al., Proc. Natl.
Acd. Sci. USA, 101:8682-8686 (2004); Thomas et. al., Expert Opin.
Biol. Ther. 5:495-505 (2005)), respiratory synctial virus (RSV)
(Bitko et. al., Nat. Med. 11:50-55 (2005)), hepatitis B virus (HBV)
(Morrissey et. al., Nat. Biotechnol. 23:1002-1007 (2005)),
hepatitis C virus (Kapadia, Proc. Natl. Acad. Sci. USA,
100:2014-2018 (2003); Wilson et. al., Proc. Natl. Acad. Sci. USA,
100:2783-2788 (2003)) and SARS coronavirus (Li et. al., Nat. Med.
11:944-951 (2005)).
[0007] Antisense methodology is the complementary hybridization of
relatively short oligonucleotides to mRNA or DNA such that the
normal, essential functions, such as protein synthesis, of these
intracellular nucleic acids are disrupted. Hybridization is the
sequence-specific hydrogen bonding via Watson-Crick base pairs of
oligonucleotides to RNA or single-stranded DNA. Such base pairs are
said to be complementary to one another.
[0008] The naturally-occurring events that alter the expression
level of the target sequence, discussed by Cohen (Oligonucleotides:
Antisense Inhibitors of Gene Expression, CRC Press, Inc., 1989,
Boca Raton, Fla.) are thought to be of two types. The first,
hybridization arrest, describes the terminating event in which the
oligonucleotide inhibitor binds to the target nucleic acid and thus
prevents, by simple steric hindrance, the binding of essential
proteins, most often ribosomes, to the nucleic acid. Methyl
phosphonate oligonucleotides (Miller et al. (1987) Anti-Cancer Drug
Design, 2:117-128), and .alpha.-anomer oligonucleotides are the two
most extensively studied antisense agents which are thought to
disrupt nucleic acid function by hybridization arrest.
[0009] Another means by which antisense oligonucleotides alter the
expression level of target sequences is by hybridization to a
target mRNA, followed by enzymatic cleavage of the targeted RNA by
intracellular RNase H. A 2'-deoxyribofuranosyl oligonucleotide or
oligonucleotide analog hybridizes with the targeted RNA and this
duplex activates the RNase H enzyme to cleave the RNA strand, thus
destroying the normal function of the RNA. Phosphorothioate
oligonucleotides are the most prominent example of an antisense
agent that operates by this type of antisense terminating
event.
[0010] The opportunity to use these and other nucleic acid based
therapies holds significant promise, providing solutions to medical
problems that could not be addressed with current, traditional
medicines. The location and sequences of an increasing number of
disease-related genes are being identified, and clinical testing of
nucleic acid-based therapeutics for a variety of diseases is now
underway.
[0011] Despite the advances in application of oligonucleotides and
oligonucleotide analogs as therapeutics, the need exists for
oligonucleotides having improved pharmacologic properties. Efforts
aimed at improving the transmembrane delivery of nucleic acids and
oligonucleotides have utilized protein carriers, antibody carriers,
liposomal delivery systems, electroporation, direct injection, cell
fusion, viral vectors, and calcium phosphate-mediated
transformation. However, many of these techniques are limited by
the types of cells in which transmembrane transport is enabled and
by the conditions needed for achieving such transport. Some
progress has been made on increasing the cellular uptake of
single-stranded oligonucleotides, including increasing the membrane
permeability via conjugates and cellular delivery of
oligonucleotides. In U.S. Pat. No. 6,656,730, M. Manoharan
describes compositions in which a ligand that binds serum,
vascular, or cellular proteins may be attached via an optional
linking moiety to one or more sites on an oligonucleotide. These
sites include one or more of, but are not limited to, the
2'-position, 3'-position, 5'-position, the internucleotide linkage,
and a nucleobase atom of any nucleotide residue.
[0012] Unlike many of the methods mentioned above, receptor
mediated endocytotic activity can be used successfully both in
vitro and in vivo. This mechanism of uptake involves the movement
of ligands bound to membrane receptors into the interior of an area
that is enveloped by the membrane via invagination of the membrane
structure. This process is initiated via activation of a
cell-surface or membrane receptor following binding of a specific
ligand to the receptor. Many receptor-mediated endocytotic systems
are known and have been studied, including those that recognize
sugars such as galactose, mannose, mannose-6-phosphate, peptides
and proteins such as transferrin, asialoglycoprotein, vitamin B12,
insulin and epidermal growth factor (EGF). The Asialoglycoprotein
receptor (ASGP-R) is a high capacity receptor, which is highly
abundant on hepatocytes. The ASGP-R shows a 50-fold higher affinity
for N-Acetyl-D-Galactosylamine (GalNAc) than D-Gal. Previous work
has shown that multivalency is required to achieve nM affinity,
while spacing among sugars is also crucial. Multivalent GalNAc
clusters and galactosylated carrier systems have been successfully
used to target small molecules to hepatocytes in vivo and in
vitro.
[0013] Receptor mediated endocytosis has been well studied and is
known to be a critical pathway for the uptake and internalization
of a variety of cellular nutrients. These are highly developed
mechanisms because of their critical role in providing nutrients to
cells and in maintaining cellular physiology. Thus many examples of
the utilization of receptor mediated endocytosis pathways for the
delivery of drugs, proteins, nucleic acids and other molecules to
cells are known.
[0014] One way in which this has been applied is the conjugation of
essential nutrients that are actively transported into cells with
the drug or molecule of interest. The transporters or receptors
involved in the uptake are capable of recognizing the nutrient
portion of the conjugate and ferrying the entire conjugate into the
cell. Examples of nutrients that are actively transported into
cells and that may be of use in conjugates include, but are not
limited to, folic acid, vitamin B6, cholesterol and vitamin B12.
Such molecules have been conjugated to macromolecules such as
nucleic acids and oligonucleotides to afford conjugates that
exhibit improved cellular penetration. Manorharan et al., PCT
Application WO 93/07883; Low et al., U.S. Pat. Nos. 5,108,921,
5,416,016.
[0015] Folic acid and its various forms, such as dihydrofolate and
tetrahydrofolate, are essential vitamins that are crucial for the
biosynthesis of nucleic acids and therefore are critical to the
survival and proliferation of cells. Folate cofactors play an
important role in the one-carbon transfers that are critical for
the biosynthesis of pyrimidine nucleosides. Cells therefore have a
sophisticated system of transporting folates into the cytoplasm.
Uptake of folates occurs by two different pathways depending on the
cell type. Cells expressing a carrier or transporter for folate
that exhibits a low affinity (Kd.about.10.sup.-6 M) for the vitamin
prefer reduced over oxidized forms of folate. Cells that express
membrane receptors called folate binding protein (FBP), in
contrast, exhibit high binding affinity (Kd.about.10.sup.-9 M) and
prefer the oxidized form of the vitamin. This latter receptor is
believed to mediate the uptake of folates into the cytoplasm via
endocytosis.
[0016] The use of biotin conjugates and also folic acid conjugates
to enhance transmembrane transport of exogenous molecules,
including oligonucleotides, has been reported by Low et al., U.S.
Pat. Nos. 5,108,921; 5,416,016; PCT Application WO 90/12096. Folic
acid was conjugated to 3'-aminoalkyl-oligonucleotides at their
3'-terminus via carbodiimide chemistry. The multiplicity of folate
receptors on membrane surfaces of most cells and the associated
receptor mediated endocytotic processes were implicated in the
enhanced transport of these oligonucleotide-folic acid conjugates
into cells. There are however, several limitations to this approach
for the conjugation of folic acid to oligonucleotides.
[0017] Folic acid and many related folates and antifolates exhibit
very poor solubility that hinders the effective conjugation of
folic acid to oligonucleotides and subsequent purification of
oligonucleotide-folic acid conjugates. Further folic acid bears two
reactive carboxylic acid groups that are just as likely to react
with the terminal amino group of the 3-aminoalkyl-oligonucleotide.
Thus conjugation will typically result in a mixture of a- and
g-conjugates arising from the reaction of the a-carboxylate and the
g-carboxylate of the glutamic acid portion of the folic acid
molecule. This poses difficulties from the standpoint of
characterizing the conjugate and further from the standpoint of
polyglutamylation of folates. Polyglutamylation of folates is a
well recognized phenomenon that has significant implications on the
transport, localization and activity of folates. Since
polyglutamylation rates differ significantly between the .alpha.-
and .gamma.-carboxylates, the use of poorly defined mixtures of
oligonucleotide-folate conjugates, as obtained from the Low et al.
procedure, U.S. Pat. No. 5,108,921, will lead to variable transport
and concentration of the conjugate. Further, the conjugation of
folates onto one end of an oligonucleotide may be a disadvantage
because of the known propensity of exonucleases to rapidly cleave
oligonucleotides by excising the terminal residues. Also, it has
been observed that oligonucleotide-folic acid conjugates prepared
in this fashion are light sensitive.
[0018] Therefore, there is a clear need for new
oligonucleotide-folate conjugates, oligonucleotide-carbohydrate
conjugates and methods for their preparation, that address the
shortcomings of oligonucleotide conjugates as described above. The
present invention is directed to this very important end.
SUMMARY OF THE INVENTION
[0019] The present invention provides compounds of formula I
##STR00003##
[0020] wherein:
[0021] X is H, a hydroxyl protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide;
[0022] Y is H, a hydroxyl protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide;
[0023] Q is a tether;
[0024] R is folate, a folate analog, a folate mimic, a folate
receptor binding ligand;
[0025] Z', Z'', Z''' and Z'''' are independently O or S;
[0026] n represent independently for each occurrence 1-20;
[0027] m represent independently for each occurrence 0-50;
[0028] L.sup.6 and L.sup.7 are each independently for each
occurrence --(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2)C(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0029] Q' is NH, O, S, CH.sub.2, C(O)O, C(O)NH,
--NH--CH(R.sup.a)--C(O)--, --C(O)--CH(R.sup.a)--NH--, CO,
##STR00004##
[0030] R.sup.a is H or amino acid side chain.
[0031] In some preferred embodiments R is folate, a folate analog,
a folate mimic or a folate receptor binding ligand.
[0032] In some embodiments, R is
##STR00005##
[0033] Embodiments can include one or more of the features
described above.
[0034] In a further aspect, this invention features an iRNA agent
having a first strand and a second strand, wherein at least one
subunit having a formula (I) is incorporated into at least one of
said strands.
[0035] In one aspect, this invention features an iRNA agent having
a first strand and a second strand, wherein at least two subunits
having a formula (I) are incorporated into at least one of said
strands.
[0036] In another aspect, this invention provides a method of
making an iRNA agent described herein having a first strand and a
second strand in which at least one subunit of formula (I) is
incorporated in the strands. The method includes contacting the
first strand with the second strand.
[0037] In a further aspect, this invention provides a method of
modulating expression of a target gene, the method includes
administering an iRNA agent described herein having a first strand
and a second strand in which at least one subunit of formula (I) is
incorporated in the strands to a subject.
[0038] In one aspect, this invention features a pharmaceutical
composition having an iRNA agent described herein having a first
strand and a second strand in which at least one subunit of formula
(I) is incorporated in the strands and a pharmaceutically
acceptable carrier.
[0039] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0040] FIG. 1. Graphical representation of folate conjugated
sequences.
[0041] FIG. 2. Graphical representation of sequences AL-3609 and
AL-3610.
[0042] FIG. 3. Graphical representation of sequences AL-3664,
AL-3665, AL-3670 and AL-3671.
[0043] FIG. 4. Graphical representation of sequences AL-3639,
AL-3640, AL-3668 and AL-3669.
[0044] FIG. 5. Folate conjugate monomer (compound 108) used in the
synthesis of oligonucleotides.
[0045] FIG. 6. Cholesterol conjugate monomer used in the synthesis
of oligonucleotides.
[0046] FIG. 7. C18 Spacer monomer used in the synthesis of
oligonucleotides.
[0047] FIG. 8. 12 Disulfide linker monomer used in the synthesis of
oligonucleotides.
[0048] FIG. 9. A schematic view of oligonucleotide conjugate design
considerations.
[0049] FIG. 10. Comparison of binding of various folate conjugates
to folate receptor.
[0050] FIG. 11. Effect of folate conjugation on the silencing
activity of siRNAs.
[0051] FIG. 12. In vivo targeting of folate conjugated siRNAs.
[0052] FIG. 13. Tissue levels of folate conjugated siRNAs in
vivo.
[0053] FIG. 14. Tumor distribution of folate conjugated siRNAs.
[0054] FIG. 15. Co-localization of folate conjugated siRNAs with
macrophages.
[0055] FIG. 16. Establishment of KB EGFP tumors in nude mice.
[0056] FIG. 17. Conjugates with endosomolytic linkers.
DETAILED DESCRIPTION
[0057] The inventor has discovered, inter alia, that attachment of
a folic acid moiety to an iRNA agent can optimize one or more
properties of the iRNA agent. In many cases, the folic acid will be
attached to a modified subunit of the iRNA agent. E.g., the ribose
sugar of one or more ribonucleotide subunits of an iRNA agent can
be replaced with another moiety, e.g., a non-carbohydrate
(preferably cyclic) carrier to which is attached a folic acid. 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.
[0058] The carriers further include (i) at least 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" 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 folic acid, folic acid analog, a folic acid mimic or a
ligand capable of binding to the folate receptor. 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.
[0059] In addition to the cyclic carriers described herein, RRMS
can include cyclic carriers described in copending co-owned U.S.
application Ser. No. 10/916,185 filed Aug. 10, 2004, and U.S.
application Ser. No. 10/946,873 filed Sep. 21, 2004, both of which
are hereby incorporated by reference.
[0060] Accordingly, in one aspect, the invention features, a
monomer having the structure shown in formula (I')
##STR00006##
wherein:
[0061] A and B are each independently for each occurrence O,
N(R.sup.N) or S;
[0062] X is H, a protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide;
[0063] Y is H, a protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotid-
e, a nucleotide, or an oligonucleotide;
[0064] R is folate, a folate analog a folate mimic or a folate
receptor binding ligand;
[0065] L.sup.6 and L.sup.7 are each independently for each
occurrence --(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0066] Q' is NH, O, S, CH.sub.2, C(O)O, C(O)NH,
--NH--CH(R.sup.a)--C(O)--, --C(O)--CH(R.sup.a)--NH--, CO,
##STR00007##
[0067] R.sup.a is H or amino acid side chain;
[0068] R' and R'' are each independently H, CH.sub.3, OH, SH,
NH.sub.2, NH(Alkyl=Me, Et, Pr, isoPr, Bu, Bn) or
N(diAlkyl=Me.sub.2, Et.sub.2, Bn.sub.2);
[0069] Z', Z'', Z''' and Z'''' are independently O or S;
[0070] n represent independently for each occurrence 1-20; and
[0071] m represent independently for each occurrence 0-50.
[0072] Accordingly, in one aspect, the invention features, a
monomer having the structure shown in formula (I).
##STR00008##
[0073] wherein:
[0074] X is H, a hydroxyl protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide;
[0075] Y is H, a hydroxyl protecting group, a phosphate group, a
phosphodiester group, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a
PEG, a steroid, a lipophile, a polymer,
--P(Z')(Z'')O-L.sup.6-Q'-L.sup.7-OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide;
[0076] Q is a tether;
[0077] R is folate, a folate analog, a folate mimic, a folate
receptor binding ligand;
[0078] n represent independently for each occurrence 1-20;
[0079] m represent independently for each occurrence 0-50;
[0080] L.sup.6 and L.sup.7 are each independently for each
occurrence --(CH.sub.2).sub.n--, --C(R')(R'')(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R')(R'')--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0081] Q' is NH, O, S, CH.sub.2, C(O)O, C(O)NH,
--NH--CH(R.sup.a)--C(O)--, --C(O)--CH(R.sup.a)--NH--, CO,
##STR00009##
[0082] R.sup.a is H or amino acid side chain.
[0083] In some embodiments, R is
##STR00010##
[0084] In some embodiments, R is one of
##STR00011##
[0085] In some embodiments, R is one of
##STR00012##
[0086] In some embodiments, R is one of
##STR00013##
[0087] In some embodiments, R is
##STR00014##
[0088] In some embodiments, the RRMS has the structure
##STR00015##
[0089] In some embodiments, the RRMS has the structure
##STR00016##
[0090] In some embodiments, RRMS has the structure
##STR00017##
[0091] In some embodiments, RRMS has the structure
##STR00018##
[0092] In some embodiments, RRMS has the structure
##STR00019##
[0093] In some embodiments, RRMS has the structure
##STR00020##
[0094] In some embodiments, RRMS has the structure
##STR00021##
[0095] In some embodiments, RRMS has the structure
##STR00022##
[0096] In some embodiments, RRMS has the
structure
##STR00023##
[0097] In one aspect, the invention features, a compound having the
structure shown in formula (CI)
##STR00024##
[0098] A and B are independently for each occurrence hydrogen,
protecting group, optionally substituted aliphatic, optionally
substituted aryl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--O-nucleoside, or
--P(Z.sup.1)(Z.sup.2)--O-oligonucleotide; wherein Z.sup.1 and
Z.sup.2 are each independently for each occurrence O, S, N(alkyl)
or optionally substituted alkyl;
[0099] J.sub.1 and J.sub.2 are independently O, S, NR.sup.N,
optionally substituted alkyl, OC(O)NH, NHC(O)O, C(O)NH, NHC(O),
OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,
OP(N(R.sup.P).sub.2)O, or OP(N(R.sup.P).sub.2); and
[0100] is 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 decalin; preferably, the acyclic group is
selected from serinol backbone or diethanolamine backbone.
[0101] In preferred embodiments, ligand is a folate or folate
analog. As used herein, the term "folate" is meant to refer to
folate and folate derivatives, including pteroic acid derivatives
and analogs. The analogs and derivatives of folic acid suitable for
use in the present invention include, but are not limited to,
antifolates, dihy-drofloates, tetrahydrofolates,
tetrahydrorpterins, folinic acid, pteropolyglutamic acid, 1-deza,
3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza,
8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic
acid derivatives. Additional folate analogs are described in
published US publication US2004/0,242,582 (published Dec. 2,
2004).
[0102] In one embodiment, the compound is a pyrroline ring system
as shown in formula (CII)
##STR00025##
[0103] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0104] R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16,
R.sup.17, and R.sup.18 are each independently for each occurrence
H, --CH.sub.2OR.sup.a, or OR.sup.b,
[0105] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--O-nucleoside,
--P(Z.sup.1)(Z.sup.2)--O-oligonucleotide,
--P(Z.sup.1)(O-linker-R.sup.L)--O-nucleoside, or
--P(Z.sup.1)(O-linker-R.sup.L)--O-oligonucleotide;
[0106] R.sup.30 is independently for each occurrence
-linker-R.sup.L or R.sup.31;
[0107] R.sup.L is hydrogen or a ligand;
[0108] R.sup.31 is
--C(O)CH(N(R.sup.32).sub.2)(CH.sub.2).sub.hN(R.sup.32).sub.2;
[0109] R.sup.32 is independently for each occurrence H, --R.sup.L,
-linker-R.sup.L or R.sup.31;
[0110] Z.sup.1 is independently for each occurrence O or S;
[0111] Z.sup.2 is independently for each occurrence O, S, N(alkyl)
or optionally substituted alkyl; and
[0112] h is independently for each occurrence 1-20.
[0113] For the pyrroline-based click-carriers, R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.3 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.9 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.11 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.15 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. In certain
embodiments, CH.sub.2OR.sup.a and OR.sup.b may be geminally
substituted. For the 4-hydroxyproline-based carriers, R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. The pyrroline- and
4-hydroxyproline-based compounds may therefore contain linkages
(e.g., carbon-carbon bonds) wherein bond rotation is restricted
about that particular linkage, e.g. restriction resulting from the
presence of a ring. Thus, CH.sub.2OR.sup.a and OR.sup.b may be cis
or trans with respect to one another in any of the pairings
delineated above Accordingly, all cis/trans isomers are expressly
included. The compounds may also contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of the compounds are expressly included
(e.g., the centers bearing CH.sub.2OR.sup.a and OR.sup.b can both
have the R configuration; or both have the S configuration; or one
center can have the R configuration and the other center can have
the S configuration and vice versa).
[0114] In one embodiment, R.sup.1 is CH.sub.2OR.sup.a and R.sup.9
is OR.sup.b.
[0115] In one embodiment, R.sup.b is a solid support.
[0116] In one embodiment, carrier of formula (CII) is a
phosphoramidite, i.e., one of R.sup.a or R.sup.b is
--P(O-alkyl)N(alkyl).sub.2, e.g.,
--P(OCH.sub.2CH.sub.2CN)N(i-propyl).sub.2. In one embodiment,
R.sup.b is --P(O-alkyl)N(alkyl).sub.2.
[0117] In embodiment, the compound is a ribose ring system as shown
in formula (CIII).
##STR00026##
[0118] wherein:
[0119] X is O, S, NR.sup.N or CR.sup.P.sub.2;
[0120] B is independently for each occurrence hydrogen, optionally
substituted natural or non-natural nucleobase, optionally
substituted natural nucleobase conjugated with -linker-R.sup.L or
optionally substituted non-natural nucleobase conjugated with
-linker-R.sup.L;
[0121] R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each
independently for each occurrence H, OR.sup.6, F, N(R.sup.N).sub.2,
or -J-linker-R.sub.L;
[0122] J is absent, O, S, NR.sup.N, OC(O)NH, NHC(O)O, C(O)NH,
NHC(O), NHSO, NHSO.sub.2, NHSO.sub.2NH, OC(O), C(O)O, OC(O)O,
NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R.sup.P).sub.2)O, or
OP(N(R.sup.P).sub.2);
[0123] R.sup.6 is independently for each occurrence hydrogen,
hydroxyl protecting group, optionally substituted alkyl, optionally
substituted aryl, optionally substituted cycloalkyl, optionally
substituted aralkyl, optionally substituted alkenyl, optionally
substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a
diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--O-nucleoside,
--P(Z.sup.1)(Z.sup.2)--O-oligonucleotide,
--P(Z.sup.1)(Z.sup.2)-formula (CIII),
--P(Z.sup.1)(O-linker-R.sup.L)--O-nucleoside,
--P(Z.sup.1)(O-linker-R.sup.L)--O-oligonucleotide, or
--P(Z.sup.1)(O-linker-R.sup.L)--O-formula (CIII);
[0124] R.sup.N is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted heteroaryl or an amino protecting group;
[0125] R.sup.P is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl or optionally substituted heteroaryl;
[0126] R.sup.L is hydrogen or a ligand;
[0127] Z.sup.1 and Z.sup.2 are each independently for each
occurrence O, SN(alkyl) or optionally substituted alkyl; and
[0128] provided that R.sup.L is present at least once and further
provided that R.sup.L is a ligand at least once.
[0129] In one embodiment, the carrier of formula (CI) is an acyclic
group and is termed an "acyclic carrier". Preferred acyclic
carriers can have the structure shown in formula (CIV) or formula
(CV) below.
[0130] In one embodiment, the compound is an acyclic carrier having
the structure shown in formula (CIV).
##STR00027##
[0131] wherein:
[0132] W is absent, O, S and N(R.sup.N), where R.sup.N is
independently for each occurrence H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted cycloalkyl,
optionally substituted aralkyl, optionally substituted heteroaryl
or an amino protecting group;
[0133] E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO,
SO.sub.2, or SO.sub.2NH;
[0134] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--O-nucleoside,
--P(Z.sup.1)(Z.sup.2)--O-oligonucleotide,
--P(Z.sup.1)(O-linker-R.sup.L)--O-nucleoside, or
--P(Z.sup.1)(O-linker-R.sup.L)--O-oligonucleotide;
[0135] R.sup.30 is independently for each occurrence
-linker-R.sup.L or R.sup.31;
[0136] R.sup.L is hydrogen or a ligand;
[0137] R.sup.31 is
--C(O)CH(N(R.sup.32).sub.2)(CH.sub.2).sub.hN(R.sup.32).sub.2;
[0138] R.sup.32 is independently for each occurrence H, --R.sup.L,
-linker-R.sup.L or R.sup.31;
[0139] Z.sup.1 is independently for each occurrence O or S;
[0140] Z.sup.2 is independently for each occurrence O, S, N(alkyl)
or optionally substituted alkyl;
[0141] h is independently for each occurrence 1-20; and
[0142] r, s and t are each independently for each occurrence 0, 1,
2 or 3.
[0143] When r and s are different, then the tertiary carbon can be
either the R or S configuration. In preferred embodiments, x and y
are one and z is zero (e.g. carrier is based on serinol). The
acyclic carriers can optionally be substituted, e.g. with hydroxy,
alkoxy, perhaloalky.
[0144] In one embodiment, the compound is an acyclic carrier having
the structure shown in formula (CV)
##STR00028##
[0145] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0146] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--O-nucleoside,
--P(Z.sup.1)(Z.sup.2)--O-oligonucleotide,
--P(Z.sup.1)(Z.sup.2)-formula (I),
--P(Z.sup.1)(O-linker-R.sup.L)--O-nucleoside, or
--P(Z.sup.1)(O-linker-R.sup.L)--O-oligonucleotide;
[0147] R.sup.30 is independently for each occurrence
-linker-R.sup.L or R.sup.31;
[0148] R.sup.L is hydrogen or a ligand;
[0149] R.sup.31 is
--C(O)CH(N(R.sup.32).sub.2)(CH.sub.2).sub.hN(R.sup.32).sub.2;
[0150] R.sup.32 is independently for each occurrence H, --R.sup.L,
-linker-R.sup.L or R.sup.31;
[0151] Z.sup.1 is independently for each occurrence O or S;
[0152] Z.sup.2 is independently for each occurrence O, S, N(alkyl)
or optionally substituted alkyl; and
[0153] h is independently for each occurrence 1-20; and
[0154] r and s are each independently for each occurrence 0, 1, 2
or 3.
[0155] Other carrier compounds amenable to the invention are
described in copending applications U.S. Ser. No. 10/916,185, filed
Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S.
Ser. No. 10/985,426, filed Nov. 9, 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. 11/119,533, filed Apr. 29, 2005, which are
incorporated by reference in their entireties for all purposes.
[0156] In some embodiments, ligand is chosen from a group
consisting of
##STR00029## ##STR00030## ##STR00031##
[0157] Embodiments can include one or more of the features
described above.
[0158] In a further aspect, this invention features an iRNA agent
having a first strand and a second strand, wherein at least one
subunit having a formula (I) is incorporated into at least one of
said strands.
[0159] In one aspect, this invention features an iRNA agent having
a first strand and a second strand, wherein at least two subunits
having a formula (I) are incorporated into at least one of said
strands.
[0160] In another aspect, this invention provides a method of
making an iRNA agent described herein having a first strand and a
second strand in which at least one subunit of formula (I) is
incorporated in the strands. The method includes contacting the
first strand with the second strand.
[0161] In a further aspect, this invention provides a method of
modulating expression of a target gene, the method includes
administering an iRNA agent described herein having a first strand
and a second strand in which at least one subunit of formula (I) is
incorporated in the strands to a subject.
[0162] In one aspect, this invention features a pharmaceutical
composition having an iRNA agent described herein having a first
strand and a second strand in which at least one subunit of formula
(I) is incorporated in the strands and a pharmaceutically
acceptable carrier.
[0163] RRMS monomers described herein may be incorporated into any
double-stranded RNA-like molecule described herein, e.g., an iRNA
agent. An iRNA agent may include a duplex comprising a hybridized
sense and antisense strand, in which the antisense strand and/or
the sense strand may include one or more of the RRMSs described
herein. An RRMS can be introduced at one or more points in one or
both strands of a double-stranded iRNA agent. An RRMS can be placed
at or near (within 1, 2, or 3 positions) of the 3' or 5' end of the
sense strand or at near (within 1, 2 or 3 positions of) the 3' end
of the antisense strand. In some embodiments it is preferred to not
have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end
of the antisense strand. An RRMS can be internal, and will
preferably be positioned in regions not critical for antisense
binding to the target.
[0164] In an embodiment, an iRNA agent may have an RRMS at (or
within 1, 2, or 3 positions of) the 3' end of the antisense strand.
In an embodiment, an iRNA agent may have an RRMS at (or within 1,
2, or 3 positions of) the 3' end of the antisense strand and at (or
within 1, 2, or 3 positions of) the 3' end of the sense strand. In
an embodiment, an iRNA agent may have an RRMS at (or within 1, 2,
or 3 positions of) the 3' end of the antisense strand and an RRMS
at the 5' end of the sense strand, in which both RRMS are located
at the same end of the iRNA agent.
[0165] In an embodiment, an iRNA agent may have an RRMS at (or
within 1, 2, or 3 positions of) the 3' end of the antisense strand
and an RRMS at the 5' end of the sense strand, in which both RRMSs
may share the same ligand (e.g., folic acid) via connection of
their individual tethers to separate positions on the ligand. A
ligand shared between two proximal RRMSs is referred to herein as a
"hairpin ligand."
[0166] In other embodiments, an iRNA agent may have an RRMS at the
3' end of the sense strand and an RRMS at an internal position of
the sense strand. An iRNA agent may have an RRMS at an internal
position of the sense strand; or may have an RRMS at an internal
position of the antisense strand; or may have an RRMS at an
internal position of the sense strand and an RRMS at an internal
position of the antisense strand.
[0167] In preferred embodiments the iRNA agent includes a first and
second sequences, which are preferably two separate molecules as
opposed to two sequences located on the same strand, have
sufficient complementarity to each other to hybridize (and thereby
form a duplex region), e.g., under physiological conditions, e.g.,
under physiological conditions but not in contact with a helicase
or other unwinding enzyme.
[0168] It is preferred that the first and second sequences be
chosen such that the double stranded iRNA agent includes a single
strand or unpaired region at one or both ends of the molecule.
Thus, a double stranded iRNA agent contains first and second
sequences, preferably paired to contain an overhang, e.g., one or
two 5' or 3' overhangs but preferably a 3' overhang of 2-3
nucleotides. Most embodiments will have a 3' overhang. Preferred
iRNA agents will have single-stranded overhangs, preferably 3'
overhangs, of 1 or preferably 2 or 3 nucleotides in length at each
end. 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. 5' ends are preferably phosphorylated.
[0169] Other modifications to sugars, bases, or backbones can be
incorporated into the iRNA agents.
[0170] The iRNA agents can take an architecture or structure
described herein. The iRNA agents can be palindromic, or double
targeting, as described herein.
[0171] The iRNA agents can have a sequence such that a
non-cannonical or other than cannonical Watson-Crick structure is
formed between two monomers of the iRNA agent or between a strand
of the iRNA agent and another sequence, e.g., a target or
off-target sequence, as is described herein.
[0172] The iRNA agent can be selected to target any of a broad
spectrum of genes, including any of the genes described herein.
[0173] In a preferred embodiment the iRNA agent has an architecture
(architecture refers to one or more of overall length, length of a
duplex region, the presence, number, location, or length of
overhangs, single strand versus double strand form) described
herein. E.g., the iRNA agent can be less than 30 nucleotides in
length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21
nucleotides in length and there is a duplex region of about 19
pairs. In one embodiment, the iRNA is 21 nucleotides in length, and
the duplex region of the iRNA is 19 nucleotides. In another
embodiment, the iRNA is greater than 30 nucleotides in length.
[0174] In some embodiment the duplex region of the iRNA agent will
have, mismatches. Preferably it will have no more than 1, 2, 3, 4,
or 5 bases, which do not form canonical Watson-Crick pairs or which
do not hybridize. Overhangs are discussed in detail elsewhere
herein but are preferably about 2 nucleotides in length. The
overhangs can be complementary to the gene sequences being targeted
or can be other sequence. TT is a preferred overhang sequence. The
first and second iRNA agent sequences can also be joined, e.g., by
additional bases to form a hairpin, or by other non-base
linkers.
[0175] In addition to the RRMS-containing bases the iRNA agents
described herein can include nuclease resistant monomers (NRMs)
described in copending co-owned U.S. Provisional application Ser.
No. 10/553,659 filed on Apr. 14, 2006 and International Application
No. PCT/US04/07070, both of which are hereby incorporated by
reference.
[0176] In some embodiments, the iRNA agent will have a monomer with
the structure shown in formula (LI) in addition to monomer of
formula (I) or formula (I').
##STR00032##
[0177] wherein X.sup.6 and Y.sup.6 are each independently H, a
hydroxyl protecting group, a phosphate group, a phosphodiester
group, an activated phosphate group, an activated phosphite group,
a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside,
--P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a
polymer,
--P(Z')(Z'')O--R.sup.1-Q'-R.sup.2--OP(Z''')(Z'''')O-oligonucleotide,
a nucleotide, or an oligonucleotide, --P(Z')(Z'')-formula (I) or
--P(Z')(Z'')--;
[0178] Q.sup.6 is absent or
--(P.sup.6-Q.sup.6-R.sup.6).sub.v-T.sup.6-;
[0179] P.sup.6 and T.sup.6 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;
[0180] Q.sup.6 is independently for each occurrence absent,
substituted alkylene wherein 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);
[0181] R.sup.6 is 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,
##STR00033##
or heterocyclyl;
[0182] R' and R'' are each independently H, C.sub.1-C.sub.6 alkyl
OH, SH, N(R.sup.N).sub.2;
[0183] R.sup.N is independently for each occurrence hydrogen,
methyl, ethyl, propyl, isopropyl, butyl or benzyl;
[0184] R.sup.a is H or amino acid side chain;
[0185] Z', Z'', Z''' and Z'''' are each independently for each
occurrence O or S;
[0186] v represent independently for each occurrence 0-20;
[0187] R.sup.L is a lipophile (e.g., cholesterol, cholic acid,
adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a
vitamin (e.g., folate, vitamin A, biotin, pyridoxal), a peptide, a
carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide,
tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic
component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene
(e.g., triterpene, e.g., sarsasapogenin, Friedelin, epifriedelanol
derivatized lithocholic acid), or a cationic lipid.
[0188] In some embodiments, one or more, e.g., 1, 2, 3, 4 or 5,
monomers of formula (LI) in addition to one or more, e.g. 1, 2, 3,
4, or 5, monomers of formula (I) or formula (I') are present in the
iRNA agent.
[0189] In some preferred embodiments only 1 monomer of formula (I)
or formula (I') and 1 monomer of formula (LI) are present in the
iRNA agent.
[0190] In some embodiments, R.sup.L is cholesterol.
[0191] In some embodiments, R.sup.L is lithocholic.
[0192] In some embodiments, R.sup.L is oleyl lithocholic.
[0193] In a preferred embodiment, the iRNA agent targets an
exogenous gene of a genetically modified cell. An exogenous gene
can be, for example, a viral or bacterial gene that derives from an
organism that has invaded or infected the cell, or the exogenous
gene can be any gene introduced into the cell by natural or
artificial means, such as by a genetic recombination event. An iRNA
agent can target a viral gene, for example, such as a hepatitis
viral gene (e.g., a gene of an HAV, HBV, or HCV). Alternatively, or
in addition, the iRNA agent can silence a reporter gene, such as
GFP or beta galatosidase and the like. These iRNA agents can be
used to silence exogenous genes in an adherent tumor cell line.
[0194] In another aspect, the invention provides, methods of
silencing a target gene by providing an iRNA agent to which a folic
acid, folic acid analog or folic acid mimic is conjugated. In a
preferred embodiment the conjugated iRNA agent can be used to
silence a target gene in an organism, e.g., a mammal, e.g., a
human, or to silence a target gene in a cell line or in cells which
are outside an organism. In the case of a whole organism, the
method can be used to silence a gene, e.g., a gene described
herein, and treat a condition mediated by the gene. In the case of
use on a cell which is not part of an organism, e.g., a primary
cell line, secondary cell line, tumor cell line, or transformed or
immortalized cell line, the iRNA agent to which a folic acid, folic
acid analog or folic acid mimic is conjugated can be used to
silence a gene, e.g., one described herein. Cells which are not
part of a whole organism can be used in an initial screen to
determine if an iRNA agent is effective in silencing a gene. A test
in cells which are not part of a whole organism can be followed by
testing the iRNA agent in a whole animal. In preferred embodiments,
the iRNA agent which is conjugated to a a carbohydrate, a steroid,
or a steroid tethered to at least one carbohydrate is conjugated is
administered to an organism, or contacted with a cell which is not
part of an organism, in the absence of (or in a reduced amount of)
other reagents that facilitate or enhance delivery, e.g., a
compound which enhances transit through the cell membrane. (A
reduced amount can be an amount of such reagent which is reduced in
comparison to what would be needed to get an equal amount of
nonconjugated iRNA agent into the target cell).
[0195] In a preferred embodiment the iRNA agent is suitable for
delivery to a cell in vivo, e.g., to a cell in an organism. In
another aspect, the iRNA agent is suitable for delivery to a cell
in vitro, e.g., to a cell in a cell line.
[0196] An iRNA agent to which a folic acid, folic acid analog or
folic acid mimic is attached can target any gene described herein
and can be delivered to any cell type described herein, e.g., a
cell type in an organism, tissue, or cell line. Delivery of the
iRNA agent can be in vivo, e.g., to a cell in an organism, or in
vitro, e.g., to a cell in a cell line.
[0197] In another aspect, the invention provides compositions of
iRNA agents described herein, and in particular compositions of an
iRNA agent to which a folate receptor binding ligand e.g. a folic
acid, folic acid analog or folic acid mimic is conjugated, e.g., a
lipophilic conjugated iRNA agent described herein. In a preferred
embodiment the composition is a pharmaceutically acceptable
composition.
[0198] In preferred embodiments, the composition, e.g.,
pharmaceutically acceptable composition, is free of, has a reduced
amount of, or is essentially free of other reagents that facilitate
or enhance delivery, e.g., compounds which enhance transit through
the cell membrane. (A reduced amount can be an amount of such
reagent which is reduced in comparison to what would be needed to
get an equal amount of nonconjugated iRNA agent into the target
cell). E.g., the composition is free of, has a reduced amount of,
or is essentially free of: an additional lipophilic moiety; a
transfection agent, e.g., concentrations of an ion or other
substance which substantially alters cell permeability to an iRNA
agent; a transfecting agent such as Lipofectamine.TM. (Invitrogen,
Carlsbad, Calif.), Lipofectamine 2000.TM., TransIT-TKO.TM. (Mirus,
Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.),
polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),
DOTAP, DOSPER, Metafectene.TM. (Biontex, Munich, Germany), and the
like.
[0199] In a preferred embodiment the composition is suitable for
delivery to a cell in vivo, e.g., to a cell in an organism. In
another aspect, the iRNA agent is suitable for delivery to a cell
in vitro, e.g., to a cell in a cell line.
[0200] The RRMS-containing iRNA agents can be used in any of the
methods described herein, e.g., to target any of the genes
described herein or to treat any of the disorders described herein.
They can be incorporated into any of the formulations, modes of
delivery, delivery modalities, kits or preparations, e.g.,
pharmaceutical preparations, described herein. E.g, a kit which
includes one or more of the iRNA aents described herein, a sterile
container in which the iRNA agent is disclosed, and instructions
for use.
[0201] The methods and compositions of the invention, e.g., the
RRSM-containing iRNA agents described herein, can be used with any
of the iRNA agents described herein. In addition, the methods and
compositions of the invention can be used for the treatment of any
disease or disorder described herein, and for the treatment of any
subject, e.g., any animal, any mammal, such as any human.
[0202] The methods and compositions of the invention, e.g., the
RRMS-containing iRNA agents described herein, can be used with any
dosage and/or formulation described herein, as well as with any
route of administration described herein.
DEFINITIONS
[0203] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0204] The term "alkyl" refers to a hydrocarbon chain that may be a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.12 alkyl indicates that
the group may have from 1 to 12 (inclusive) carbon atoms in it. The
term "haloalkyl" refers to an alkyl in which one or more hydrogen
atoms are replaced by halo, and includes alkyl moieties in which
all hydrogens have been replaced by halo (e.g., perfluoroalkyl).
Alkyl and haloalkyl groups may be optionally inserted with O, N, or
S. The terms "aralkyl" refers to an alkyl moiety in which an alkyl
hydrogen atom is replaced by an aryl group. Aralkyl includes groups
in which more than one hydrogen atom has been replaced by an aryl
group. Examples of "aralkyl" include benzyl, 9-fluorenyl,
benzhydryl, and trityl groups.
[0205] The term "alkenyl" refers to a straight or branched
hydrocarbon chain containing 2-8 carbon atoms and characterized in
having one or more double bonds. Examples of a typical alkenyl
include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl
and 3-octenyl groups. The term "alkynyl" refers to a straight or
branched hydrocarbon chain containing 2-8 carbon atoms and
characterized in having one or more triple bonds. Some examples of
a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and
propargyl. The sp.sup.2 and sp.sup.3 carbons may optionally serve
as the point of attachment of the alkenyl and alkynyl groups,
respectively.
[0206] The terms "alkylamino" and "dialkylamino" refer to
--NH(alkyl) and --N (alkyl).sub.2 radicals respectively. The term
"aralkylamino" refers to a --NH(aralkyl) radical. The term "alkoxy"
refers to an --O-alkyl radical, and the terms "cycloalkoxy" and
"aralkoxy" refer to an --O-cycloalkyl and O-aralkyl radicals
respectively. The term "siloxy" refers to a R.sub.3SiO-- radical.
The term "mercapto" refers to an SH radical. The term "thioalkoxy"
refers to an --S-alkyl radical.
[0207] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--), e.g., --CH.sub.2--, --CH.sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2--. The term "alkylenedioxo" refers to a
divalent species of the structure --O--R--O--, in which R
represents an alkylene.
[0208] The term "aryl" refers to an aromatic monocyclic, bicyclic,
or tricyclic hydrocarbon ring system, wherein any ring atom can be
substituted. Examples of aryl moieties include, but are not limited
to, phenyl, naphthyl, anthracenyl, and pyrenyl.
[0209] The term "cycloalkyl" as employed herein includes saturated
cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups
having 3 to 12 carbons, wherein any ring atom can be substituted.
The cycloalkyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkyl moieties include, but are not limited to, cyclohexyl,
adamantyl, and norbornyl.
[0210] The term "heterocyclyl" refers to a nonaromatic 3-10
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3,
1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or
tricyclic, respectively), wherein any ring atom can be substituted.
The heterocyclyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
heterocyclyl include, but are not limited to tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and
pyrrolidinyl.
[0211] The term "cycloalkenyl" as employed herein includes
partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or
polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5
to 8 carbons, wherein any ring atom can be substituted. The
cycloalkenyl groups herein described may also contain fused rings.
Fused rings are rings that share a common carbon-carbon bond or a
common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkenyl moieties include, but are not limited to cyclohexenyl,
cyclohexadienyl, or norbornenyl.
[0212] The term "heterocycloalkenyl" refers to a partially
saturated, nonaromatic 5-10 membered monocyclic, 8-12 membered
bicyclic, or 11-14 membered tricyclic ring system having 1-3
heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9
heteroatoms if tricyclic, said heteroatoms selected from O, N, or S
(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S
if monocyclic, bicyclic, or tricyclic, respectively), wherein any
ring atom can be substituted. The heterocycloalkenyl groups herein
described may also contain fused rings. Fused rings are rings that
share a common carbon-carbon bond or a common carbon atom (e.g.,
spiro-fused rings). Examples of heterocycloalkenyl include but are
not limited to tetrahydropyridyl and dihydropyran.
[0213] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein any ring atom can be substituted. The
heteroaryl groups herein described may also contain fused rings
that share a common carbon-carbon bond.
[0214] The term "oxo" refers to an oxygen atom, which forms a
carbonyl when attached to carbon, an N-oxide when attached to
nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0215] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents.
[0216] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl,
heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any
atom of that group. Suitable substituents include, without
limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano,
nitro, amino, SO.sub.3H, sulfate, phosphate, perfluoroalkyl,
perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo,
thioxo, imino (alkyl, aryl, aralkyl), S(O).sub.nalkyl (where n is
0-2), S(O).sub.n aryl (where n is 0-2), S(O).sub.n heteroaryl
(where n is 0-2), S(O).sub.n heterocyclyl (where n is 0-2), amine
(mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and
combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide
(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations
thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl,
and combinations thereof), unsubstituted aryl, unsubstituted
heteroaryl, unsubstituted heterocyclyl, and unsubstituted
cycloalkyl. In one aspect, the substituents on a group are
independently any one single, or any subset of the aforementioned
substituents.
[0217] The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and
uracilyl" and the like refer to radicals of adenine, cytosine,
guanine, thymine, and uracil.
[0218] A "protected" moiety refers to a reactive functional group,
e.g., a hydroxyl group or an amino group, or a class of molecules,
e.g., sugars, having one or more functional groups, in which the
reactivity of the functional group is temporarily blocked by the
presence of an attached protecting group. Protecting groups useful
for the monomers and methods described herein can be found, e.g.,
in Greene, T. W., Protective Groups in Organic Synthesis (John
Wiley and Sons: New York), 1981, which is hereby incorporated by
reference.
Ligand-Conjugated Monomers
[0219] Cyclic sugar replacement-based monomers, e.g., sugar
replacement-based ligand-conjugated monomers, are also referred to
herein as ribose replacement monomer subunit (RRMS) monomer
compounds. Preferred carriers have the general formula (I) provided
below. The carriers are an entity which can be incorporated into a
strand. Thus, it is understood that the structures also encompass
the situations wherein one (in the case of a terminal position) or
two (in the case of an internal position) of the attachment points,
e.g., either X or Y in case of terminal position and both X and Y
in case of internal position, is connected to the phosphate, or
modified phosphate, e.g., sulfur containing, backbone.
Tethers
[0220] In certain embodiments, a moiety, e.g., a ligand may be
connected indirectly to the carrier via the intermediacy of an
intervening tether. Tethers are connected to the carrier at a
tethering attachment point (TAP) and may include any
C.sub.1-C.sub.100 carbon-containing moiety, (e.g. C.sub.1-C.sub.75,
C.sub.1-C.sub.50, C.sub.1-C.sub.20, C.sub.1-C.sub.10; C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8,
C.sub.9, or C.sub.10), preferably having at least one nitrogen
atom. In preferred embodiments, the nitrogen atom forms part of a
terminal amino or amido (NHC(O)--) group on the tether, which may
serve as a connection point for the ligand. Preferred tethers
(underlined) include TAP-(CH.sub.2--NH--; TAP-C(O)(CH.sub.2)NH--;
TAP-NR''''(CH--).sub.nNH--, TAP-C(O)--(CH.sub.2).sub.n--C(O)--;
TAP-C(O)--(CH.sub.2).sub.n--C(O)O--; TAP-C(O)--O--;
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--; TAP-C(O)--(CH.sub.2).sub.n;
TAP-C(O)--NH--; TAP-C(O)--; TAP-(CH.sub.2).sub.n--C(O)--;
TAP-(CH.sub.2).sub.n--C(O)O--; TAP-(CH.sub.2).sub.n; or
TAP-(CH.sub.2).sub.n--NH--C(O)--;
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH--;
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.n--C(R')(R'')--SS--(-
CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH--;
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.n--SS--(CH.sub.2).su-
b.nCH(R''')--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH--;
TAP-(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nC(R')(R'')--SS--(CH.sub.2-
).sub.n--; in which each n is independently 1-20 (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), R',
R'' and R''' are described elsewhere and R'''' is C.sub.1-C.sub.6
alkyl. Preferably, n is 2, 5, 6, or 11. In other embodiments, the
nitrogen may form part of a terminal oxyamino group, e.g.,
--ONH.sub.2, or hydrazino group, --NHNH.sub.2. The tether may
optionally be substituted, e.g., with hydroxy, alkoxy,
perhaloalkyl, and/or optionally inserted with one or more
additional heteroatoms, e.g., N, O, or S. Preferred tethered
ligands may include, e.g., TAP-(CH.sub.2).sub.nNH(LIGAND);
TAP-C(O)(CH.sub.2).sub.nNH(LIGAND);
TAP-NR''''(CH.sub.2).sub.nNH(LIGAND);
TAP-(CH.sub.2).sub.nONH(LIGAND);
TAP-C(O)(CH.sub.2).sub.nONH(LIGAND);
TAP-NR''''(CH.sub.2).sub.nONH(LIGAND);
TAP-(CH.sub.2).sub.nNHNH.sub.2(LIGAND),
TAP-C(O)(CH.sub.2).sub.nNHNH.sub.2(LIGAND);
TAP-NR''''(CH.sub.2).sub.nNHNH.sub.2(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--C(O)(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--C(O)O(LIGAND); TAP-C(O)--O(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n(LIGAND); TAP-C(O)--NH(LIGAND);
TAP-C(O)(LIGAND); TAP-(CH.sub.2).sub.n--C(O) (LIGAND);
TAP-(CH.sub.2)--C(O)O(LIGAND); TAP-(CH.sub.2).sub.n(LIGAND);
TAP-(CH.sub.2).sub.n--NH--C(O)(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nC(R')(R')--SS--(CH.-
sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.n--SS--(CH.sub.2).su-
b.nCH(R''')--NH--C(O)--(CH.sub.2).sub.nCH(R''')NH(LIGAND);
TAP-(CH.sub.2).sub.n--NH--C(O)--(CH.sub.2).sub.nC(R')(R'')--SS--(CH.sub.2-
).sub.n(LIGAND). In some embodiments, amino terminated tethers
(e.g., NH.sub.2, ONH.sub.2, NH.sub.2NH.sub.2) can form an imino
bond (i.e., C.dbd.N) with the ligand. In some embodiments, amino
terminated tethers (e.g., NH.sub.2, ONH.sub.2, NH.sub.2NH.sub.2)
can be acylated, e.g., with C(O)CF.sub.3.
[0221] In some embodiments, the tether can terminate with a
mercapto group (i.e., SH) or an olefin (e.g., CH.dbd.CH.sub.2). For
example, the tether can be TAP-(CH.sub.2).sub.n--SH,
TAP-C(O)(CH.sub.2).sub.nSH,
TAP-(CH.sub.2).sub.n--(CH.dbd.CH.sub.21--, or
TAP-C(O)(CH.sub.2).sub.n(CH.dbd.CH.sub.2--, in which n can be as
described elsewhere. The tether may optionally be substituted,
e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally
inserted with one or more additional heteroatoms, e.g., N, O, or S.
The double bond can be cis or trans or E or Z.
[0222] In other embodiments the tether may include an electrophilic
moiety, preferably at the terminal position of the tether.
Preferred electrophilic moieties include, e.g., an aldehyde, alkyl
halide, mesylate, tosylate, nosylate, or brosylate, or an activated
carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl
ester. Preferred tethers (underlined) include
TAP-(CH.sub.2).sub.nCHO; TAP-C(O)(CH.sub.2).sub.nCHO; or
TAP-NR''''(CH.sub.2).sub.nCHO, in which n is 1-6 and R'''' is
C.sub.1-C.sub.6 alkyl; or TAP-(CH.sub.2).sub.nC(O)ONHS;
TAP-C(O)(CH.sub.2).sub.nC(O)ONHS; or
TAP-NR''''(CH.sub.2).sub.nC(O)ONHS, in which n is 1-6 and R'''' is
C.sub.1-C.sub.6 alkyl; TAP-(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5;
TAP-C(O)(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5; or
TAP-NR''''(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5, in which n is 1-11
and R'''' is C.sub.1-C.sub.6 alkyl; or
--(CH.sub.2).sub.nCH.sub.2LG; TAP-C(O)(CH.sub.2).sub.nCH.sub.2LG;
or TAP-NR''''(CH.sub.2).sub.nCH.sub.2LG, in which n can be as
described elsewhere and R'''' is C.sub.1-C.sub.6 alkyl (LG can be a
leaving group, e.g., halide, mesylate, tosylate, nosylate,
brosylate). Tethering can be carried out by coupling a nucleophilic
group of a ligand, e.g., a thiol or amino group with an
electrophilic group on the tether.
[0223] In other embodiments, it can be desirable for the monomer to
include a phthalimido group (K) at the terminal position of the
tether.
##STR00034##
[0224] In other embodiments, other protected amino groups can be at
the terminal position of the tether, e.g., alloc, monomethoxy
trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the
aryl portion can be ortho-nitrophenyl or ortho,
para-dinitrophenyl).
[0225] Any of the tethers described herein may further include one
or more additional linking groups, e.g., --O--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.n--SS--, --(CH.sub.2)--, or --(CH.dbd.CH)--.
Ligands
[0226] A wide variety of entities, e.g., ligands, can be tethered
to an iRNA agent, e.g., to the carrier of a ligand-conjugated
monomer subunit. Examples are described below in the context of a
ligand-conjugated monomer subunit but that is only preferred,
entities can be coupled at other points to an iRNA agent.
[0227] Preferred moieties are ligands, which are coupled,
preferably covalently, either directly or indirectly via an
intervening tether, to the carrier. In preferred embodiments, the
ligand is attached to the carrier via an intervening tether. As
discussed above, the ligand or tethered ligand may be present on
the ligand-conjugated monomer when the ligand-conjugated monomer is
incorporated into the growing strand. In some embodiments, the
ligand may be incorporated into a "precursor" ligand-conjugated
monomer subunit after a "precursor" ligand-conjugated monomer
subunit has been incorporated into the growing strand. For example,
a monomer having, e.g., an amino-terminated tether, e.g.,
TAP-(CH.sub.2)NH.sub.2 may be incorporated into a growing sense or
antisense strand. In a subsequent operation, i.e., after
incorporation of the precursor monomer subunit into the strand, a
ligand having an electrophilic group, e.g., a pentafluorophenyl
ester or aldehyde group, can subsequently be attached to the
precursor ligand-conjugated monomer by coupling the electrophilic
group of the ligand with the terminal nucleophilic group of the
precursor ligand-conjugated monomer subunit tether.
[0228] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of an iRNA agent 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, 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.
[0229] Preferred 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.
[0230] 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;
nuclease-resistance conferring moieties; and natural or unusual
nucleobases.
[0231] General examples include lipids, steroids (e.g.,
cholesterol, uvaol, hecigenin, diosgenin), terpenes (e.g.,
triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol
derivatized lithocholic acid), vitamins (e.g., folic acid, folic
acid mimics, folate receptor binding ligands, vitamin A, vitamin E,
biotin, pyridoxal), carbohydrates, proteins, protein binding
agents, integrin targeting molecules, CCR5 receptor antagonists,
CCR5 receptor binding ligands, polycationics (e.g., porphyrins),
peptides, polyamines, peptide mimics, PEG. In some embodiments, the
ligand can be one of the following:
##STR00035##
[0232] 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 certain
embodiments, 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 GAL4 peptide (Subbarao
et al., Biochemistry, 1987, 26: 2964-2972), the EALA (SEQ ID NO: 1)
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 certain embodiments, 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.
Exemplary primary sequences of peptide based endosomolytic ligands
are shown in Table 1.
TABLE-US-00001 TABLE 1 List of peptides with endosomolytic
activity. SEQ ID Name Sequence (N to C) Ref. NO: GALA
AALEALAEALEALAEALEALAEAAAAGGC 1 2 EALA
AALAEALAEALAEALAEALAEALAAAAGGC 2 3 (SEQ ID NO: 1) ALEALAEALEALAEA 3
4 INF-7 GLFEAIEGFIENGWEGMIWDYG 4 5 Inf GLFGAIAGFIENGWEGMIDGWYG 5 6
HA-2 diINF- GLF EAI EGFI ENGW EGMI DGWYGC 5 7 7 GLF EAI EGFI ENGW
EGMI DGWYGC diINF3 GLF EAI EGFI ENGW EGMI DGGC 6 8 GLF EAI EGFI
ENGW EGMI DGGC GLF GLFGALAEALAEALAEHLAEALAEALEALAAGGS 6 9 C GALA-
GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 10 INF3 INF-5 GLF EAI EGFI
ENGW EGnI DG K 4 11 GLF EAI EGFI ENGW EGnI DG 12 n, norleucine
References 1. Subbarao et al., Biochemistry, 1987, 26: 2964-2972.
2. Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586 3. Turk,
M. J., Reddy, J. A. et al. (2002). Characterization of a novel
pH-sensitive peptide that enhances drug release from
folate-targeted liposomes at endosomal pHs. Biochim. Biophys. Acta
1559, 56-68. 4. Plank, C. Oberhauser, B. Mechtler, K. Koch, C.
Wagner, E. (1994). The influence of endosome-disruptive peptides on
gene transfer using synthetic virus-like gene transfer systems, J.
Biol. Chem. 269 12918-12924. 5. Mastrobattista, E., Koning, G. A.
et al. (2002). Functional characterization of an
endosome-disruptive peptide and its application in cytosolic
delivery of immunoliposome-entrapped proteins. J. Biol. Chem. 277,
27135-43. 6. Oberhauser, B., Plank, C. et al. (1995). Enhancing
endosomal exit of nucleic acids using pH-sensitive viral fusion
peptides. Deliv. Strategies Antisense Oligonucleotide Ther.
247-66.
[0233] 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.
[0234] 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.
[0235] 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); an
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.
[0236] 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. Table 2 shows some examples of targeting ligands and their
associated receptors.
TABLE-US-00002 TABLE 2 Targeting Ligands and their associated
receptors Liver Cells Ligand Receptor Negatively charged Scavenger
receptors molecules 1) Parenchymal Cell (PC) Galactose ASGP-R
(Hepatocytes) (Asiologlycoprotein receptor) Gal NAc ASPG-R
(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin
ASPG-r 2) Sinusoidal Endothelial Hyaluronan Hyaluronan receptor
Cell (SEC) Procollagen Procollagen receptor Mannose Mannose
receptors N-acetyl Glucosamine Scavenger receptors Immunoglobulins
Fc Receptor LPS CD14 Receptor Insulin Receptor mediated
transcytosis Transferrin Receptor mediated transcytosis Albumins
Non-specific Sugar-Albumin conjugates Mannose-6-phosphate
Mannose-6-phosphate receptor 3) Kupffer Cell (KC) Mannose Mannose
receptors Fucose Fucose receptors Albumins Non-specific
Mannose-albumin conjugates
[0237] 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 (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, bomeol, 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.
[0238] 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.
[0239] 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.
[0240] The ligand can increase the uptake of the iRNA agent into
the cell by activating an inflammatory response, for example.
Exemplary ligands that would have such an effect include tumor
necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma
interferon.
[0241] 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, neproxin 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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 are B
vitamin, 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).
[0246] 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.
[0247] 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
(see Table 3, for example).
TABLE-US-00003 TABLE 3 Exemplary Cell Permeation Peptides. Cell SEQ
Permeation ID Peptide Amino acid Sequence Reference NO: Penetratin
RQIKIWFQNRRMKWKK Derossi et al., J. Biol. 13 Chem. 269: 10444, 1994
Tat fragment GRKKRRQRRRPPQC Vives et al., J. Biol. 14 (48-60)
Chem., 272: 16010, 1997 Signal GALFLGWLGAAGSTMGAWSQP Chaloin et
al., 15 Sequence- KKKRKV Biochem. Biophys. based peptide Res.
Commun., 243: 601, 1998 PVEC LLIILRRRIRKQAHAHSK Elmquist et al.,
Exp. 16 Cell Res., 269: 237, 2001 Transportan
GWTLNSAGYLLKINLKALAALA Pooga et al., FASEB J., 17 KKIL 12: 67, 1998
Amphiphilic KLALKLALKALKAALKLA Oehlke et al., Mol. 18 model peptide
Ther., 2: 339, 2000 Arg.sub.9 RRRRRRRRR Mitchell et al., J. Pept.
19 Res., 56: 318, 2000 Bacterial cell KFFKFFKFFK 20 wall permeating
LL-37 LLGDFFRKSKEKIGKEFKRIVQR 21 IKDFLRNLVPRTES Cecropin P1
SWLSKTAKKLENSAKKRISEGIA 22 IAIQGGPR .alpha.-defensin
ACYCRIPACIAGERRYGTCIYQG 23 RLWAFCC b-defensin
DHYNCVSSGGQCLYSACPIFTKI 24 QGTCYRGKAKCCK Bactenecin RKCRIVVIRVCR 25
PR-39 RRRPRPPYLPRPRPPPFFPPRLPP 26 RIPPGFPPRFPPRFPGKR-NH2
Indolicidin ILPWKWPWWPWRR-NH2 27
[0248] 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: 28). An
RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:
29)) 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: 14)) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 13)) 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.
[0249] 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).
[0250] Peptides that target markers enriched in proliferating cells
can be used. E.g., RGD containing peptides and peptidomimetics can
target cancer cells, in particular cells that exhibit an
I.sub.v.theta..sub.3 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 I.sub.v-.theta..sub.3 integrin
ligand. Generally, such ligands can be used to control
proliferating cells and angiogeneis. Preferred conjugates of this
type lignads that targets PECAM-1, VEGF, or other cancer gene,
e.g., a cancer gene described herein.
[0251] 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).
[0252] 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, H.sub.2A 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.
[0253] 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.
[0254] 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.
[0255] Endosomal release agents include imidazoles, poly or
oligoimidazoles, PEIs, peptides, fusogenic peptides,
polycarboxylates, 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.
[0256] PK modulator stands for pharmacokinetic modulator. PK
modulator include lipophiles, bile acids, steroids, phospholipid
analogues, peptides, protein binding agents, PEG, vitamins etc.
Examplary PK modulator 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 of phosphorothioate linkages in the backbaone
are also amenable to the present invention as ligands (e.g. as PK
modulating ligands).
[0257] In addition, aptamers that bind serum components (e.g. serum
proteins) are also amenable to the present invention as PK
modulating ligands.
[0258] Other ligands amenable to the invention are described in
copending applications U.S. 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.
[0259] 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.
[0260] 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.
[0261] The ligand or tethered ligand may be present on a monomer
when said monomer is incorporated into the growing strand. In some
embodiments, the ligand may be incorporated via coupling to a
"precursor" monomer after said "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)NH.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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] There are numerous methods for preparing conjugates of
oligomeric compounds. Generally, an oligomeric compound is attached
to a conjugate moiety by contacting a reactive group (e.g., OH, SH,
amine, carboxyl, aldehyde, and the like) on the oligomeric compound
with a reactive group on the conjugate moiety. In some embodiments,
one reactive group is electrophilic and the other is
nucleophilic.
[0266] For example, an electrophilic group can be a
carbonyl-containing functionality and a nucleophilic group can be
an amine or thiol. Methods for conjugation of nucleic acids and
related oligomeric compounds with and without linking groups are
well described in the literature such as, for example, in Manoharan
in Antisense Research and Applications, Crooke and LeBleu, eds.,
CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is
incorporated herein by reference in its entirety.
[0267] Representative United States patents that teach the
preparation of oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904, 582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254, 469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;
5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599,923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737;
6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806;
6,486,308; 6,525,031; 6,528,631; 6,559,279; each of which is herein
incorporated by reference.
[0268] The monomers and methods described herein can be used in the
preparation of modified RNA, e.g., an iRNA agent, which
incorporates a RRMS, such as those described herein and those
described in copending co-owned U.S. application Ser. Nos.
10/916,185 filed Aug. 10, 2004, 10/946,873 filed Sep. 21, 2004,
10/985,426 filed Nov. 9, 2004 and 11/833,934 filed Aug. 3, 2007,
all of which are hereby incorporated by reference.
[0269] Modified RNA molecules include e.g. those molecules
containing a chemically or stereochemically modified nucleoside
(e.g., having one or more backbone modifications, e.g.,
phosphorothioate or P-alkyl; having one or more sugar
modifications, e.g., 2'-OCH.sub.3 or 2'-F; and/or having one or
more base modifications, e.g., 5-alkylamino or 5-allylamino) or a
nucleoside surrogate.
[0270] Coupling of 5'-hydroxyl groups with phosphoramidites forms
phosphite ester intermediates, which in turn are oxidized e.g.,
with iodine, to the phosphate diester. Alternatively, the
phosphites may be treated with e.g., sulfur, selenium, amino, and
boron reagents to form modified phosphate backbones. Linkages
between the monomers described herein and a nucleoside or
oligonucleotide chain can also be treated with iodine, sulfur,
selenium, amino, and boron reagents to form unmodified and modified
phosphate backbones respectively. Similarly, the monomers described
herein may be coupled with nucleosides or oligonucleotides
containing any of the modifications or nucleoside surrogates
described herein.
[0271] The synthesis and purification of oligonucleotide ligand
conjugates can be performed by established methods. See, for
example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan,
"Oligonucleotide Conjugates in Antisense Technology," in Antisense
Drug Technology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001.
iRNA Agent Structure
[0272] The monomers described herein can be used to make
oligonucleotides which are useful as iRNA agents, e.g., RNA
molecules, (double-stranded; single-stranded) that mediate RNAi,
e.g., with respect to an endogenous gene of a subject or to a gene
of a pathogen. In most cases the iRNA agent will incorporate
momomers described herein together with naturally occurring
nucleosides or nucleotides or with other modified nucleosides or
nucleotides. The modified monomers can be present at any position
in the iRNA agent, e.g., at the terminii or in the middle region of
an iRNA agent or in a duplex region or in an unpaired region. In a
preferred embodiment iRNA agent can have any architecture, e.g.,
architecture described herein. e.g., it can be incorporated into an
iRNA agent having an overhang structure, a hairpin or other single
strand structure or a two-strand structure, as described
herein.
[0273] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogate, all of which are defined
herein (see, e.g., the section below entitled RNA Agents). While
numerous modified RNAs and nucleoside surrogates are described,
preferred examples include those which have greater resistance to
nuclease degradation than do unmodified RNAs. Preferred examples
include those which have a 2' sugar modification, a modification in
a single strand overhang, preferably a 3' single strand overhang,
or, particularly if single stranded, a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group.
[0274] An "iRNA agent" as used herein, is an RNA agent which can,
or which can be cleaved into an RNA agent which can, down regulate
the expression of a target gene, preferably an endogenous or
pathogen target RNA. While not wishing to be bound by theory, an
iRNA agent may act by one or more of a number of mechanisms,
including post-transcriptional cleavage of a target mRNA sometimes
referred to in the art as RNAi, or pre-transcriptional or
pre-translational mechanisms. An iRNA agent can include a single
strand or can include more than one strands, e.g., it can be a
double stranded iRNA agent. If the iRNA agent is a single strand it
is particularly preferred that it include a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group.
[0275] The RRMS-containing iRNA agent should include a region of
sufficient homology to the target gene, and be of sufficient length
in terms of nucleotides, such that the iRNA agent, or a fragment
thereof, can mediate down regulation of the target gene. (For ease
of exposition the term nucleotide or ribonucleotide is sometimes
used herein in reference to one or more monomeric subunits of an
RNA agent. It will be understood herein that the usage of the term
"ribonucleotide" or "nucleotide", herein can, in the case of a
modified RNA or nucleotide surrogate, also refer to a modified
nucleotide, or surrogate replacement moiety at one or more
positions.) Thus, the iRNA agent is or includes a region which is
at least partially, and in some embodiments fully, complementary to
the target RNA. It is not necessary that there be perfect
complementarity between the iRNA agent and the target, but the
correspondence must be sufficient to enable the iRNA agent, or a
cleavage product thereof, to direct sequence specific silencing,
e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
[0276] As discussed elsewhere herein, an iRNA agent will often be
modified or include nucleoside surrogates in addition to the ribose
replacement modification subunit (RRMS). Single stranded regions of
an iRNA agent will often be modified or include nucleoside
surrogates, e.g., the unpaired region or regions of a hairpin
structure, e.g., a region which links two complementary regions,
can have modifications or nucleoside surrogates. Modification to
stabilize one or more 3'- or 5'-terminus of an iRNA agent, e.g.,
against exonucleases, or to favor the antisense strand of the iRNA
agent to enter into RISC are also favored. Modifications can
include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl
linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic,
triethylene glycol, hexaethylene glycol), special biotin or
fluorescein reagents that come as phosphoramidites and that have
another DMT-protected hydroxyl group, allowing multiple couplings
during RNA synthesis.
[0277] In addition to homology to target RNA and the ability to
down regulate a target gene, an iRNA agent will preferably have one
or more of the following properties: [0278] (1) if single stranded
it will preferably have a 5' modification which includes one or
more phosphate groups or one or more analogs of a phosphate group;
[0279] (2) it will, despite modifications, even to a very large
number, or all of the nucleosides, have an antisense strand that
can present bases (or modified bases) in the proper three
dimensional framework so as to be able to form correct base pairing
and form a duplex structure with a homologous target RNA which is
sufficient to allow down regulation of the target, e.g., by
cleavage of the target RNA; [0280] (3) it will, despite
modifications, even to a very large number, or all of the
nucleosides, still have "RNA-like" properties, i.e., it will
possess the overall structural, chemical and physical properties of
an RNA molecule, even though not exclusively, or even partly, of
ribonucleotide-based content. For example, an iRNA agent can
contain, e.g., a sense and/or an antisense strand in which all of
the nucleotide sugars contain e.g., 2' fluoro in place of 2'
hydroxyl. This deoxyribonucleotide-containing agent can still be
expected to exhibit RNA-like properties; [0281] (4) regardless of
the nature of the modification, and even though the RNA agent can
contain deoxynucleotides or modified deoxynucleotides, particularly
in overhang or other single strand regions, it is preferred that
DNA molecules, or any molecule in which more than 50, 60, or 70% of
the nucleotides in the molecule, or more than 50, 60, or 70% of the
nucleotides in a duplexed region are deoxyribonucleotides, or
modified deoxyribonucleotides which are deoxy at the 2' position,
are excluded from the definition of RNA agent.
[0282] A "single strand iRNA agent" as used herein, is an iRNA
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand iRNA
agents are preferably antisense with regard to the target molecule.
In preferred embodiments single strand iRNA agents are 5'
phosphorylated or include a phosphoryl analog at the 5' prime
terminus. 5'-phosphate modifications include those which are
compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO).sub.2(O)P--O-5');
5'-diphosphate ((HO).sub.2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO).sub.2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO).sub.2(O)P--S-5'); any additional
combination of oxygen/sulfur replaced monophosphate, diphosphate
and triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO).sub.2(O)P--NH-5', (HO)(NH.sub.2)(O)P--O-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH.sub.2--),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl
(MeOCH.sub.2--), ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-). (These
modifications can also be used with the antisense strand of a
double stranded iRNA.)
[0283] It may be desirable to modify one or both of the antisense
and sense strands of a double strand iRNA agent. In some cases they
will have the same modification or the same class of modification
but in other cases the sense and antisense strand will have
different modifications, e.g., in some cases it is desirable to
modify only the sense strand. It may be desirable to modify only
the sense strand, e.g., to inactivate it, e.g., the sense strand
can be modified in order to inactivate the sense strand and prevent
formation of an active sRNA/protein or RISC. This can be
accomplished by a modification which prevents 5'-phosphorylation of
the sense strand, e.g., by modification with a 5'-O-methyl
ribonucleotide (see Nykanen et al., (2001) ATP requirements and
small interfering RNA structure in the RNA interference pathway.
Cell 107, 309-321.) Other modifications which prevent
phosphorylation can also be used, e.g., simply substituting the
5'-OH by H rather than O-Me. Alternatively, a large bulky group may
be added to the 5'-phosphate turning it into a phosphodiester
linkage, though this may be less desirable as phosphodiesterases
can cleave such a linkage and release a functional sRNA 5'-end.
Antisense strand modifications include 5' phosphorylation as well
as any of the other 5' modifications discussed herein, particularly
the 5' modifications discussed above in the section on single
stranded iRNA molecules.
[0284] In some cases the sense and the antisense strands will
include different modifications. Multiple different modifications
can be included on the sense and antisense strands. The
modifications on each strand may differ from each other, and may
also differ from the various modifications on the other strand. For
example, the sense strand may have a modification, e.g., a
modification described herein, and the antisense strand may have a
different modification, e.g., a different modification described
herein. In other cases, one strand, such as the sense strand may
have two different modifications, and the antisense strand may
include a modification that differs from the at least two
modifications on the sense strand.
[0285] It is preferred that the sense and antisense strands be
chosen such that the double stranded iRNA agent includes a single
strand or unpaired region at one or both ends of the molecule.
Thus, a double-stranded iRNA agent contains sense and antisense
strands, preferable paired to contain an overhang, e.g., one or two
5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides.
Most embodiments will have a 3' overhang. Preferred sRNA agents
will have single-stranded overhangs, preferably 3' overhangs, of 1
or preferably 2 or 3 nucleotides in length at each end. 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. 5' ends are preferably phosphorylated.
[0286] Preferred lengths for the duplexed region is between 15 and
30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the sRNA agent range discussed above. sRNA agents
can resemble in length and structure the natural Dicer processed
products from long dsRNAs. Embodiments in which the two strands of
the sRNA agent are linked, e.g., covalently linked are also
included. Hairpin, or other single strand structures which provide
the required double stranded region, and preferably a 3' overhang
are also within the invention.
[0287] The monomers and methods described herein can also be used
in the preparation of oligonucleotides other than iRNA agents. In
the context of this invention, the term "oligonucleotide" refers to
a polymer or oligomer of nucleotide or nucleoside monomers
consisting of naturally occurring bases, sugars and intersugar
(backbone) linkages. The term "oligonucleotide" also includes
polymers or oligomers comprising non-naturally occurring monomers,
or portions thereof, which function similarly. Such modified or
substituted oligonucleotides are often preferred over native forms
because of properties such as, for example, enhanced cellular
uptake and increased stability in the presence of nucleases.
[0288] The oligonucleotides used herein can be single-stranded DNA
or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples
of double-stranded DNA include structural genes, genes including
control and termination regions, and self-replicating systems such
as viral or plasmid DNA. Examples of double-stranded RNA include
siRNA, iRNA agents and other RNA interference reagents.
Single-stranded nucleic acids include, e.g., antisense
oligonucleotides, ribozymes, microRNA, and triplex-forming
oligonucleotides. The oligonucleotides of this invention may
include one or more of the oligonucleotide modifications described
herein.
[0289] Nucleic acids of the present invention may be of various
lengths, generally dependent upon the particular form of nucleic
acid. For example, in particular embodiments, plasmids or genes may
be from about 1,000 to 100,000 nucleotide residues in length. In
particular embodiments, oligonucleotides may range from about 10 to
100 nucleotides in length. In various related embodiments,
oligonucleotides, single-stranded, double-stranded, and
triple-stranded, may range in length from about 10 to about 50
nucleotides, from about 20 o about 50 nucleotides, from about 15 to
about 30 nucleotides, from about 20 to about 30 nucleotides in
length.
[0290] In particular embodiments, an oligonucleotide (or a strand
thereof) of the present invention specifically hybridizes to or is
complementary to a target polynucleotide. "Specifically
hybridizable" and "complementary" are terms which are used to
indicate a sufficient degree of complementarity such that stable
and specific binding occurs between the DNA or RNA target and the
oligonucleotide. It is understood that an oligonucleotide need not
be 100% complementary to its target nucleic acid sequence to be
specifically hybridizable. An oligonucleotide is specifically
hybridizable when binding of the oligonucleotide to the target
interferes with the normal function of the target molecule to cause
a loss of utility or expression therefrom, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment,
or, in the case of in vitro assays, under conditions in which the
assays are conducted. Thus, in other embodiments, this
oligonucleotide includes 1, 2, or 3 base substitutions, e.g.
mismatches, as compared to the region of a gene or mRNA sequence
that it is targeting or to which it specifically hybridizes. Some
exemplary oligonucleotides include antisense oligonucleotide,
ribozymes, apatamers, microRNAs and antagomirs.
[0291] As nucleic acids are polymers of subunits or monomers, many
of the modifications described below occur at a position which is
repeated within a nucleic acid, e.g., a modification of a base, or
a phosphate moiety, or the 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, and infact in most 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 regions,
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 an RNA
or may only occur in a single strand region of an RNA. E.g., a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, may only occur in a terminal regions,
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.
[0292] In some embodiments it is particularly preferred, 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. E.g.,
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
will be modified, e.g., with a modification described herein.
Modifications can include, e.g., the use of modifications at the 2'
OH group of the ribose sugar, e.g., the use of
deoxyribonucleotides, e.g., deoxythymidine, instead of
ribonucleotides, and modifications in the phosphate group, e.g.,
phosphothioate modifications. Overhangs need not be homologous with
the target sequence.
[0293] Unmodified oligoribonucleotides may be less than optimal in
some applications, e.g., unmodified oligoribonucleotides can be
prone to degradation by e.g., cellular nucleases. Nucleases can
hydrolyze nucleic acid phosphodiester bonds. However, chemical
modifications to one or more of the RNA components can confer
improved properties, and, e.g., can render oligoribonucleotides
more stable to nucleases. Unmodified oligoribonucleotides may also
be less than optimal in terms of offering tethering points for
attaching ligands or other moieties to an iRNA agent.
[0294] Modified nucleic acids and nucleotide surrogates can include
one or more of: [0295] (i) alteration of the backbone, e.g.,
replacement, of one or both of the non-linking phosphate oxygens
and/or of one or more of the linking phosphate oxygens in the
backbone. For simplicity of terminology, except where otherwise
noted, one of the three non-linking oxygens at the 5' end of a
nucleic acid and at the 3' end of a nucleic acid, are within the
term "linking phosphate oxygens" as used herein.); [0296] (ii)
alteration, e.g., replacement, of a constituent of the ribose
sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale
replacement of the ribose sugar with a structure other than ribose,
e.g., as described herein; [0297] (iii) wholesale replacement of
the phosphate diester with "dephospho" linkers; [0298] (iv)
modification or replacement of a naturally occurring base; [0299]
(v) replacement or modification of the ribose-phosphate backbone
(bracket II); [0300] (vi) modification of the 3' end or 5' end of
the RNA, e.g., removal, modification or replacement of a terminal
phosphate group or conjugation of a moiety, e.g. a fluorescently
labeled moiety, to either the 3' or 5' end of RNA.
[0301] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid bur rather modified simply
indicates a difference from a naturally occurring molecule.
The Phosphate Group
[0302] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-linking oxygen
atoms. However, the phosphate group can be modified by replacing
one or both of the oxygens with a different substituent. One result
of this modification to RNA phosphate backbones can be increased
resistance of the oligoribonucleotide to nucleolytic breakdown.
Thus while not wishing to be bound by theory, it can be desirable
in some embodiments to introduce alterations which result in either
an uncharged linker or a charged linker with unsymmetrical charge
distribution.
[0303] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. Phosphorodithioates have
both non-linking oxygens replaced by sulfur.
[0304] The phosphate group can be replaced by non-phosphorus
containing connectors While not wishing to be bound by theory, it
is believed that since the charged phosphodiester group is the
reaction center in nucleolytic degradation, its replacement with
neutral structural mimics should impart enhanced nuclease
stability. Again, while not wishing to be bound by theory, it can
be desirable, in some embodiment, to introduce alterations in which
the charged phosphate group is replaced by a neutral moiety.
[0305] Examples of moieties which can replace the phosphate group
include siloxane, carbonate, carboxymethyl, carbamate, amide,
thioether, ethylene oxide linker, sulfonate, sulfonamide,
thioformacetal, formacetal, oxime, methyleneimino,
methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo
and methyleneoxymethylimino. Preferred replacements include the
methylenecarbonylamino and methylenemethylimino groups.
The Sugar Group
[0306] A modified RNA can include modification of all or some of
the sugar groups of the ribonucleic acid. For example, the 2'
hydroxyl group (OH) can be modified or replaced with a number of
different "oxy" or "deoxy" substituents. While not being bound by
theory, enhanced stability is expected since the hydroxyl can no
longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide
can catalyze degradation by intramolecular nucleophilic attack on
the linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0307] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0308] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality. Preferred substitutents are 2'-methoxyethyl,
2'-OCH3, 2'-O-allyl, 2'-C-- allyl, and 2'-fluoro.
[0309] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, a modified RNA can include
nucleotides containing e.g., arabinose, as the sugar.
[0310] Modified RNAs can also include "abasic" sugars, which lack a
nucleobase at C-1'. These abasic sugars can also be further contain
modifications at one or more of the constituent sugar atoms.
[0311] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0312] The modification can also entail the wholesale replacement
of a ribose structure with another entity at one or more sites in
the iRNA agent.
Replacement of Ribophosphate Backbone
[0313] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates. While not
wishing to be bound by theory, it is believed that the absence of a
repetitively charged backbone diminishes binding to proteins that
recognize polyanions (e.g. nucleases). Again, while not wishing to
be bound by theory, it can be desirable in some embodiment, to
introduce alterations in which the bases are tethered by a neutral
surrogate backbone.
[0314] Examples include the mophilino, cyclobutyl, pyrrolidine and
peptide nucleic acid (PNA) nucleoside surrogates. A preferred
surrogate is a PNA surrogate.
Terminal Modifications
[0315] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. For example, the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a spacer. The
terminal atom of the spacer can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' O, N, S or C group
of the sugar. Alternatively, the spacer can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs). These
spacers or linkers can include e.g., --(CH.sub.2).sub.n--,
--(CH.sub.2)N--, --(CH.sub.2).sub.nO--, --(CH.sub.2)S--,
O(CH.sub.2CH.sub.2O)CH.sub.2CH.sub.2OH (e.g., n=3 or 6), abasic
sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,
disulfide, thiourea, sulfonamide, or morpholino, or biotin and
fluorescein reagents. When a spacer/phosphate-functional molecular
entity-spacer/phosphate array is interposed between two strands of
iRNA agents, this array can substitute for a hairpin RNA loop in a
hairpin-type RNA agent. The 3' end can be an --OH group. While not
wishing to be bound by theory, it is believed that conjugation of
certain moieties can improve transport, hybridization, and
specificity properties. Again, while not wishing to be bound by
theory, it may be desirable to introduce terminal alterations that
improve nuclease resistance. Other examples of terminal
modifications 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 (e.g. EDTA),
lipophilic carriers (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).
[0316] Terminal modifications can be added for a number of reasons,
including as discussed elsewhere herein to modulate activity or to
modulate resistance to degradation. Terminal modifications useful
for modulating activity include modification of the 5' end with
phosphate or phosphate analogs. E.g., in preferred embodiments iRNA
agents, especially antisense strands, are 5' phosphorylated or
include a phosphoryl analog at the 5' prime terminus. 5'-phosphate
modifications include those which are compatible with RISC mediated
gene silencing.
[0317] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa
488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include cholesterol. Terminal
modifications can also be useful for cross-linking an RNA agent to
another moiety; modifications useful for this include mitomycin
C.
The Bases
[0318] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. For example, nuclease
resistant oligoribonucleotides can be prepared with these bases or
with synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases, e.g., "unusual
bases" and "universal bases" described herein, can be employed.
Examples include without limitation 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine,
5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles,
2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil,
uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,
5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil,
3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine,
5-methylcytosine, N.sup.4-acetyl cytosine, 2-thiocytosine,
N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases. Further purines and pyrimidines include those
disclosed in U.S. Pat. No. 3,687,808, those disclosed in the
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613.
[0319] Generally, base changes are less preferred for promoting
stability, but they can be useful for other reasons, e.g., some,
e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent.
Modified bases can reduce target specificity. This should be taken
into consideration in the design of iRNA agents.
Exemplary Modifications and Placement within an iRNA Agent
[0320] Some modifications may preferably be included on an iRNA
agent at a particular location, e.g., on the sense strand or
antisense strand, or on the 5' or 3' end of the sense or antisense
strand of an iRNA agent. A preferred location of a modification on
an iRNA agent, may confer preferred properties on the agent. For
example, preferred locations of particular modifications may confer
optimum gene silencing properties, or increased resistance to
endonuclease or exonuclease activity. A modification described
herein and below may be the sole modification, or the sole type of
modification included on multiple ribonucleotides, or a
modification can be combined with one or more other modifications
described herein and below. For example, a modification on a sense
strand of a dsRNA agent can be different than a modification on the
antisense strand of an iRNA agent. Similarly, two different
modifications on the sense strand can differ from a modification on
the antisense strand. Other additional unique modifications,
without limitation, can be incorporates into the sense and
antisense strands.
[0321] An iRNA agent may include a backbone modification to any
nucleotide on an iRNA strand. For example, an iRNA agent may
include a phosphorothioate linkage or P-alkyl modification in the
linkages between one or more nucleotides of an iRNA agent. The
nucleotides can be terminal nucleotides, e.g., nucleotides at the
last position of a sense or antisense strand, or internal
nucleotides.
[0322] An iRNA agent can include a sugar modification, e.g., a 2'
or 3' sugar modification. Exemplary sugar modifications include,
for example, a 2'-O-methylated nucleotide, a 2'-deoxy nucleotide,
(e.g., a 2'-deoxyfluoro nucleotide), a 2'-O-methoxyethyl
nucleotide, a 2'-O-NMA, a 2'-DMAEOE, a 2'-aminopropyl, 2'-hydroxy,
or a 2'-ara-fluoro or a locked nucleic acid (LNA), extended nucleic
acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid
(CeNA). A 2' modification is preferably 2'OMe, and more preferably,
2'-deoxyfluoro. When the modification is 2'OMe, the modification is
preferably on the sense strand. When the modification is a 2'
fluoro, and the modification may be on the sense or antisense
strand, or on both strands. A 2'-ara-fluoro modification will
preferably be on the sense strand of the iRNA agent. An LNA
modification will preferably be on the sense strand of the iRNA
agent or on the
[0323] An iRNA agent may include a 3' sugar modification, e.g., a
3'OMe modification. Preferably a 3'OMe modification is on the sense
strand of the iRNA agent.
[0324] An iRNA agent may includes a 5'-methyl-pyrimidine (e.g., a
5'-methyl-uridine modification or a 5'-methyl-cytodine)
modification.
[0325] The modifications described herein can be combined onto a
single iRNA agent. For example, an iRNA agent may have a
phosphorothioate linkage and a 2' sugar modification, e.g., a 2'OMe
or 2'F modification. In another example, an iRNA agent may include
at least one 5' Me-pyrimidine and a 2' sugar modification, e.g., a
2'F or 2'OMe modification.
[0326] An iRNA agent may include a nucleobase modification, such as
a cationic modification, such as a 3'-abasic cationic modification.
The cationic modification can be e.g., an alkylamino-dT (e.g., a C6
amino-dT), an allylamino conjugate, a pyrrolidine conjugate, a
pthalamido, a porphyrin, or a hydroxyprolinol conjugate, on one or
more of the terminal nucleotides of the iRNA agent. When an
alkylamino-dT conjugate is attached to the terminal nucleotide of
an iRNA agent, the conjugate is preferably attached to the 3' end
of the sense or antisense strand of an iRNA agent. When a
pyrrolidine linker is attached to the terminal nucleotide of an
iRNA agent, the linker is preferably attached to the 3' or 5' end
of the sense strand, or the 3' end of the antisense strand. When a
pyrrolidine linker is attached to the terminal nucleotide of an
iRNA agent, the linker is preferably on the 3' or 5' end of the
sense strand, and not on the 5' end of the antisense strand.
[0327] One or more nucleotides of an iRNA agent may have a 2'-5'
linkage. Preferably, the 2'-5' linkage is on the sense strand. When
the 2'-5' linkage is on the terminal nucleotide of an iRNA agent,
the 2'-5' linkage occurs on the 5' end of the sense strand.
[0328] The iRNA agent may include an L-sugar, preferably on the
sense strand, and not on the antisense strand.
[0329] The iRNA agent may include a methylphosphonate modification.
When the methylphosphonate is on the terminal nucleotide of an iRNA
agent, the methylphosphonate is at the 3' end of the sense or
antisense strands of the iRNA agent.
[0330] An iRNA agent may be modified by replacing one or more
ribonucleotides with deoxyribonucleotides. Preferably, adjacent
deoxyribonucleotides are joined by phosphorothioate linkages, and
the iRNA agent does not include more than four consecutive
deoxyribonucleotides on the sense or the antisense strands.
[0331] An iRNA agent may include a difluorotoluoyl (DFT)
modification, e.g., 2,4-difluorotoluoyl uracil, or a guanidine to
inosine substitution.
[0332] The iRNA agent may include at least one
5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide, or a terminal 5'-uridine-guanine-3'
(5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2'-modified
nucleotide, or a terminal 5'-cytidine-adenine-3' (5'-CA-3')
dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide,
or a terminal 5'-uridine-uridine-3' (5'-UU-3') dinucleotide,
wherein the 5'-uridine is a 2'-modified nucleotide, or a terminal
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide. The chemically modified
nucleotide in the iRNA agent may be a 2'-O-methylated nucleotide.
In some embodiments, the modified nucleotide can be a 2'-deoxy
nucleotide, a 2'-deoxyfluoro nucleotide, a 2'-O-methoxyethyl
nucleotide, a 2'-O-NMA, a 2'-DMAEOE, a 2'-aminopropyl, 2'-hydroxy,
or a 2'-ara-fluoro, or a locked nucleic acid (LNA), extended
nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene
nucleic acid (CeNA). The iRNA agents including these modifications
are particularly stabilized against exonuclease activity, when the
modified dinucleotide occurs on a terminal end of the sense or
antisense strand of an iRNA agent, and are otherwise particularly
stabilized against endonuclease activity.
[0333] An iRNA agent may have a single overhang, e.g., one end of
the iRNA agent has a 3' or 5' overhang and the other end of the
iRNA agent is a blunt end, or the iRNA agent may have a double
overhang, e.g., both ends of the iRNA agent have a 3' or 5'
overhang, such as a dinucleotide overhang. In another alternative,
both ends of the iRNA agent may have blunt ends.
[0334] The iRNA agent may further include a sense RNA strand and an
antisense RNA strand, wherein the antisense RNA strand is 25 or
fewer nucleotides in length, and includes an antisense nucleotide
sequence having 18-25 nucleotides in length. The iRNA agent may
further include a nucleotide overhang having 1 to 4 unpaired
nucleotides, which may be at the 3'-end of the antisense RNA
strand, and the nucleotide overhang may have the nucleotide
sequence 5'-GC-3' or 5'-CGC-3'. The unpaired nucleotides may have
at least one phosphorothioate dinucleotide linkage, and at least
one of the unpaired nucleotides may be chemically modified in the
2'-position. The doublestrand region of the iRNA agent may include
phosphorothioate dinucleotide linkages on one or both of the sense
and antisense strands. The antisense RNA strand and the sense RNA
strand may be connected with a linker, e.g., a chemical linker such
as hexaethylene glycol linker, a
poly-(oxyphosphinico-oxy-1,3-propandiol) linker, an allyl linker,
or a polyethylene glycol linker.
REFERENCES
General References
[0335] The oligoribonucleotides and oligoribonucleosides used in
accordance with this invention may be with solid phase synthesis,
see for example "Oligonucleotide synthesis, a practical approach",
Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A
Practical Approach", Ed. F. Eckstein, IRL Press, 1991 (especially
Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide
synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter
3,2'-O-Methyloligoribonucleotide-s: synthesis and applications,
Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis
of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of
oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7,
Oligodeoxynucleotides containing modified bases. Other particularly
useful synthetic procedures, reagents, blocking groups and reaction
conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,
486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,
2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993,
49, 6123-6194, or references referred to therein.
[0336] Modification described in WO 00/44895, WO01/75164, or
WOO02/44321 can be used herein.
[0337] The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
Phosphate Group References
[0338] The preparation of phosphinate oligoribonucleotides is
described in U.S. Pat. No. 5,508,270. The preparation of alkyl
phosphonate oligoribonucleotides is described in U.S. Pat. No.
4,469,863. The preparation of phosphoramidite oligoribonucleotides
is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
The preparation of phosphotriester oligoribonucleotides is
described in U.S. Pat. No. 5,023,243. The preparation of borano
phosphate oligoribonucleotide is described in U.S. Pat. Nos.
5,130,302 and 5,177,198. The preparation of 3'-Deoxy-3'-amino
phosphoramidate oligoribonucleotides is described in U.S. Pat. No.
5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is
described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
Preparation of sulfur bridged nucleotides is described in Sproat et
al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0339] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
Replacement of the Phosphate Group References
[0340] Methylenemethylimino linked oligoribonucleosides, also
identified herein as MMI linked oligoribonucleosides,
methylenedimethylhydrazo linked oligoribonucleosides, also
identified herein as MDH linked oligoribonucleosides, and
methylenecarbonylamino linked oligonucleosides, also identified
herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified
herein as amide-4 linked oligoribonucleosides as well as mixed
backbone compounds having, as for instance, alternating MMI and PO
or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications
PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and
92/20823, respectively). Formacetal and thioformacetal linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
Nos. 5,264,562 and 5,264,564. Ethylene oxide linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
No. 5,223,618. Siloxane replacements are described in Cormier, J.
F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements
are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933.
Carboxymethyl replacements are described in Edge, M. D. et al. J.
Chem. Soc. Perkin Trans. 11972, 1991. Carbamate replacements are
described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0341] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They may also be prepared in accordance with U.S.
Pat. No. 5,539,083.
Terminal Modification References
[0342] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Bases References
[0343] N-2 substituted purine nucleoside amidites can be prepared
as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine
nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can
be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908. Additional references can be disclosed in
the above section on base modifications.
Nuclease Resistant Monomers
[0344] An iRNA agent can include monomers which have been modified
so as to inhibit degradation, e.g., by nucleases, e.g.,
endonucleases or exonucleases, found in the body of a subject.
These monomers are referred to herein as NRMs, or nuclease
resistance promoting monomers or modifications. In many cases these
modifications will modulate other properties of the iRNA agent as
well, e.g., the ability to interact with a protein, e.g., a
transport protein, e.g., serum albumin, or a member of the
RISC(RNA-induced Silencing Complex), or the ability of the first
and second sequences to form a duplex with one another or to form a
duplex with another sequence, e.g., a target molecule.
[0345] The monomers and methods described herein can be used to
prepare an RNA, e.g., an iRNA agent, that incorporates a nuclease
resistant monomer (NRM), such as those described herein and those
described in copending, co-owned U.S. Provisional application Ser.
No. 10/553,659 filed on Apr. 14, 2006, and International
Application No. PCT/US04/07070, both of which are hereby
incorporated by reference.
Linkers
[0346] The term "linker" means an organic moiety that connects two
parts of a compound. Linkers typically comprise a direct bond or an
atom such as oxygen or sulfur, a unit such as NR.sup.1, C(O),
C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of atoms, such as
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, arylalkyl,
arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,
heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,
heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,
cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,
alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,
alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,
alkylheteroarylalkyl, alkylheteroarylalkenyl,
alkylheteroarylalkynyl, alkenylheteroarylalkyl,
alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
alkynylheteroarylalkyl, alkynylheteroarylalkenyl,
alkynylheteroarylalkynyl, alkylheterocyclylalkyl,
alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,
alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,
alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,
alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,
alkynylhereroaryl, where one or more methylenes can be interrupted
or terminated by O, S, S(O), SO.sub.2, N(R.sup.1).sub.2, C(O),
cleavable linking group, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclic; where R.sup.1 is hydrogen, acyl,
aliphatic or substituted aliphatic. It is further understood that
the term "linker" also encompasses tethers. In one embodiment, the
linker is --[(P-Q''--R).sub.q--X--(P'-Q'''-R').sub.q'].sub.q''-T-,
wherein:
[0347] P, R, T, P', R' and T are each independently for each
occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2,
CH.sub.2NH, CH.sub.2OO; NHCH(R.sup.a)C(O),
--C(O)--CH(R.sup.a)--NH--, CH.dbd.N--O,
##STR00036##
or heterocyclyl;
[0348] Q'' and Q''' are each independently for each occurrence
absent, --(CH.sub.2).sub.n--,
--C(R.sup.1)(R.sup.2)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.1)(R.sup.2)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0349] X is absent or a cleavable linking group;
[0350] R.sup.a is H or an amino acid side chain;
[0351] R.sup.1 and R.sup.2 are each independently for each
occurrence H, CH.sub.3, OH, SH or N(R.sup.N).sub.2;
[0352] R.sup.N is independently for each occurrence H, methyl,
ethyl, propyl, isopropyl, butyl or benzyl;
[0353] q, q' and q'' are each independently for each occurrence
0-20 and wherein the repeating unit can be the same or
different;
[0354] n is independently for each occurrence 1-20; and
[0355] m is independently for each occurrence 0-50.
[0356] In one embodiment, the linker comprises at least one
cleavable linking group.
[0357] In certain embodiments, the linker is a branched linker. The
branchpoint of the branched linker may be at least trivalent, but
may be a tetravalent, pentavalent or hexavalent atom, or a group
presenting such multiple valencies. In certain embodiments, the
branchpoint is, --N, --N(O)--C, --O--C, --S--C, --SS--C,
--C(O)N(O)--C, --OC(O)N(O)--C, --N(O)C(O)--C, or --N(O)C(O)O--C;
wherein Q is independently for each occurrence H or optionally
substituted alkyl. In other embodiment, the branchpoint is glycerol
or glycerol derivative.
[0358] In certain embodiments, the linker is a branched linker. The
branchpoint of the branched linker may be at least trivalent, but
may be a tetravalent, pentavalent or hexavalent atom, or a group
presenting such multiple valencies. In certain embodiments, the
branchpoint is, --N, --N(O)--C, --O--C, --S--C, --SS--C,
--C(O)N(O)--C, --OC(O)N(O)--C, --N(O)C(O)--C, or --N(O)C(O)O--C;
wherein Q is independently for each occurrence H or optionally
substituted alkyl. In other embodiment, the branchpoint is glycerol
or glycerol derivative.
Cleavable Linking Groups
[0359] A cleavable linking group is one which is sufficiently
stable outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least 10 times or more, preferably at least 100 times faster in the
target cell or under a first reference condition (which can, e.g.,
be selected to mimic or represent intracellular conditions) than in
the blood of a subject, or under a second reference condition
(which can, e.g., be selected to mimic or represent conditions
found in the blood or serum).
[0360] Cleavable linking groups are susceptible to cleavage agents,
e.g., pH, redox potential or the presence of degradative molecules.
Generally, cleavage agents are more prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples
of such degradative agents include: redox agents which are selected
for particular substrates or which have no substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents
such as mercaptans, present in cells, that can degrade a redox
cleavable linking group by reduction; esterases; endosomes or
agents that can create an acidic environment, e.g., those that
result in a pH of five or lower; enzymes that can hydrolyze or
degrade an acid cleavable linking group by acting as a general
acid, peptidases (which can be substrate specific), and
phosphatases.
[0361] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0362] A linker can include a cleavable linking group that is
cleavable by a particular enzyme. The type of cleavable linking
group incorporated into a linker can depend on the cell to be
targeted. For example, liver targeting ligands can be linked to the
cationic lipids through a linker that includes an ester group.
Liver cells are rich in esterases, and therefore the linker will be
cleaved more efficiently in liver cells than in cell types that are
not esterase-rich. Other cell-types rich in esterases include cells
of the lung, renal cortex, and testis.
[0363] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0364] In general, the suitability of a candidate cleavable linking
group can be evaluated by testing the ability of a degradative
agent (or condition) to cleave the candidate linking group. It will
also be desirable to also test the candidate cleavable linking
group for the ability to resist cleavage in the blood or when in
contact with other non-target tissue. Thus one can determine the
relative susceptibility to cleavage between a first and a second
condition, where the first is selected to be indicative of cleavage
in a target cell and the second is selected to be indicative of
cleavage in other tissues or biological fluids, e.g., blood or
serum. The evaluations can be carried out in cell free systems, in
cells, in cell culture, in organ or tissue culture, or in whole
animals. It may be useful to make initial evaluations in cell-free
or culture conditions and to confirm by further evaluations in
whole animals. In preferred embodiments, useful candidate compounds
are cleaved at least 2, 4, 10 or 100 times faster in the cell (or
under in vitro conditions selected to mimic intracellular
conditions) as compared to blood or serum (or under in vitro
conditions selected to mimic extracellular conditions).
Redox Cleavable Linking Groups
[0365] One class of cleavable linking groups are redox cleavable
linking groups that are cleaved upon reduction or oxidation. An
example of reductively cleavable linking group is a disulphide
linking group (--S--S--). To determine if a candidate cleavable
linking group is a suitable "reductively cleavable linking group,"
or for example is suitable for use with a particular iRNA moiety
and particular targeting agent one can look to methods described
herein. For example, a candidate can be evaluated by incubation
with dithiothreitol (DTT), or other reducing agent using reagents
know in the art, which mimic the rate of cleavage which would be
observed in a cell, e.g., a target cell. The candidates can also be
evaluated under conditions which are selected to mimic blood or
serum conditions. In a preferred embodiment, candidate compounds
are cleaved by at most 10% in the blood. In preferred embodiments,
useful candidate compounds are degraded at least 2, 4, 10 or 100
times faster in the cell (or under in vitro conditions selected to
mimic intracellular conditions) as compared to blood (or under in
vitro conditions selected to mimic extracellular conditions). The
rate of cleavage of candidate compounds can be determined using
standard enzyme kinetics assays under conditions chosen to mimic
intracellular media and compared to conditions chosen to mimic
extracellular media.
Phosphate-Based Cleavable Linking Groups
[0366] Phosphate-based cleavable linking groups are cleaved by
agents that degrade or hydrolyze the phosphate group. An example of
an agent that cleaves phosphate groups in cells are enzymes such as
phosphatases in cells. Examples of phosphate-based linking groups
are --O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--,
--S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--,
--O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)--O--,
--O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--,
--S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are
--O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--,
--S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--,
--O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--,
--O--P(S)(H)--O--, --S--P(O)(H)--O--, --S--P(S)(H)--O--,
--S--P(O)(H)--S--, --O--P(S)(H)--S--. A preferred embodiment is
--O--P(O)(OH)--O--. These candidates can be evaluated using methods
analogous to those described above.
Acid Cleavable Linking Groups
[0367] Acid cleavable linking groups are linking groups that are
cleaved under acidic conditions. In preferred embodiments acid
cleavable linking groups are cleaved in an acidic environment with
a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower),
or by agents such as enzymes that can act as a general acid. In a
cell, specific low pH organelles, such as endosomes and lysosomes
can provide a cleaving environment for acid cleavable linking
groups. Examples of acid cleavable linking groups include but are
not limited to hydrazones, esters, and esters of amino acids. Acid
cleavable groups can have the general formula --C.dbd.NN--, C(O)O,
or --OC(O). A preferred embodiment is when the carbon attached to
the oxygen of the ester (the alkoxy group) is an aryl group,
substituted alkyl group, or tertiary alkyl group such as dimethyl
pentyl or t-butyl. These candidates can be evaluated using methods
analogous to those described above.
Ester-Based Linking Groups
[0368] Ester-based cleavable linking groups are cleaved by enzymes
such as esterases and amidases in cells. Examples of ester-based
cleavable linking groups include but are not limited to esters of
alkylene, alkenylene and alkynylene groups. Ester cleavable linking
groups have the general formula --C(O)O--, or --OC(O)--. These
candidates can be evaluated using methods analogous to those
described above.
Peptide-Based Cleaving Groups
[0369] Peptide-based cleavable linking groups are cleaved by
enzymes such as peptidases and proteases in cells. Peptide-based
cleavable linking groups are peptide bonds formed between amino
acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)
and polypeptides. Peptide-based cleavable groups do not include the
amide group (--C(O)NH--). The amide group can be formed between any
alkylene, alkenylene or alkynelene. A peptide bond is a special
type of amide bond formed between amino acids to yield peptides and
proteins. The peptide based cleavage group is generally limited to
the peptide bond (i.e., the amide bond) formed between amino acids
yielding peptides and proteins and does not include the entire
amide functional group. Peptide-based cleavable linking groups have
the general formula --NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where
R.sup.A and R.sup.B are the R groups of the two adjacent amino
acids. These candidates can be evaluated using methods analogous to
those described above.
Synthesis
[0370] As can be appreciated by the skilled artisan, methods of
synthesizing the compounds of the formulae herein will be evident
to those of ordinary skill in the art. The synthesized compounds
can be separated from a reaction mixture and further purified by a
method such as column chromatography, high pressure liquid
chromatography, or recrystallization. Additionally, the various
synthetic steps may be performed in an alternate sequence or order
to give the desired compounds. Synthetic chemistry transformations
and protecting group methodologies (protection and deprotection)
useful in synthesizing the compounds described herein are known in
the art and include, for example, those such as described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser
and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of
Reagents for Organic Synthesis, John Wiley and Sons (1995), and
subsequent editions thereof.
Pharmaceutical Compositions
[0371] In one embodiment, the invention relates to a pharmaceutical
composition containing a modified iRNA agent, as described in the
preceding sections, and a pharmaceutically acceptable carrier, as
described below. A pharmaceutical composition including the
modified iRNA agent is useful for treating a disease caused by
expression of a target gene. In this aspect of the invention, the
iRNA agent of the invention is formulated as described below. The
pharmaceutical composition is administered in a dosage sufficient
to inhibit expression of the target gene.
[0372] The pharmaceutical compositions of the present invention are
administered in dosages sufficient to inhibit the expression or
activity of the target gene. Compositions containing the iRNA agent
of the invention can be administered at surprisingly low dosages. A
maximum dosage of 5 mg iRNA agent per kilogram body weight per day
may be sufficient to inhibit or completely suppress the expression
or activity of the target gene.
[0373] In general, a suitable dose of modified iRNA agent will be
in the range of 0.001 to 500 milligrams per kilogram body weight of
the recipient per day (e.g., about 1 microgram per kilogram to
about 500 milligrams per kilogram, about 100 micrograms per
kilogram to about 100 milligrams per kilogram, about 1 milligrams
per kilogram to about 75 milligrams per kilogram, about 10
micrograms per kilogram to about 50 milligrams per kilogram, or
about 1 microgram per kilogram to about 50 micrograms per
kilogram). The pharmaceutical composition may be administered once
per day, or the iRNA agent may be administered as two, three, four,
five, six or more sub-doses at appropriate intervals throughout the
day. In that case, the iRNA agent contained in each sub-dose must
be correspondingly smaller in order to achieve the total daily
dosage. The dosage unit can also be compounded for delivery over
several days, e.g., using a conventional sustained release
formulation which provides sustained release of the iRNA agent over
a several day period. Sustained release formulations are well known
in the art.
[0374] In this embodiment, the dosage unit contains a corresponding
multiple of the daily dose.
[0375] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the infection
or disease, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual iRNA
agent encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0376] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases. For example, mouse
repositories can be found at The Jackson Laboratory, Charles River
Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource
Centers (MMRRC) National Network and at the European Mouse Mutant
Archive. Such models may be used for in vivo testing of iRNA agent,
as well as for determining a therapeutically effective dose.
[0377] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), ocular, rectal, vaginal and topical (including buccal
and sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection. The pharmaceutical
compositions can also be administered intraparenchymally,
intrathecally, and/or by stereotactic injection.
[0378] For oral administration, the iRNA agent useful in the
invention will generally be provided in the form of tablets or
capsules, as a powder or granules, or as an aqueous solution or
suspension.
[0379] Tablets for oral use may include the active ingredients
mixed with pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0380] Capsules for oral use include hard gelatin capsules in which
the active ingredient is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil.
[0381] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, the pharmaceutical compositions of the invention
will generally be provided in sterile aqueous solutions or
suspensions, buffered to an appropriate pH and isotonicity.
Suitable aqueous vehicles include Ringer's solution and isotonic
sodium chloride. In a preferred embodiment, the carrier consists
exclusively of an aqueous buffer. In this context, "exclusively"
means no auxiliary agents or encapsulating substances are present
which might affect or mediate uptake of iRNA agent in the cells
that harbor the target gene or virus. Such substances include, for
example, micellar structures, such as liposomes or capsids, as
described below. Although microinjection, lipofection, viruses,
viroids, capsids, capsoids, or other auxiliary agents are required
to introduce iRNA agent into cell cultures, surprisingly these
methods and agents are not necessary for uptake of iRNA agent in
vivo. The iRNA agent of the present invention are particularly
advantageous in that they do not require the use of an auxiliary
agent to mediate uptake of the iRNA agent into the cell, many of
which agents are toxic or associated with deleterious side effects.
Aqueous suspensions according to the invention may include
suspending agents such as cellulose derivatives, sodium alginate,
polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such
as lecithin. Suitable preservatives for aqueous suspensions include
ethyl and n-propyl p-hydroxybenzoate.
[0382] The pharmaceutical compositions can also include
encapsulated formulations to protect the iRNA agent against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811; PCT publication
WO 91/06309; and European patent publication EP-A-43075, which are
incorporated by reference herein.
[0383] Toxicity and therapeutic efficacy of iRNA agent can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. iRNA agents that
exhibit high therapeutic indices are preferred.
[0384] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosages of compositions of the invention are preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any iRNA agent used in the method of
the invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
of the iRNA agent or, when appropriate, of the polypeptide product
of a target sequence (e.g., achieving a decreased concentration of
the polypeptide) that includes the IC.sub.50 (i.e., the
concentration of the test iRNA agent which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0385] In addition to their administration individually or as a
plurality, as discussed above, iRNA agents relating to the
invention can be administered in combination with other known
agents effective in treating viral infections and diseases. In any
event, the administering physician can adjust the amount and timing
of iRNA agent administration on the basis of results observed using
standard measures of efficacy known in the art or described
herein.
[0386] For oral administration, the iRNA agent useful in the
invention will generally be provided in the form of tablets or
capsules, as a powder or granules, or as an aqueous solution or
suspension.
Methods for Treating Diseases Caused by Expression of a Target
Gene
[0387] In one embodiment, the invention relates to a method for
treating a subject having a disease or at risk of developing a
disease caused by the expression of a target gene. In this
embodiment, iRNA agents can act as novel therapeutic agents for
controlling one or more of cellular proliferative and/or
differentiative disorders, disorders associated with bone
metabolism, immune disorders, hematopoietic disorders,
cardiovascular disorders, liver disorders, viral diseases, or
metabolic disorders. The method includes administering a
pharmaceutical composition of the invention to the patient (e.g., a
human), such that expression of the target gene is silenced.
Because of their high efficiency and specificity, the iRNA agent of
the present invention specifically target mRNA of target genes of
diseased cells and tissues, as described below, and at surprisingly
low dosages. The pharmaceutical compositions are formulated as
described in the preceding section, which is hereby incorporated by
reference herein.
[0388] Examples of genes which can be targeted for treatment
include, without limitation, an oncogene (Hanahan, D. and R. A.
Weinberg, Cell (2000) 100:57; and Yokota, J., Carcinogenesis (2000)
21(3):497-503); a cytokine gene (Rubinstein, M., et al., Cytokine
Growth Factor Rev. (1998) 9(2):175-81); a idiotype (Id) protein
gene (Benezra, R., et al., Oncogene (2001) 20(58):8334-41; Norton,
J. D., J. Cell Sci. (2000) 113(22):3897-905); a prion gene
(Prusiner, S. B., et al., Cell (1998) 93(3):337-48; Safar, J., and
S. B. Prusiner, Prog. Brain Res. (1998) 117:421-34); a gene that
expresses molecules that induce angiogenesis (Gould, V. E. and B.
M. Wagner, Hum. Pathol. (2002) 33(11):1061-3); adhesion molecules
(Chothia, C. and E. Y. Jones, Annu. Rev. Biochem. (1997) 66:823-62;
Parise, L. V., et al., Semin. Cancer Biol. (2000) 10(6):407-14);
cell surface receptors (Deller, M. C., and Y. E. Jones, Curr. Opin.
Struct. Biol. (2000) 10(2):213-9); genes of proteins that are
involved in metastasizing and/or invasive processes (Boyd, D.,
Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J.,
Carcinogenesis (2000) 21(3):497-503); genes of proteases as well as
of molecules that regulate apoptosis and the cell cycle (Matrisian,
L. M., Curr. Biol. (1999) 9(20):R776-8; Krepela, E., Neoplasma
(2001) 48(5):332-49; Basbaum and Werb, Curr. Opin. Cell Biol.
(1996) 8:731-738; Birkedal-Hansen, et al., Crit. Rev. Oral Biol.
Med. (1993) 4:197-250; Mignatti and Rifkin, Physiol. Rev. (1993)
73:161-195; Stetler-Stevenson, et al., Annu. Rev. Cell Biol. (1993)
9:541-573; Brinkerhoff, E., and L. M. Matrisan, Nature Reviews
(2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem. (2000)
69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.
(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)
488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)
2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari,
R., Bioassays (2001) 23(7):563-5); genes that express the EGF
receptor; Mendelsohn, J. and J. Baselga, Oncogene (2000)
19(56):6550-65; Normanno, N., et al., Front. Biosci. (2001)
6:D685-707); and the multi-drug resistance 1 gene, MDR1 gene
(Childs, S., and V. Ling, Imp. Adv. Oncol. (1994) 21-36).
[0389] In the prevention of disease, the target gene may be one
which is required for initiation or maintenance of the disease, or
which has been identified as being associated with a higher risk of
contracting the disease. In the treatment of disease, the iRNA
agent can be brought into contact with the cells or tissue
exhibiting the disease. For example, iRNA agent substantially
identical to all or part of a mutated gene associated with cancer,
or one expressed at high levels in tumor cells, may be brought into
contact with or introduced into a cancerous cell or tumor gene.
[0390] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., a carcinoma, sarcoma, metastatic
disorder or hematopoietic neoplastic disorder, such as a leukemia.
A metastatic tumor can arise from a multitude of primary tumor
types, including but not limited to those of prostate, colon, lung,
breast and liver origin. As used herein, the terms "cancer,"
"hyperproliferative," and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state or
condition characterized by rapidly proliferating cell growth. These
terms are meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. Proliferative disorders also include
hematopoietic neoplastic disorders, including diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof.
[0391] The pharmaceutical compositions of the present invention can
also be used to treat a variety of immune disorders, in particular
those associated with overexpression or aberrant expression of a
gene or expression of a mutant gene. Examples of hematopoietic
disorders or diseases include, without limitation, autoimmune
diseases (including, for example, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,
automimmune thyroiditis, dermatitis (including atopic dermatitis
and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's
disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing, loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung
fibrosis), graft-versus-host disease, cases of transplantation, and
allergy.
[0392] In another embodiment, the invention relates to methods for
treating viral diseases, including but not limited to hepatitis C,
hepatitis B, herpes simplex virus (HSV), HIV-AIDS, poliovirus, and
smallpox virus. iRNA agent of the invention are prepared as
described herein to target expressed sequences of a virus, thus
ameliorating viral activity and replication. The iRNA agents can be
used in the treatment and/or diagnosis of viral infected tissue,
both animal and plant. Also, such iRNA agent can be used in the
treatment of virus-associated carcinoma, such as hepatocellular
cancer.
[0393] For example, the iRNA agent of the present invention are
useful for treating a subject having an infection or a disease
associated with the replication or activity of a (+) strand RNA
virus having a 3'-UTR, such as HCV. In this embodiment, the iRNA
agent can act as novel therapeutic agents for inhibiting
replication of the virus. The method includes administering a
pharmaceutical composition of the invention to the patient (e.g., a
human), such that viral replication is inhibited. Examples of (+)
strand RNA viruses which can be targeted for inhibition include,
without limitation, picornaviruses, caliciviruses, nodaviruses,
coronaviruses, arteriviruses, flaviviruses, and togaviruses.
Examples of picornaviruses include enterovirus (poliovirus 1),
rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus),
cardiovirus (encephalomyocarditis virus), aphthovirus
(foot-and-mouth disease virus O), and parechovirus (human echovirus
22). Examples of caliciviruses include vesiculovirus (swine
vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease
virus), "Norwalk-like viruses" (Norwalk virus), "Sapporo-like
viruses" (Sapporo virus), and "hepatitis E-like viruses" (hepatitis
E virus). Betanodavirus (striped jack nervous necrosis virus) is
the representative nodavirus. Coronaviruses include coronavirus
(avian infections bronchitis virus) and torovirus (Berne virus).
Arterivirus (equine arteritis virus) is the representative
arteriviridus. Togavirises include alphavirus (Sindbis virus) and
rubivirus (Rubella virus). Finally, the flaviviruses include
flavivirus (Yellow fever virus), pestivirus (bovine diarrhea
virus), and hepacivirus (hepatitis C virus). In a preferred
embodiment, the virus is hepacivirus, the hepatitis C virus.
Although the foregoing list exemplifies vertebrate viruses, the
present invention encompasses the compositions and methods for
treating infections and diseases caused by any (+) strand RNA virus
having a 3'-UTR, regardless of the host. For example, the invention
encompasses the treatment of plant diseases caused by sequiviruses,
comoviruses, potyviruses, sobemovirus, luteoviruses, tombusviruses,
tobavirus, tobravirus, bromoviruses, and closteroviruses.
[0394] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to, oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), ocular, rectal, vaginal, and topical (including buccal
and sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection. The pharmaceutical
compositions can also be administered intraparenchymally,
intrathecally, and/or by stereotactic injection.
Methods for Inhibiting Expression of a Target Gene.
[0395] In yet another aspect, the invention relates to a method for
inhibiting the expression of a target gene in a cell or organism.
In one embodiment, the method includes administering the inventive
iRNA agent or a pharmaceutical composition containing the iRNA
agent to a cell or an organism, such as a mammal, such that
expression of the target gene is silenced. Because of their
surprisingly improved stability and bioavailability, the iRNA agent
of the present invention effectively inhibit expression or activity
of target genes at surprisingly low dosages. Compositions and
methods for inhibiting the expression of a target gene using iRNA
agent can be performed as described in the preceding sections,
particularly Sections 4 and 5.
[0396] In this embodiment, a pharmaceutical composition containing
the iRNA agent may be administered by any means known in the art
including, but not limited to oral or parenteral routes, including
intravenous, intramuscular, intraperitoneal, subcutaneous,
transdermal, airway (aerosol), ocular, rectal, vaginal, and topical
(including buccal and sublingual) administration. In preferred
embodiments, the pharmaceutical compositions are administered by
intravenous or intraparenteral infusion or injection. The
pharmaceutical compositions can also be administered
intraparenchymally, intrathecally, and/or by stereotactic
injection.
[0397] The methods for inhibiting the expression of a target gene
can be applied to any gene one wishes to silence, thereby
specifically inhibiting its expression, provided the cell or
organism in which the target gene is expressed includes the
cellular machinery which effects RNA interference. Examples of
genes which can be targeted for silencing include, without
limitation, developmental genes including but not limited to
adhesion molecules, cyclin kinase inhibitors, Wnt family members,
Pax family members, Winged helix family members, Hox family
members, cytokines/lymphokines and their receptors,
growth/differentiation factors and their receptors, and
neurotransmitters and their receptors; (2) oncogenes including but
not limited to ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA,
ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS,
LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET,
SRC, TALl, TCL3 and YES; (3) tumor suppresser genes including but
not limited to APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53
and WT 1; and (4) enzymes including but not limited to ACP
desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases,
alcohol dehydrogenases, amylases, amyloglucosidases, catalases,
cellulases, cyclooxygenases, decarboxylases, dextrinases, DNA and
RNA polymerases, galactosidases, glucanases, glucose oxidases,
GTPases, helicases, hemicellulases, integrases, invertases,
isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,
pectinesterases, peroxidases, phosphatases, phospholipases,
phosphorylases, polygalacturonases, proteinases and peptideases,
pullanases, recombinases, reverse transcriptases, topoisomerases,
and xylanases.
[0398] In addition to in vivo gene inhibition, the skilled artisan
will appreciate that the iRNA agent of the present invention are
useful in a wide variety of in vitro applications. Such in vitro
applications, include, for example, scientific and commercial
research (e.g., elucidation of physiological pathways, drug
discovery and development), and medical and veterinary diagnostics.
In general, the method involves the introduction of the iRNA agent
into a cell using known techniques (e.g., absorption through
cellular processes, or by auxiliary agents or devices, such as
electroporation and lipofection), then maintaining the cell for a
time sufficient to obtain degradation of an mRNA transcript of the
target gene.
[0399] In one aspect the invention provides a method of modulating
the expression of a target gene in a cell, comprising providing to
said cell an iRNA agent of this invention.
[0400] In one embodiment, the target gene is selected from the
group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR,
RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS
gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene,
MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene,
EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin
gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene,
Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,
mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene,
mutations in the p27(KIP1) gene, mutations in the PPM1D gene,
mutations in the RAS gene, mutations in the caveolin I gene,
mutations in the MIB I gene, mutations in the MTAI gene, mutations
in the M68 gene, mutations in tumor suppressor genes, and mutations
in the p53 tumor suppressor gene.
[0401] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Synthesis of Folate Conjugates 108 and 109
[0402] In order to conjugate the Folic acid to the siRNA the
following strategy was used. Folic acid was conjugated to the 3'
end using the solid support, 108 and to the 5' end using the
amidite 109 as follows.
##STR00037## ##STR00038##
[0403] Preparation of 102:
[0404] Glutamate 100 (2.05 g, 6.95 mmol) and amine 101 (4.50 g,
6.95 mmol) was taken together in a mixture of DCM/DMF (50 mL, 2:1).
To this reaction mixture HBTU (3.42 g, 1.3 eq.) and DIEA (2.43 ml,
2 eq.) was added and stirred for 30 minutes at ambient temperature.
The progress of the reaction was monitored by TLC (Ethyl acetate);
solvents are removed under reduced pressure and the residue was
extracted with dichloromethane, washed with water (2 times) and
brine. The organic layer was dried over anhydrous sodium sulfate.
Solvents were removed and residue was purified by chromatography
(during the packing of column please add few drops of TEA, First
elute with 1:1 ethyl acetate/hexane, followed ethyl acetate) to get
the product 102 as yellow gum (Yield, 6.35 g, 97%). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta.=7.94 (bs, 1H), 7.70-7.77 (m, 1H),
7.15-7.40 (m, 14H), 6.80-6.90 (m, 4H), 5.01 (s, 2H), 4.69-4.50 (m,
1H), 4.01-4.15 (m, 1H), 3.71 (s, 6H), 3.61 (s, 3H), 2.85-3.40 (m,
5H), 2.46-2.52 (m, 1H), 1.80-2.22 (m, 7H), 0.89-1.50 (m, 6H), 0.83
(s, 9H), 0.04-0.01 (m, 6H). .sup.13C NMR DMSO-d.sub.6)
.delta.=172.68, 170.91, 170.82, 170.80, 170.28, 164.56, 162.25,
158.11, 157.99, 156.05, 145.06, 144.74, 136.86, 135.77, 135.59,
135.33, 129. 57, 128.89, 128.31, 127.78, 127.74, 127.69, 127.62,
127.51, 126.58, 113.08, 112.71, 85.49, 85.24, 70.32, 65.47, 63.36,
59.71, 54.99, 54.96, 54.86, 54.60, 53.48, 51.78, 38.33, 38.20,
36.78, 35.73, 33.93, 31.45, 30.71, 29.02, 26.65, 26.65, 26.14,
25.62, 24.12, 20.70, 17.65, 17.62, 14.04, -4.91, -4.93, -4.97. MS.
Molecular weight calculated for C.sub.52H.sub.67N.sub.3O.sub.9Si,
Cal. 906.19. Found 907.2 (MH.sup.+).
[0405] Preparation of 103:
[0406] The Cbz protected amine 102 (6.30 g 6.80 mmol) was dissolved
in a mixture of MeOH/Ethyl acetate (1:3, 75 mL) and degassed with
argon. Pd/C (0.80 g, 10 wt % Degussa wet type) was added and
degassed and purged with hydrogen. The mixture was stirred under
hydrogen (Balloon pressure) for 6-9 hrs. Reaction was monitored by
TLC (Ethyl acetate, PMA stain). The TLC of the reaction mixture
showed the complete disappearance of the starting Cbz protected
amine. The reaction mixture was filtered through a pad of celite,
washed with a mixture of MeOH/EtOAc (100 mL) and the combined
filtrates were concentrated. The residue was dried under high
vacuum overnight to provide the product 103 as pale yellow foam
(5.30 g, 98%). It was directly used for the next reaction with out
further purification. .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta.=7.94 (bs, 1H), 7.70-7.75 (m, 1H), 7.12-7.33 (m, 9H),
6.81-6.88 (m, 4H), 4.50-4.64 (m, 1H), 4.05-4.17 (m, 1H), 3.71 (m,
6H), 3.59 (s, 3H), 3.19-3.40 (m, 2H), 2.85-3.05 (m, 2H), 2.47-2.52
(m, 1H), 1.80-2.22 (m, 7H), 0.89-1.50 (m, 6H), 0.83 (s, 9H),
0.04-0.01 (m, 6H). .sup.13C NMR DMSO-d.sub.6) .delta.=176.23,
176.11, 171.74, 171.53, 170.98, 165.15, 162.96, 158.46, 158.33,
158.15, 145.37, 145.04, 140.04, 136.10, 135.94, 135.74, 135.64,
129.92, 129.92, 128.55, 128.24, 128.13, 127.83, 127.17, 127.01,
113.52, 113.42, 113.09, 86.25, 85.62, 70.67, 69.67, 65.50, 63.65,
60.22, 56.06, 55.44, 55.35, 55.32, 55.06, 53.96, 53.55, 51.86,
37.13, 36.22, 34.31, 32.78, 31.93, 31.17, 30.67, 29.21, 26.42,
25.94, 24.84, 24.46, 21.04, 17.81, 17.95, 14.35, -4.60, -4.65 MS.
Molecular weight calculated for
C.sub.44H.sub.63F.sub.3N.sub.3O.sub.8Si, Cal. 789.44. Found 790.5
(MH.sup.+).
[0407] Preparation of 105:
[0408] N.sup.10-(Trifluoroacetyl)pteroic acid 104 (2.30 g, 6.00
mmol) and amine 103 (5.75 g, 1.2 eq) were dissolved in DMF (60 mL,
it takes about 15-20 minutes to dissolve the compounds in
solution). HBTU (3.01 g, 1.3 eq.) followed by DIEA (3 mL, 3 eq.)
were added and stirred for 30 minutes. Reaction was monitored by
TLC (8% MeOH/DCM, PMA stain). TLC of the reaction mixture showed
completion of reaction. Solvents were removed under reduced
pressure and the residue extracted with DCM, washed with water and
brine. The organic layer was dried over anhydrous sodium sulfate.
Solvents were removed and residue was purified by chromatography
(3-8% MeOH/DCM) to get the product 105 a pale yellow solid
(Yield=6.77 g, 93%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta.=8.88-8.90 (d, J=7.07 Hz, 1H), 8.60 (bs, 1H), 7.90 (d, J=8.4
Hz, 2H), 7.80 (t, J=8.4 Hz, 1H), 7.60 (d, J=8.06 Hz, 2H), 7.12-7.30
(m, 9H), 6.80-6.89 (m, 4H), 5.09 (bs, 2H), 4.52-4.68 (m, 1H),
4.32-4.40 (m, 1H), 4.05-1.12 (m, 1H), 3.71 (s, 6H), 3.62 (s, 3H),
2.86-3.30 (m, 6H), 1.75-2.23 (m, 8H), 1.10-145 (m, 6H), 0.82 (s,
9H), 0.04-0.01 (m, 6H). .sup.13C NMR DMSO-d.sub.6) .delta.=172.38,
170.96, 170.88, 165.60, 158.11, 157.99, 145.08, 141.66, 135.77,
135.60, 134.19, 129.59, 128.74, 128.74, 128.45, 128.13, 127.78,
127.52, 113.20, 113.11, 85.24, 55.01, 54.98, 54.91, 52.46, 51.90,
45.72, 40.12, 39.92, 39.71, 39.50, 39.29, 39.08, 38.87, 25.65,
17.69, 8.68, -4.88, -4.94. MS. Molecular weight calculated for
C.sub.60H.sub.72F.sub.3N.sub.9O.sub.11Si, Cal. 1179.51. Found
1181.0 (MH.sup.+).
[0409] Preparation of 106:
[0410] To a solution of 105 (6.75 g, 5.72 mmol) in a mixture of
DCM/Pyridine (100 mL, 1:1) DMAP (1.00 g, 1. 5 eq.) was added and
cooled in an ice-water bath. Isobutyric anhydride (10 mL, excess)
was added and stirred overnight. The reaction was monitored by TLC.
Reaction mixture was quenched with MeOH. Solvents were removed and
the residue extracted with DCM, washed with water and brine; dried
over anhydrous sodium sulfate. Solvents were removed and the
residue was purified by chromatography (first ethyl acetate then
3-5% MeOH/DCM) to get the product 106 as pale yellow solid
(Yield=6.70 g, 94%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta.=11.90-12.32 (m, 3H), 8.84-8.90 (m, 2H), 7.96-7.90 (m, 3H),
7.60-7.84 (m, 3H), 7.10-7.35 (m, 9H), 6.80-6.90 (m, 4H), 5.20 (bs,
2H), 4.50-4.67 (m, 1H), 4.34-4.42 (m, 1H), 4.05-4.12 (m, 1H), 3.71
(s, 6H), 3.61 (s, 3H), 2.86-3.30 (m, 6H), 1.75-2.23 (m, 6H), 1.17
(d, J=7.08 Hz, 3H), 1.05 (d, J=7.08 Hz, 3H), 1.10-1.45 (m, 6H),
0.82 (s, 9H), 0.04-0.01 (m, 6H). .sup.13C NMR DMSO-d.sub.6)
.delta.=180.81, 177.83, 172.37, 170.98, 170.94, 170.87, 165.56,
162.28, 159.89, 158.12, 157.99, 155.90, 155.55, 149.99, 149.87,
147.79, 145.10, 144.78, 141.79, 135.77, 135.61, 135.44, 135.35,
134.25, 130.55, 129.60, 128.71, 128.51, 127.88, 127.78, 127.53,
126.60, 117.54, 114.66, 113.19, 113.09, 85.86, 85.25, 70.35, 63.37,
59.75, 55.00, 54.97, 54.62, 54.02, 52.49, 51.89, 45.75, 38.37,
38.22, 36.79, 35.76, 35.00, 33.94, 33.12, 31.69, 30.74, 29.04,
26.30, 26.16, 25.63, 24.14, 20.74, 18.89, 18.74, 17.68, 17.65,
14.07, 8.67, -4.89, -4.96. MS. Molecular weight calculated for
C.sub.64H.sub.78F.sub.3N.sub.9O.sub.12Si, Cal. 1249.55. Found:
1248.4 (M-H).sup.-.
[0411] Preparation of 107:
[0412] Compound 106 (6.70 g, 5.36 mmol) was dissolved in
acetonitrile (50 mL) and Triethylamine (20 mL). To that HF/TEA (20
mL) was added and stirred for 3 hrs. The reaction was monitored by
TLC (5% MeOH/DCM). TLC showed complete disappearance of starting
material. Water and sodium bicarbonate solution was added to the
reaction mixture and extracted with DCM. The organic layer was
dried over sodium sulfate. Solvents were removed and the residue
was purified by chromatography (2-5% MeOH/DCM) to get the product
107 as pale yellow solid (Yield=5.10 g, 84%). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta.=11.45-11.95 (m, 2H), 8.81-8.95 (m,
2H), 7.60-7.94 (m, 5H), 7.10-7.36 (m, 9H), 6.78-6.91 (m, 4H), 5.19
(bs, 2H), 4.85-5.01 (bs, 1H), 4.34-4.42 (m, 1H), 3.71 (s, 6H), 3.61
(s, 3H), 2.86-3.55 (m, 9H), 1.75-2.23 (m, 6H), 1.16 (d, J=7.08 Hz,
3H), 1.10-1.45 (m, 6H), 0.95 (d, J=7.08 Hz, 3H). .sup.13C NMR
DMSO-d.sub.6) .delta.=180.36, 172.39, 170.98, 170.87, 170.34,
165.60, 160.60, 158.11, 157.98, 155.89, 155.54, 154.97, 151.03,
149.68, 147.30, 145.10, 144.76, 141.76, 135.88, 135.74, 135.49,
135.44, 134.24, 130.50, 129.62, 129.56, 128.74, 128.50, 127.88,
127.78, 127.59, 126.59, 117.54, 114.67, 113.19, 113.10, 85.78,
85.11, 68.59, 67.47, 65.18, 63.34, 59.76, 55.01, 54.98, 52.48,
51.90, 38.38, 36.29, 34.91, 34.15, 32.51, 31.67, 29.05, 26.29,
26.19, 24.18, 20.75, 18.84, 14.08, 11.07. MS. Molecular weight
calculated for C.sub.58H.sub.64F.sub.3N.sub.9O.sub.12, Cal.
1135.46. Found 1135.0 (M-H).sup.-.
[0413] Preparation of Long alkyl chain CPG 108:
[0414] Hydroxy derivative 107 (0.500 g, 0.441 mmol) was dissolved
in DCM (10 mL) to that Succinic anhydride (0.088 g, 2 eq) and DMAP
(0.160 g, 3 eq.) were added and stirred overnight. TLC showed
completion of reaction. The reaction mixture was diluted with DCM
(20 mL), washed successively with cold dilute citric acid and water
(2 times), dried over sodium sulfate. Solvents were removed and
dried under high vacuum to get the succinate. PPh.sub.3 (0.150 g,
1.3 eq.), DMAP (0.080 g, 1.5 eq.) and the succinate from the
previous step were dissolved in a mixture of acetonitrile and DCM
(6 mL). A solution of DTNP (0.143 g, 1.05 eq.) in DCM (1 mL) was
added to the above solution. The mixture was slowly shaken for 3-4
minutes. Long chain alkyl amine-CPG (lcaa CPG, 2.05 g, 133
.mu.mol/g) was added to the mixture and gently shaken for 2 h. The
CPG was filtered, successively washed with DCM, mixture of MeOH/DCM
(1:9) and DCM until filtrate remained colorless and dried. The
dried CPG was transferred into another flask treated with Ac.sub.2O
in pyridine (25%) in the presence of TEA (1 mL) for 15 min. under
gentle shaking. Finally the CPG was filtered, washed with DCM,
DCM:MeOH (9:1), followed by DCM and ether. The CPG 108 was dried
under vacuum overnight and the loading was measured as reported
(2.14 g, loading 53.3 .mu.mol/g).
[0415] Preparation of Phosphoramidite 109:
[0416] To a solution of 107 (0.20 g, 0.176 mmol) in EtOAc/DCM (4:1,
10 mL) chlorophosphonate reagent (0.086 mL, 2 eq.) was added,
followed by TEA (0.53 mL, 2 eq.). After 10 minutes the solution
becomes cloudy. The reaction was monitored by TLC. TLC showed
reaction was complete in 30 minutes, diluted with DCM washed with
sodium bicarbonate solution and dried over sodium sulfate. Solvents
were removed and the residue was dissolved small amount of
DCM/EtOAc mixture and precipitated the product with hexane
(Yield=195 mg, 82%). MS. Molecular weight calculated for
C.sub.67H.sub.81F.sub.3N.sub.11O.sub.13P, Cal. 1335.57. Found.
1136.60 (M+H).
Example 2
Synthesis of Pteroic Acid Precursor 110
[0417] In another method the appropriately substituted pteroic acid
precursor 110, amenable for RNA synthesis was prepared as
follows.
##STR00039##
Synthesis of
4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-trifl-
uoroacetyl)-amino]benzoic acid 110
[0418] To a suspension of pteroic acid (25 g, 61.2 mmol) and DMAP
(11.25 g, 92 mmol) in anhydrous pyridine (400 mL), TBDPS chloride
(42 g, 153 mmol) was added. The reaction mixture was stirred at
room temperature for 30 h after which isobutric anhydride (14.6 g,
92 mmol) was added and the mixture was slightly warmed. An
additional 60 mL of pyridine was also added and the reaction
mixture was stirred at room temperature overnight. The reaction
mixture became homogenous after which pyridine and other volatiles
were concentrated in a rotary evaporator. The residue was stirred
with EtOAc (1 L) and acetic acid (100 mL) and water (500 mL) for 24
h. The thus obtained slurry was filtered, the residue was washed
with water (500 mL), EtOAc (1 L) and dried to obtain the pure
product as a white solid (26.1 g, 89%). .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta.=8.87 (s, 1H), 7.95 (d, J=8.6 Hz, 2H), 7.67 (d,
J=8.6 Hz, 2H), 5.21 (s, 2H), 2.79-2.74 (m, 1H), 1.12 (d, J=6.83 Hz,
6H), .sup.13C NMR (DMSO-d.sub.6) .delta.=180.72, 166.49, 159.25,
149.87, 147.68, 142.69, 136.34, 134.45, 130.54, 129.16, 128.86,
127.49, 34.96, 33.09, 26.52, 18.88, 18.74. .sup.19F NMR
(DMSO-d.sub.6) .delta.-64.32. MS. Molecular weight calculated for
C.sub.20H.sub.17F.sub.3N.sub.6O.sub.5, Cal. 478.12. Found 479.12
(MH.sup.+).
Example 3
Synthesis of Disulfide Precursor 112
[0419] In order to conjugate Folic acid to siRNA via a cleavable
disulfide linker the following strategy was used. The pyridyl
disulfide precursor 112 was synthesized by conjugating
hydroxyprolinol derivative 101 with the NHS ester 111 as
follows.
##STR00040##
Synthesis of 4-methyl-4-(pyridine-2-yldisulfanyl)-pentanoic
acid{6-[2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-dim-
ethyl-silanyloxy)-pyrrolidin-1-yl]-6-oxo-hexyl}-amide 112
[0420] To a solution of the amine 101 (3.23 g, 5 mmol) in
dichloromethane (50 mL), diisopropylethylamine (1.27 g, 10 mmol)
and the NHS ester 111 (1.8 g, 5 mmol) was added and the mixture was
stirred at room temperature overnight. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (200 mL) and the organic layer was
washed with satd. NaHCO.sub.3 (100 mL), water (100 mL), brine (100
mL) and dried (Na.sub.2SO.sub.4). Solvents were removed and the
residue was purified by chromatography (2-5% MeOH/DCM) to get the
product 112 as pale yellow foam (Yield=4.10 g, 92%). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta.=8.39 (d, J=4.6 Hz, 1H), 7.80-7.73
(m, 3H), 7.33-7.24 (m, 4H), 7.22-7.16 (m, 5H), 6.88-6.84 (m, 4H),
4.66-4.62 (m, 0.7H), 4.58-4.50 (m, 0.3H), 4.16-4.06 (m, 1H),
3.78-3.66 (m, 2H), 3.26-3.18 (m, 2H), 3.12-2.89 (m, 4H), 2.24-2.12
(m, 4H), 2.02-1.86 (m, 1H), 1.84-1.76 (m, 3H), 1.50-1.10 (m, 6H),
1.20 (s, 3H), 0.83 (s, 9H), 0.81 (s, 3H), 0.04-0.01 (m, 6H).
.sup.13C NMR (DMSO-d.sub.6) .delta.=171.23, 170.91, 170.81, 160.02,
158.09, 157.96, 149.14, 145.05, 144.73, 137.51, 135.75, 135.58,
135.42, 135.32, 129.56, 127.85, 127.75, 127.50, 126.73, 126.58,
120.98, 119.22, 113.18, 113.09, 85.83, 85.23, 70.31, 69.31, 65.17,
63.34, 54.99, 54.96, 54.60, 52.19, 38.30, 36.77, 36.43, 33.92,
32.41, 31.01, 28.94, 26.87, 26.11, 25.64, 24.08, 17.66, -4.88,
-4.91, -4.94. MS. Molecular weight calculated for
C.sub.49H.sub.67N.sub.3O.sub.6S.sub.2Si, Cal. 885.42. Found 886.42
(MH.sup.+).
Example 4
Synthesis of Folate Conjugates 121 and 122
[0421] The pyridyl disulfide 112 is treated with the thiol 114 to
get the disulfide 115 which on treatment with the glutamate 116
provided the amide 117. The Fmoc disulfide 117 is treated with
piperidine to remove the Fmoc protecting group to give the amine
118 which on treatment with the protected pteroic acid 110 provided
the couple product 119. The TBDMS protecting group in 119 is
removed using HF:NEt.sub.3 complex to provide the hydroxyprolinol
precursor 120. The hydroxyprolinol 120 is converted to the solid
support 122 via the succinate and the amidite 121 as follows.
##STR00041## ##STR00042## ##STR00043##
Example 5
Synthesis of Disulfide Precursor 124
[0422] In another example the folate conjugate without the
stabilizing gem dimethyl group next to the disulfide link was
probed. In a procedure similar to that described above for compound
112 (example 3) is used to synthesize 124 as follows.
##STR00044##
Example 6
Synthesis of Folate Conjugates 130 and 131
[0423] In another approach the Folic acid was conjugated to the
siRNA via a disulfide link through cystine as follows.
##STR00045## ##STR00046## ##STR00047##
Example 7
[0424] In another embodiment the disulfide linkage was placed
closer to the siRNA as follows.
##STR00048## ##STR00049##
Example 8
Synthesis of Compounds Folic Acid Precursors Amenable to
Oligonucleotide Synthesis
[0425] In order to synthesize an appropriately substituted more
versatile precursor of Folic acid amenable for RNA synthesis, the
following strategy was used. In this method the protected Folic
acid 110 was treated with the .gamma.-tert-butyl, .alpha.-Me ester
of glutamic acid, 142 to obtain the ester 143 which on treatment
with TFA/CH.sub.2Cl.sub.2 provided the precursor 144. The precursor
144 was coupled with various amines like 145, 151, 155, 161, 165,
172 and 176 provided the couple products 146, 152, 156, 162, 166,
173 and 177 respectively. These products are then converted to
solid-supports 147, 153, 157, 163, 167, 174 and 178, and
phosphoramidites 148, 154, 158, 164, 168 and 175.
[0426] Synthesis of 147
##STR00050## ##STR00051##
Synthesis of
2-{4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-tr-
ifluoroacetyl)-amino}-pentanedioic acid 5-tert-butyl ester 1-methyl
ester 143
[0427] In a representative procedure, the pteroic acid precursor
110 (2.4 g, 5 mmol) was dissolved in anhydrous DMF (20 mL), HBTU
(1.9 g, 1 eq.) followed by DIEA (1 mL, 5 eq.) were added and
stirred for 20 minutes. To this reaction mixture the amine
hydrochloride 142 (1.2 g, 1 eq) was added as a solution in DMF (6
mL). Reaction was monitored by TLC (8% MeOH/DCM, PMA stain). TLC of
the reaction mixture showed completion of the reaction. The
reaction mixture was slowly poured in ice with vigorous stirring.
The precipitated product was filtered to get the product 143 as a
white solid (Yield=2.85 g, 86%). .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta.=12.33 (s, 1H), 11.94 (s, 1H), 8.88 (s, 1H), 8.82 (d,
J=7.3 Hz, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 5.22
(s, 2H), 4.46-4.40 (m, 1H), 3.62 (s, 3H), 2.86-2.73 (m, 1H), 2.32
(t, J=7.4 Hz, 2H) 2.05-1.90 (m, 2H), 1.35 (m, 9H), 1.12 (d, J=6.8
Hz, 6H). .sup.13C NMR DMSO-d.sub.6) .delta.=180.75, 172.13, 171.45,
165.64, 159.10, 154.80, 149.97, 149.79, 147.72, 141.75, 134.15,
130.53, 128.70, 128.49, 117.50, 114.64, 79.79, 51.96, 51.91, 34.96,
31.22, 27.68, 25.71, 18.72. MS. Molecular weight calculated for
C.sub.30H.sub.34F.sub.3N.sub.7O.sub.8, Cal. 677.63. Found 676.72
(M-H.sup.-).
Synthesis of
2-{4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-tr-
ifluoroacetyl)-amino}-pentanedioic acid 1-methyl ester 144
[0428] The ester 143 (2 g, 2. 9 mmol) was dissolved in 20 mL of 50%
TFA in dichloromethane and the solution was stirred at room
temperature for 30 min. after which the TLC showed the complete
disappearance of the starting ester. The reaction mixture was
concentrated and the residue was crystallized from
CH.sub.2Cl.sub.2:Hexanes (2:3) and crystallized product was
filtered off and dried to obtain the pure product 144 (1.76 g, 96%)
as off white powder. .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta.=12.32 (bs, 1H), 11.94 (s, 1H), 8.88 (s, 1H), 8.84 (d, J=7.4
Hz, 1H), 7.90 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.3 Hz, 2H), 5.22 (s,
2H), 4.45-4.41 (m, 1H), 3.62 (s, 3H), 2.78-2.75 (m, 1H), 2.35 (t,
J=7.4 Hz, 2H) 2.07-1.92 (m, 2H), 1.12 (d, J=6.8 Hz, 6H). .sup.13C
NMR DMSO-d.sub.6) .delta.=180.77, 173.70, 172.19, 165.70, 159.21,
155.54, 149.93, 149.84, 147.75, 141.78, 134.18, 130.53, 128.71,
128.49, 117.51, 114.64, 53.98, 52.06, 51.93, 34.97, 30.11, 25.68,
18.73. MS. Molecular weight calculated for
C.sub.26H.sub.26F.sub.3N.sub.7O.sub.8, Cal. 621.18. Found 620.18
(M-H.sup.-).
[0429] Preparation of 146:
[0430] N.sup.10-(Trifluoroacetyl)pteroic acid 144 (2.30 g, 6.00
mmol) is dissolved in DMF (60 mL, it takes about 15-20 minutes to
dissolve the compounds in solution). HBTU (3.01 g, 1.3 eq.)
followed by DIEA (3 mL, 3 eq.) are added and stirred for 30 minutes
then the amine 145 (5.75 g, 1.2 eq) is added and the reaction
mixture is stirred at room temperature. Reaction is monitored by
TLC (8% MeOH/DCM, PMA stain). TLC of the reaction mixture showed
the completion of reaction. Solvents are removed under reduced
pressure and the residue extracted with DCM, washed with water and
brine. The organic layer is dried over anhydrous sodium sulfate.
Solvents are removed and residue was purified by chromatography
(3-8% MeOH/DCM) to get the product 146 as a foam (Yield=6.77 g,
93%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=8.88-8.90 (d,
J=7.07 Hz, 1H), 8.60 (bs, 1H), 7.90 (d, J=8.4 Hz, 2H), 7.80 (t,
J=8.4 Hz, 1H), 7.60 (d, J=8.06 Hz, 2H), 7.12-7.30 (m, 9H),
6.80-6.89 (m, 4H), 5.09 (bs, 2H), 4.52-4.68 (m, 1H), 4.32-4.40 (m,
1H), 4.05-1.12 (m, 1H), 3.71 (s, 6H), 3.62 (s, 3H), 2.86-3.30 (m,
6H), 1.75-2.23 (m, 8H), 1.10-145 (m, 6H), 0.82 (s, 9H), 0.04-0.01
(m, 6H). .sup.13C NMR DMSO-d.sub.6) .delta.=172.38, 170.96, 170.88,
165.60, 158.11, 157.99, 145.08, 141.66, 135.77, 135.60, 134.19,
129.59, 128.74, 128.74, 128.45, 128.13, 127.78, 127.52, 113.20,
113.11, 85.24, 55.01, 54.98, 54.91, 52.46, 51.90, 45.72, 40.12,
39.92, 39.71, 39.50, 39.29, 39.08, 38.87, 25.65, 17.69, 8.68,
-4.88, -4.94. MS. Molecular weight calculated for
C.sub.60H.sub.68F.sub.3N.sub.9O.sub.12S.sub.2, Cal. 1228.36. Found
1227.3 (M-H).sup.-.
[0431] Preparation of Long alkyl chain CPG 147:
[0432] Hydroxy derivative 146 (0.7 g, 0.441 mmol) was dissolved in
DCM (10 mL) to that Succinic anhydride (0.088 g, 2 eq) and DMAP
(0.160 g, 3 eq.) were added and stirred overnight. TLC showed
completion of reaction. The reaction mixture was diluted with DCM
(20 mL), washed successively with cold dilute citric acid and water
(2 times), dried over sodium sulfate. Solvents were removed and
dried under high vacuum to get the succinate. PPh.sub.3 (0.150 g,
1.3 eq.), DMAP (0.080 g, 1.5 eq.) and the succinate from the
previous step were dissolved in a mixture of acetonitrile and DCM
(6 mL). A solution of DTNP (0.143 g, 1.05 eq.) in DCM (1 mL) was
added to the above solution. The mixture was slowly shaken for 3-4
minutes. Long chain alkyl amine-Polystyrene (5.05 g, 250 mol/g) was
added to the mixture and gently shaken for 2 h. The CPG was
filtered, successively washed with DCM, mixture of MeOH/DCM (1:9)
and DCM until filtrate remained colorless and dried. The dried
support was transferred into another flask treated with Ac.sub.2O
in pyridine (25%) in the presence of TEA (1 mL) for 15 min. under
gentle shaking. Finally the CPG was filtered, washed with DCM,
DCM:MeOH (9:1), followed by DCM and ether. The support 147 was
dried under vacuum overnight and the loading was measured as
reported (5.14 g, loading 78 .mu.mol/g).
[0433] Synthesis of Compound 153
##STR00052## ##STR00053##
[0434] Using a similar procedure to that described above, a folate
conjugated solid support with a cleavable disulfide linkage was
synthesized as follows. Treatment of the protected folic acid 144
with the amine 151 provided the coupled precursor 152 as white
foam. The amine 151 was prepared by coupling the amine 145 with the
azido acid 149 followed by the reduction of the azide group using
triphenyl phosphine in the presence of water.
[0435] Synthesis of Azide 150:
[0436] Using a similar procedure to that used for the synthesis of
146, coupling of the amine 145 (2 g, 3.2 mmol) with the azido acid
149 (0.9 g, 3.2 mmol) provided the coupled azide 150 (1.86 g, 64%)
as a foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=7.94-7.90
(m, 2H), 7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H),
5.22 (d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H),
3.30 (s, 3H), 3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H), 1.16 (d, J=7.08
Hz, 3H), 1.10-1.45 (m, 6H), 0.95 (d, J=7.08 Hz, 3H). .sup.13C NMR
DMSO-d.sub.6) .delta.=172.22, 170.42, 162.27, 158.06, 157.96,
145.04, 135.81, 135.71, 135.34, 129.56, 127.75, 127.56, 126.57,
113.09, 85.09, 68.55, 63.27, 54.97, 54.88, 50.57, 50.45, 35.74,
35.29, 30.74, 28.99, 28.96, 28.89, 28.86, 28.71, 28.55, 28.47,
28.19, 27.29, 26.08, 25.16. MS. Molecular weight calculated for
C.sub.50H.sub.73N.sub.5O.sub.6S.sub.2, Cal. 904.27. Found 905.3
(MH.sup.+).
[0437] Synthesis of the Amine 151.
[0438] Treatment of the azide 150 (1.86 g, 2.06 mmol) with
triphenyl phosphine (0.54 g, 2.1 mmol) with THF (60 mL) and water
(5 mL) at room temperature followed by usual workup and column
chromatography provided the pure amine 151 (1.45 g, 82%) as a foam.
.sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=7.93-7.89 (m, 1H),
7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H), 5.22 (d,
0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H), 3.30 (s,
3H), 3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H), 1.16 (d, J=7.08 Hz, 3H),
1.10-1.45 (m, 6H), 0.95 (d, J=7.08 Hz, 3H). .sup.13C NMR
DMSO-d.sub.6) .delta.=172.22, 170.42, 162.27, 158.06, 157.96,
145.04, 135.81, 135.71, 135.34, 129.56, 127.75, 127.56, 126.57,
113.09, 85.09, 68.55, 63.27, 54.97, 54.88, 50.57, 50.45, 35.74,
35.29, 30.74, 28.99, 28.96, 28.89, 28.86, 28.71, 28.55, 28.47,
28.19, 27.29, 26.08, 25.16. MS. Molecular weight calculated for
C.sub.50H.sub.75N.sub.3O.sub.6S.sub.2, Cal. 878.28. Found 879.26
(MH.sup.+).
[0439] Synthesis of compound 152
[0440] Using a similar procedure to that used for the synthesis of
146, coupling of the amine 151 (1.45 g, 1.65 mmol) with the acid
144 (1.02 g, 1.65 mmol) provided the coupled product 152 (2 g, 81%)
as a foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=11.65 (bs,
1H), 8.90-8.85 (m, 1H), 7.93 (d, J=6 Hz, 2H), 7.85 (m, H), 7.78 (d,
J=76 Hz, 2H), 7.40-7.19 (m, 7H), 6.90-6.87 (m, 6H), 5.21 (s, 2H),
4.80-4.56 (m, 1H), 4.42-4.32 (m, 1H), 4.26-4.16 (m, 1H), 3.96-3.79
(m, 1H), 3.72 (s, 3H), 3.30-2.60 (m, 7H), 2.23-1.65 (m, 6H),
1.36-1.15 (m, 24H), 1.12 (d, J=7 Hz, 6H). .sup.13C NMR
DMSO-d.sub.6) .delta.=180.44, 172.32, 170.98, 170.80, 170.58,
165.51, 162.27, 159.10, 158.21, 158.11, 155.53, 154.80, 149.82,
147.75, 145.60, 145.48, 141.75, 136.45, 136.38, 136.33, 136.20,
134.21, 130.51, 129.68, 128.67, 128.45, 127.89, 127.66, 126.70,
114.63, 113.27, 104.26, 85.94, 71.80, 61.70, 57.32, 56.92, 5502,
53.99, 52.57, 52.46, 51.87, 50.45, 45.67, 38.63, 38.44, 38.21,
35.74, 35.29, 30.74, 28.99, 28.96, 28.89, 28.86, 28.71, 28.55,
28.47, 28.19, 27.29, 26.08, 25.16, 18.79, 11.16. MS. Molecular
weight calculated for
C.sub.76H.sub.99F.sub.3N.sub.10O.sub.13S.sub.2, Cal. 1481.78. Found
1481.0 (M-H.sup.-).
[0441] The hydroxyl compound 152 on treatment with succinic
anhydride followed by treatment with polystyrene linked resin
provided the folate coupled solid support 153 in 84 .mu.M/g
loading.
[0442] Synthesis of Compound 157
[0443] Using a similar procedure to that described above, a folate
conjugated solid support with a cleavable disulfide linkage was
synthesized as follows. Treatment of the protected folic acid 144
with the amine 155 provided the coupled precursor 156 as white
foam.
##STR00054## ##STR00055##
[0444] Synthesis of Compound 156.
[0445] Using a similar procedure to that used for the synthesis of
146, coupling of the amine 155 (0.84 g, 1.45 mmol) with the acid
144 (0.875 g, 1.41 mmol) provided the coupled product 156 (1.4 g,
83%) as a foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta.=8.89-8.85 (m, 2H), 8.08-7.62 (m, 5H), 7.40-7.12 (m, 7H),
6.90-6.87 (m, 6H), 5.20 (s, 2H), 4.60-4.36 (m, 2H), 4.20-4.12 (m,
1H), 3.72 (s, 6H), 3.30-2.60 (m, 7H), 2.23-1.65 (m, 6H), 1.10 (d,
J=7 Hz, 6H). .sup.13C NMR DMSO-d.sub.6) .delta.=180.35, 172.28,
171.32, 170.18, 165.57, 160.52, 158.08, 157.96, 154.92, 149.65,
147.27, 145.04, 144.72, 141.73, 135.83 135.70, 135.48, 134.28,
130.48, 129.57, 128.69, 128.46, 127.85, 127.75, 127.55, 126.56,
114.63, 113.19, 113.09, 109.30, 85.10, 68.55, 63.22, 54.97, 53.99,
52.38, 51.87, 45.67, 37.92, 37.18, 37.05, 36.21, 34.87, 32.50,
31.58, 26.19, 24.01, 18.80, 11.08. MS. Molecular weight calculated
for C.sub.58H.sub.64F.sub.3N.sub.9O.sub.12S.sub.2, Cal. 1200.31.
Found 1200.10 (M-H.sup.-).
[0446] The hydroxyl compound 156 on treatment with succinic
anhydride followed by treatment with polystyrene linked resin
provided the folate coupled solid support 157 in 74 .mu.M/g
loading.
[0447] Synthesis of Compounds 163 and 164
##STR00056## ##STR00057## ##STR00058##
[0448] Using a similar procedure to that described above, compounds
163 and 164 are prepared. Treatment of the protected folic acid 144
with the amine 161 provided the coupled precursor 162. The
precursor 162 is then converted into a phosphoramidite, 164 and
solid-support 163.
[0449] Synthesis of Compound 167 and 168
##STR00059## ##STR00060##
[0450] Using a similar procedure to that described above, compounds
167 and 168 are prepared. Treatment of the protected folic acid 144
with the amine 165 provided the coupled precursor 166. The
precursor 166 is then converted into a phosphoramidite, 168 and
solid-support 167.
[0451] Synthesis of Compounds 177 and 178
##STR00061## ##STR00062## ##STR00063##
[0452] Using a similar procedure to that described above, compounds
174 and 175 are prepared. Treatment of the protected folic acid 144
with the amine 172 provided the coupled precursor 173. The
precursor 173 is then converted into a phosphoramidite, 175 and
solid-support 174.
[0453] Synthesis of Compounds 178
##STR00064##
Synthesis of
4(6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-
-1-yl}-[6-oxo-hexadecylcarbamoyl)-2-{4[(2-isobutrylamino-4-oxo-3,4-dihydro-
-pteridin-6-ylmethyl)-(2,2,2-trifluoro-acetyl)-amino]-benzoylamino}-butyri-
c acid methyl ester 177
[0454] The pteroic acid precursor 144 (4.3 g, 6.92 mmol) was
dissolved in anhydrous DMF (80 mL), HBTU (2.62 g, 1 eq.) followed
by DIEA (2.7 g, 3 eq.) were added and stirred for 20 minutes. To
this reaction mixture the amine 176 (4.65 g, 6.92 mmol) was added
as a solution in DMF (20 mL). Reaction was monitored by TLC (5%
MeOH/DCM, PMA stain). TLC of the reaction mixture showed completion
of the reaction after 1 h. The reaction mixture was slowly poured
in ice with vigorous stirring. The mixture was extracted with ethyl
acetate and the combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated to give the crude product. The
thus obtained crude product was further purified by column
chromatography (silica gel, 0-5% MeOH in DCM in the presence of 1%
NEt.sub.3) to obtain 177 as a white foam (Yield=5.1 g, 58%).
.sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=11.95 (bs, 1H),
8.91-8.89 (m, 2H), 7.92 (d, J=6 Hz, 2H), 7.65 (m, H), 7.68 (d, J=76
Hz, 2H), 7.40-7.38 (m, 2H), 7.36-7.19 (m, 7H), 6.90-6.87 (m, 6H),
5.21 (s, 2H), 4.80-4.56 (m, 1H), 4.42-4.32 (m, 1H), 4.26-4.16 (m,
1H), 3.96-3.79 (m, 1H), 3.72 (s, 3H), 3.30-2.60 (m, 7H), 2.23-1.65
(m, 6H), 1.36-1.15 (m, 24H), 1.12 (d, J=7 Hz, 6H). .sup.13C NMR
DMSO-d.sub.6) .delta.=180.76, 172.32, 170.98, 170.80, 170.58,
165.51, 162.27, 159.10, 158.21, 158.11, 155.53, 154.80, 149.82,
147.75, 145.60, 145.48, 141.75, 136.45, 136.38, 136.33, 136.20,
134.21, 130.51, 129.68, 128.67, 128.45, 127.89, 127.66, 126.70,
114.63, 113.27, 104.26, 85.94, 71.80, 61.70, 57.32, 56.92, 5502,
53.99, 52.57, 52.46, 51.87, MS. Molecular weight calculated for
C.sub.30H.sub.34F.sub.3N.sub.7O.sub.8, Cal. 1276.44. Found 1276.0
(M-H.sup.-).
[0455] Preparation of Solid Support 178:
[0456] Hydroxy derivative 177 (5 g, 3.91 mmol) was dissolved in DCM
(100 mL) to that Succinic anhydride (0.782 g, 2 eq) and DMAP (1.43
g, 3 eq.) were added and stirred overnight. TLC showed completion
of reaction. The reaction mixture was diluted with DCM (100 mL),
washed successively with cold dilute citric acid and water (2
times), dried over sodium sulfate. Solvents were removed and dried
under high vacuum to get the crude succinate. The thus obtained
crude succinate was purified by column chromatography (silica gel,
0-10% MeOH in DCM in the presence of 1% NEt.sub.3 to isolate the
pure succinate as a white foam (4.7 g, 93%). This succinate was
dissolved in acetonitrile (100 mL) and to it PPh.sub.3 (0.96 g, 1.1
eq.), DMAP (0.61 g, 1.5 eq.) were added after which a solution of
DTNP (3.1 g, 1.05 eq.) in ACN (10 mL) was added to the above
solution. The mixture was slowly shaken for 3-4 minutes. Long chain
alkyl amine-polystyrene (30 g, 250 mol/g) was added to the mixture
and gently shaken for 2 h. The solid support was filtered,
successively washed with DCM, mixture of MeOH/DCM (1:9) and DCM
until filtrate remained colorless and dried. The dried support was
transferred into another flask treated with Ac.sub.2O in pyridine
(25%) in the presence of TEA (1 mL) for 15 min. under gentle
shaking. Finally the solid support was filtered, washed with DCM,
DCM:MeOH (9:1), followed by DCM and ether. The solid support 116
was dried under vacuum overnight and the loading was measured as
reported (30 g, loading 72 mol/g).
Example 9
Synthesis of Folate Conjugates with PEG Spacer
[0457] In order to introduce a hydrophilic PEG spacer the following
route was used. Treatment of the commercially available PEGazido
acid 179 with the hydroxyprolinol derivative 180 provided the
coupled azide 181 which on treatment with triphenyl phosphine in
the presence of water provided the amine 182. The coupling of the
amine 182 with the pteroic acid 113 to give the coupled product
183. The hydroxyl compound 183 on treatment with succinic anhydride
followed by treatment with polystyrene linked resin provided the
folate coupled solid support 184 in 88 .mu.M/g loading.
##STR00065##
[0458] Preparation of Compound 181
[0459] Using a similar procedure to that used for the synthesis of
112, coupling of the amine 180 (2.1 g, 5 mmol) with the azido acid
179 (3.2 g, 5 mmol) provided the coupled azide 181 (3.26 g, 64%) as
a foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=7.94-7.90 (m,
2H), 7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H), 5.22
(d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H),
3.56-3.54 (m, 46H), 2.23-1.75 (m, 2H), 1.71-1.66 (m, 2H). MS.
Molecular weight calculated for C.sub.53H.sub.80N.sub.4O.sub.17,
Cal. 1044.55. Found 1045.6 (MH.sup.+).
[0460] Synthesis of the Amine 182.
[0461] Treatment of the azide 181 (3.26 g, 3.12 mmol) with
triphenyl phosphine (0.78 g, 3.1 mmol) with THF (60 mL) and water
(5 mL) at room temperature followed by usual workup and column
chromatography provided the pure amine 182 (3.45 g, 82%) as a foam.
.sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=7.93-7.89 (m, 1H),
7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H), 5.22 (d,
0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H), 3.30 (s,
3H), 3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H), 1.16 (d, J=7.08 Hz, 3H),
1.10-1.45 (m, 6H), 0.95 (d, J=7.08 Hz, 3H). MS. Molecular weight
calculated for C.sub.53H.sub.82N.sub.2O.sub.17, Cal. 1119.22. Found
1120.26 (MH.sup.+).
##STR00066##
[0462] Synthesis of Compound 183.
[0463] Using a similar procedure to that used for the synthesis of
112, coupling of the amine 182 (2.04 g, 2 mmol) with the acid 113
(1.24 g, 2 mmol) provided the coupled product 183 (1.46 g, 46%) as
a foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=8.85 (s, 1H),
7.93 (d, J=6 Hz, 2H), 7.85 (m, H), 7.78 (d, J=76 Hz, 2H), 7.40-7.19
(m, 7H), 6.90-6.87 (m, 6H), 5.21 (s, 2H), 4.80-4.56 (m, 1H),
4.42-4.32 (m, 1H), 4.26-4.16 (m, 1H), 3.96-3.79 (m, 1H), 3.72 (s,
3H), 3.62-3.46 (m, 48H), 2.23-1.65 (m, 6H), 1.36-1.15 (m, 24H),
1.12 (d, J=7 Hz, 6H). MS. Molecular weight calculated for
C.sub.76H.sub.106F.sub.3N.sub.9O.sub.24, Cal. 1622.73. Found 1622.0
(M-H.sup.-).
Example 10
Synthesis of a Folate Analogue Conjugate 199
[0464] In order to evaluate the targeting ability of the
pyrrolo[2,3-d]pyrimidine-based folate analogue, the multitargeted
antifolate, Alimta [(a) Bunn, P. A., Jr.; Smith, I. E., Guest
Editors Seminars Oncol. 2002, 29 (6), Suppl. 18, 1-75. (b) Bertino,
J.; Allegra, C.; Calvert, H., Guest Editors Seminars Oncol. 1999,
26 (2), Suppl. 6, 1-111. (c) Hanauske, A.-R.; Chen, V.; Paoletti,
P.; Niyikiza, C. The Oncologist 2001, 6, 363-373. (d) Taylor, E.
C.; Kuhnt, D.; Shih, C.; Rinzel S. M.; Grindey, G. B.; Barredo, J.;
Jannatipour, M.; Moran, R. G. J. Med. Chem. 1992, 35, 4450-4454.]
the following approach was undertaken.
[0465] The necessary building block for the conjugation to the
pyrrolo[2,3-d]pyrimidine was synthesized as follows.
##STR00067##
[0466] Synthesis of Compound 187
[0467] Caproic Acid (100 g, 0.7623 moles) was dissolved in 250 ml
NaOH (2N) and cooled in a round bottom flask (2 L). Benzyl
Chloroformate (273 mL, 0.8004 moles) and 250 ml NaOH (2N) were
added simultaneously at 0.degree. C. with stirring. The progress of
the reaction was monitored by TLC (about 2 hr). After completion of
the reaction, the mixture was acidified with dilute HCl (10%) and
extracted with EtOAc (750 mL.times.3). The organic layer was
separated, washed well with water and dried over Na.sub.2SO.sub.4.
Removal of the solvent under vacuum afforded crude 2 which was
purified by column chromatography (ethyl acetate:hexane). Yield: 80
g (40%). .sup.1H NMR (DMSO, 400 MHz)=7.33 (s, 5H), 5.09 (s, 2H),
4.88 (br, 1H), 3.18 (m, 2H), 2.23 (m, 2H), 1.62 (m, 2H), 1.50 (m,
2H), 1.34 (m, 2H).
[0468] Synthesis of Compound 188
[0469] Compound 187 (57.59 g, 0.2173 moles) was dissolved in 500 mL
dry DCM and cooled to 0.degree. C. in a round bottom flask (2 L).
This was followed with the addition of EDC.HCl (50 g, 0.2608 moles)
and HOBt (33 g, 0.2173 moles). After 15 minutes of stirring at
0.degree. C., 186 (39.46 g, 0.2173 moles) in 500 ml of dry DCM was
added at 0.degree. C. DIPEA was then added till the reaction
mixture showed basic to pH. Reaction was stirred overnight at room
temperature, quenched with water (500 mL) and extracted with DCM
(500 mL.times.3). The organic layer was washed with sat. NH.sub.4Cl
and dried over anhydrous Na.sub.2SO.sub.4. Removal of solvent
afforded the crude compound which was purified by column
chromatography (4% MeOH/DCM) using 100-200 mesh silica gel. Yield
50 g (87%). .sup.1H NMR (DMSO, 400 MHz), .delta.=7.38 (m, 5H), 7.25
(m, 1H), 5.18 (s, 1H), 4.99 (s, 2H), 4.32-4.25 (m, 2H), 3.67 (s,
3H), 3.37 (m, 1H), 2.97 (m, 2H), 2.21 (m, 2H), 2.08 (m, 2H), 1.85
(m, 1H), 1.46 (m, 2H), 1.39 (m, 2H), 1.25 (m, 2H). MS (MH).sup.+:
393.35.
[0470] Synthesis of Compound 189
[0471] Compound 188 (135 g, 0.3440 moles) was dissolved in 800 mL
THF in 3 L RBF. The reaction mixture was cooled to 0.degree. C. and
LiBH.sub.4 (9.74 g, 0.4475 moles) in 200 mL of THF was added in
portion over a span of half hour. Reaction was stirred for
additional half an hr at this temperature and then at room
temperature for 1.5 hr. The completion of the reaction was
monitored by TLC. Reaction mixture was then cooled to 00.degree. C.
and diluted with 600 mL of water. 600 mL HCl (2N) was then added
till pH was acidic. Excess of THF was removed under reduced
pressure and residue was extracted with EtOAc (700 mL.times.3).
Organic layer was separated, washed with brine and dried over
Na.sub.2SO.sub.4. The solvent was stripped under reduced pressure
to furnish 189 which was used directly for further reactions.
Yield: 120 g (96%) .sup.1H NMR (DMSO, 400 MHz), .delta.=7.35 (m,
5H), 7.31 (m, 1H), 4.98 (s, 2H), 4.27 (m, 1H), 4.02-3.95 (m, 1H),
3.47-3.22 (m, 6H) 2.98-2.93 (m, 2H), 2.15 (m, 2H), 1.88 (m, 1H),
1.76 (m, 1H), 1.47 (m, 2H), 1.40 (m, 2H), 1.17 (m, 2H). MS
(MH).sup.+: 365.2.
[0472] Synthesis of Compound 190
[0473] To a stirring solution of 189 (2 g, 0.005494 mole),
triethylamine (1.5 mL, 0.01153 moles) in DCM (10 mL), DMTr-Cl
(2.047 gm, 0.006043 moles) in 10 mL DCM was added dropwise and the
reaction mixture stirred overnight (14 h). Reaction mixture was
concentrated and the product was purified by column chromatography
(EtOAc/hexane) using 100-200 silica gel.
[0474] (Note: Few drops of TEA were added while loading the silica
gel onto the column to reduce the acidic nature of silica gel).
Yield 1.5 gm (41%). .sup.1H NMR (DMSO, 400 MHz), .delta.=7.35-7.16
(m, 15H), 6.87 (m, 4H), 4.99 (s, 2H), 4.38 (m, 1H), 4.13 (m, 1H),
3.72 (s, 6H), 3.57 (m, 1H), 3.15 (m, 1H), 2.97 (m, 3H), 2.19 (t,
2H), 2.01 (m, 1H), 1.83 (m, 1H), 1.46-1.23 (m, 7H). MS (MH).sup.+:
667.50.
[0475] Synthesis of Compound 191
[0476] The Cbz protected amine 190 (38 g, 0.05705 moles) was
dissolved in 250 mL EtOAc/MeOH (3:1). After degassing,
triethylamine (Catalytic) was added. This was followed by the
addition of Pd/C (5.5 g, 15 Wt %, 10 Wt % Degussa type) and the
reaction mixture was stirred for 3 hr under hydrogen atmosphere.
After completion of reaction (monitored by TLC), the mixture was
filtered through celite bed and washed with 300 mL (3:1
EtOAc:MeOH). Filtrate was concentrated to give crude 191 which was
purified by column chromatography (5% MeOH/DCM) on 100-200 silica
gel. (Note: Few drops of TEA were added while loading the silica
gel onto the column to reduce the acidic nature of silica gel).
Yield: 28 g (93%)_H NMR (DMSO, 400 MHz), .delta.=7.32-7.17 (m, 9H),
6.85-6.89 (m, 4H), 4.38 (m, 1H), 4.45 (m, 1H), 4.14 (m, 1H), 3.76
(s, 6H), 3.58 (m, 1H), 3.34-2.97 (m, 7H), 2.22-2.18 (m, 2H),
2.02-1.98 (m, 2H), 1.83 (m, 1H), 1.48-1.14 (m, 4H). MS (MH).sup.+:
533.37.
[0477] Synthesis of Compound 193
[0478] To a stirring solution of 192 (1 g, 0.003386, moles) in 7 mL
DCM, EDC.HCl (0.778 g, 0.004763 moles) was added at 0.degree. C.
under nitrogen atmosphere. After stirring for 5 minutes HOBt (0.518
g, 0.03385 moles) was added and the reaction mixture was stirred
additionally for 15 mins. 191 (1.8 g, 0.003386 moles) in 7 mL of
DCM was then added to reaction mixture at the same temperature and
stirring was continued. This was followed by DIPEA till reaction
mixture showed basic on pH paper (.about.0.7 mL). Stirring was
continued overnight at room temperature. After completion of
reaction, it was quenched with ice, extracted with DCM (15
mL.times.3) and washed with 20 mL NH.sub.4Cl solution. The organic
layer was separated, dried over Na.sub.2SO.sub.4 and concentrated
under reduced pressure. Purification by column chromatography (5%
MeOH-DCM) using 100-200 silica gel furnished pure 193 in
respectable yield. Yield: 2.3 g (85%). .sup.1H NMR (DMSO, 400 MHz),
.delta.=7.78 (m, 2H), 7.36-7.16 (m, 14H), 6.85 (m, 4H), 4.99 (m,
2H), 4.37 (m, 1H), 4.08 (m, 1H), 3.72 (s, 6H), 3.62-3.55 (m, 4H),
3.14 (m, 1H), 2.98 (m, 3H), 2.50 (m, 2H) 2.20-1.92 (m, 8H),
1.45-1.23 (m, 7H). MS (MH).sup.+: 810.49.
[0479] Synthesis of Compound 194
[0480] The Cbz protected amine 193 (63 mg, 0.077 mmoles) was
dissolved in 4 mL, EtOAc:MeOH (3:1). After degassing, triethylamine
(catalytic) was added. This was followed by the addition of Pd/C
(9.4 mg, 15 Wt %, 10 Wt % Degussa type) and the reaction was
stirred for 3 hr under hydrogen atmosphere. The progress of the
reaction was monitored by TLC. After completion (5 hrs) the
reaction was filtered through celite bed and washed with 15 mL (3:1
EtOAc:MeOH). The filtrate was concentrated to afford the crude
product which was column purified (5% MeOH-DCM) on 100-200 silica
gel. Yield: 40 mg (78%) .sup.1H NMR (DMSO, 400 MHz), .delta.=7.77
(m, 1H), 7.30 (m, 4H), 7.21-7.16 (m, 5H), 6.88 (m, 4H), 4.38 (m,
1H), 4.25 (m, 1H), 3.72 (s, 6H), 3.60 (m, 4H), 3.33-3.16 (m, 2H),
3.15 (m, 1H), 2.98 (m, 3H), 2.2 (t, 2H), 2.12 (t, 2H), 2.0 (m, 2H)
1.83 (m, 2H), 1.6 (m, 1H), 1.46 (m, 2H), 1.37 (m, 2H), 1.25 (m,
4H). MS (MH).sup.+: 676.41.
##STR00068## ##STR00069##
[0481] Synthesis of Compound 196
[0482] To suspension of Compound 195 (0.2 g, 0.000671 moles) in
anhydrous pyridine (5 mL) was added DMAP (0.13 g, 0.0010 moles),
followed by isobuytric anhydride (0.6 mL, 0.0040 moles) at room
temperature. The resulting mixture was then refluxed for 4 hr.
[0483] After completion of reaction (by TLC), the mixture was
poured onto ice-HCl/hexane and stirred well. The resulting solid
was filtered, washed with hexane and used directly for further
reactions. Yield (0.1 g, 34%). .sup.1H NMR (DMSO, 400 MHz):
.delta.=12.08 (s, 1H), 11.48 (s, 1H), 7.85 (d, 2H), 7.35 (d, 2H),
7.22 (s, 1H), 4.33 (m, 1H), 2.98 (m, 2H), 2.96 (m, 2H), 2.81 (m,
1H), 1.19 (d, 6H), 1.14 (d, 6H). .sup.13C NMR (DMSO): 179.85,
175.21, 167.00, 156.64, 147.81, 147.34, 146.73, 129.08, 128.32,
128.14, 121.27, 116.00, 106.07, 35.02, 34.51, 33.37, 26.90, 18.72,
18.64. MS (MH): 439.40.
[0484] Synthesis of Compound 197
[0485] To a stirring solution of 196 (0.4 g, 0.00091 moles) in 5 ml
MeOH, DIPEA (0.036 mL, 0.00278 moles), was added at room
temperature. (Note: After addition of DIPEA reaction mixture
becomes clear). After 10 minutes DMAP (catalytic) was added to the
mixture. The completion of reaction was monitored by TLC (&
LCMS). MeOH was then concentrated and the residue was diluted with
water (5 mL). Acidification with dilute HCl was followed by
extraction with ethyl acetate. Organic layer was separated, dried
over Na.sub.2SO.sub.4 and concentrated. The crude mixture was pure
enough and was used directly for further reactions. Yield: 260 mg
(54%). .sup.1H NMR (DMSO, 400 MHz), .delta.=12.77 (bs, 1H), 11.68
(s, 1H), 11.34 (s, 2H), 7.84 (d, 2H), 7.32 (d, 2H), 6.64 (s, 1H),
3.01 (m, 2H), 2.94 (m, 2H), 2.74 (m, 1H), 1.09 (d, 6H). MS
(MH).sup.+: 369.10.
[0486] Synthesis of Compound 198
[0487] To a solution of 197 (4 g, 0.01086 moles) in 15 mL dry DMF,
HBTU (4.11 g, 0.01086 moles) and DIPEA (3.87 mL, 0.02173 moles)
were added at room temperature under stirring. After half an hour,
194 (7.33 g, 0.01086 moles) in dry DMF (10 mL) was added to the
solution and stirring was continued. The progress of the reaction
was monitored by TLC (4 hr). After completion, the reaction mixture
was poured into crushed ice and was extracted with EtOAc (100
mL.times.3). Organic layer was separated and dried over
Na.sub.2SO.sub.4. Removal of solvent under reduced pressured
afforded 198, which was purified by column chromatography (3%
MeOH/DCM). Yield: 3.6 g (33%). .sup.1H NMR (DMSO, 400 MHz),
.delta.=11.68 (s, 1H), 11.34 (bs, 2H), 8.73 (bs, 1H), 7.83-7.77 (m,
3H), 7.29-7.16 (m, 11H), 6.87 (m, 4H), 6.63 (s, 1H), 4.99 (m, 1H),
4.37 (m, 2H), 4.12 (bs, 1H), 3.71 (bs, 6H), 3.62 (broad, 3H), 3.57
(m, 1H), 3.50 (m, 2H), 3.15 (m, 3H), 2.99 (m, 8H), 2.74 (m, 2H),
2.18 (m, 4H), 1.44-1.22 (m, 6H), 1.11 (d, 6H) 1.09 (m, 2H), 1.02
(m, 1H). MS (MH).sup.+: 1026.60
[0488] The hydroxyl compound 198 on treatment with succinic
anhydride followed by treatment with polystyrene linked resin
provided the folate coupled solid support 199 in 72 .mu.M/g
loading.
Example 11
Folate Building Blocks for Click-Chemistry
[0489] In order to synthesize azido functional group containing
folate conjugates the following strategy was used. The azido amine
tether 204 was synthesized starting from the commercially available
diamine 201 as shown below.
##STR00070##
[0490] Synthesis of Amine 202
[0491] To a solution of the diamine (22 g, 0.1 mol) in
dichloromethane (300 mL), triethylamine (15 mL) was added and the
mixture was cooled in ice bath. To this cold solution a solution of
(Boc).sub.2O in CH.sub.2Cl.sub.2 (100 mL) was added dropwise and
the mixture was stirred overnight. The reaction mixture was washed
with satd. NaHCO.sub.3 (200 mL), water (300 mL), brine (300 mL) and
dried (Na.sub.2SO.sub.4). Concentration of this organic layer
followed by column purification provided the pure mono Boc amine
202 in 55% yield. MS: MW Calc. for C.sub.15H.sub.32N.sub.2O.sub.5:
320.42. Found 321.41 (MH.sup.+).
[0492] Synthesis of Azide 203:
[0493] The triflic azide stock solution was prepared as reported in
Tetrahedron Letters 47 (2006) 2382-2385. The amine (0.96 g, 3
mmol), sodium bicarbonate (0.85 mg, 10 mmol) and copper (II)
sulfate pentahydrate (22 mg, 0.1 mmol) were dissolved in water (3
mL). Triflic azide stock solution (5 mL) was added, followed by the
addition of methanol (20 mL) to yield a homogeneous system. The
blue mixture was stirred for 30 min after which the TLC and MS
showed the complete disappearance of starting amine. The reaction
mixture was concentrated in a rotary evaporator and the residue was
purified by chromatography on silica gel (eluent:
dichloromethane-methanol) to obtain the pure azide 203 (1 g, 96%)
as an oil. MS: MW Calc. for C.sub.15H.sub.30N.sub.4O.sub.5: 346.42.
Found 347.41 (MH.sup.+). .sup.1HNMR (CDCl.sub.3, 400 MHz)
.delta.=4.68 (bs, 1H), 3.40-3.30 (m, 12H), 3.16 (t, J=6.4 Hz, 2H),
3.00-2.95 (m, 2H), 1.68-1.54 (m, 4H), 1.04 (s, 9H).
[0494] Synthesis of 204:
[0495] The azide 203 (1 g, 2.88 mmol) was dissolved in ethanol (10
mL) and to this a 2M solution of HCl in ether was added and the
mixture was stirred at room temperature overnight. The MS showed
the absence of starting material. The reaction mixture was
concentrated and the thus obtained oil was used as such for the
next reaction without further purification. MS: MW Calc. for
C.sub.10H.sub.23ClN.sub.4O.sub.3: 246.17. Found 247.17 (MH.sup.+).
.sup.1HNMR (DMSO-d.sub.6 400 MHz) .delta.=8.96 (bs, 1H), 7.92 (bs,
2H), 3.52-3.40 (m, 12H), 3.37 (t, J=6.8 Hz, 2H), 2.85-2.77 (m, 2H),
1.81-1.70 (m, 4H).
[0496] Synthesis of 205:
##STR00071##
[0497] Coupling of the amine 204 (0.6 g) with the acid 144 (1.2 g)
using a similar procedure to that used for the synthesis of 146
provided the coupled azide 205 (1.68 g, 93%) as a light yellow
foam. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.=12.34 (s, 1H),
11.95 (s, 1H), 8.89 (s, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.81 (m, 1H),
7.70 (d, J=8.4 Hz, 2H), 5.22 (s, 2H), 4.40-4.34 (m, 1H), 3.62 (s,
3H), 3.50-3.31 (m, 15H), 3.09-3.00 (m, 2H), 2.80-2.72 (m, 1H), 2.20
(t, J=7.4 Hz, 2H) 2.10-1.89 (m, 2H), 1.76-1.54 (m, 4H), 1.12 (d,
J=6.8 Hz, 6H). MS. Molecular weight calculated for
C.sub.36H.sub.46F.sub.3N.sub.11O.sub.10, Cal. 849.81. Found 850.2
(MH.sup.+).
[0498] Synthesis of 206:
[0499] The azide 205 (1 g) was dissolved in THF (20 mL) and to it
an aqueous solution of lithium hydroxide (100 mg in 2 mL of water)
was added and the solution was stirred at room temperature for 4 h
after which the MS showed the complete disappearance of SM. The
reaction mixture was acidified to pH 5 using acetic acid and the RM
was diluted with ethyl acetate (100 mL). The precipitated product
was filtered off and washed with water and ethyl acetate and dried
under vacuo at 40.degree. C. overnight to get the pure azide 206
(0.455 g 55%) as an orange solid. .sup.1H NMR (DMSO-d.sub.6, 400
MHz) .delta.=8.59 (s, 1H), 7.85 (bs, 1H), 7.72 (bs, 1H), 7.56 (d,
J=8.4 Hz, 2H), 6.88 (bs, 1H), 6.65 (d, J=8.4 Hz, 2H), 4.45 (s, 2H),
4.00-4.02 (m, 1H), 3.50-3.33 (m, 14H), 3.04-3.00 (m, 2H), 2.07-1.83
(m, 4H), 1.76-1.54 (m, 4H). MS. Molecular weight calculated for
C.sub.29H.sub.39N.sub.11O.sub.8, Cal. 669.69. Found 668.2
(M-H.sup.-).
[0500] In another embodiment, the alkyne containing folic acid is
synthesized as follows. In this case the protected pteroic acid 144
was coupled with the protected lysine 207 to get the coupled
product 208 which on Cbz deprotection provided the amine 209.
Coupling of the amine 209 with the acid 210 provided the coupled
product 211 which after purification and deprotection provided the
product 212 as described below.
##STR00072##
[0501] Synthesis of 208:
[0502] Using a similar procedure to that used for the synthesis of
205, coupling of the acid 144 with the lysine derivative 207
provided the coupling product 208 as a white solid in 95%
yield.
[0503] Synthesis of 209:
[0504] The compound 208 on hydrogenation with Pd/C provided the
deprotected amine 209 as a yellow solid.
[0505] Synthesis of 210:
[0506] Coupling of the amine 209 with the acid 210 using a
procedure to that used for the synthesis of 205 provided the couple
product 210 in high yields.
[0507] Synthesis of 212:
[0508] The deprotection of the protecting groups is achieved using
a similar procedure as described for the synthesis of 206 to
isolate the fully deprotected alkyne 212.
[0509] The synthesis of the building block 213 is then carried out
as shown below.
##STR00073##
[0510] The building block 213 is then converted to solid-support
214 or phosphoramidite 215.
##STR00074##
Example 12
Synthesis of Folate Conjugate 216
[0511] In order to conjugate folic acid to the 5' end of the oligo,
post synthetically, the following route was developed. Treatment of
folic acid 215 with DCC followed by N-hydroxysuccinimide provided
the activated ester 216 in 80% yield. In a typical procedure, folic
acid (5 g, 11.33 mmol) was dissolved in anhydrous DMSO (100 mL) and
to this solution was added, triethyamine (2.5 mL), DCC (4.7 g, 22.6
mmol) and N-hydroxysuccinimide (2.6 g, 22.6 mmol) and the solution
was stirred at room temperature in dark for 18 h. The reaction
mixture was filtered and to the filtrate EtOAc (1 L) was added and
the precipitated product was filtered, washed with ethyl acetate
(500 mL), ether (200 mL) and dried under vacuum to isolate the
product as a yellow powder. The purity of the product was found to
be 83% by HPLC. This product was used as such for the coupling
steps without further purification.
##STR00075##
In another embodiment a cleavable acetal linkage was used in order
to facilitate the release of siRNA from the targeting folate as
follows.
##STR00076## ##STR00077##
[0512] The ketal 217 was synthesized using a reported procedure
(Paramonov, S. E.; Bachelder, E. M.; Beaudette, T. T.; Standley, S.
M.; Lee, C. C.; Dashe, J.; Frechet, Jean M. J. Fully
Acid-Degradable Biocompatible Polyacetal Microparticles for Drug
Delivery. Bioconjugate Chemistry (2008), 19 (4), 911-919). The
transient protection of the ketal was carried out in two steps in
one pot first by treating the diamine with one equivalent of
ethyltrifluoroacetate followed by one equivalent of Cbz-OSu to
provide the di protected derivative 218 in 80% yield after column
purification. The protected amine 218 on treatment with aqueous
LiOH provided the amine 219 in quantitative yield. Coupling of this
amine 219 (0.5 g) with the protected folic acid 144 (1 g) provided
the coupled product 220 (1.1 g) which on hydrogenation provided the
amine 221 in quantitative yield.
[0513] Coupling of amine 221 was carried out with the
maleimidopropionic acid 222 to give the coupled product 223 in good
yields. The final deprotection of all the protecting group in 223
is carried out using ice-cold aqueous LiOH in THF to afford the
precursor 224 as an orange solid.
Example 13
Synthesis of Conjugated siRNAs
[0514] Synthesis of Folate Conjugated siRNA:
[0515] The 3'-folate and C6-S--S--C6-folate siRNA conjugates with
and without fluorophore label and its controls and corresponding
antisense strands used in this study are shown in Tables 4-7. These
were individually synthesized using commercially available
5'-O-(4,4'-dimethoxytrityl)-2'-O-t-butyldimethylsilyl-3'-O-(2-cyanoethyl--
N,N-diisopropyl) RNA phosphoramidite monomers of
6-N-benzoyladenosine (A.sup.Bz), 4-N-acetylcytidine (C.sup.Ac),
2-N-isobutyrylguanosine (G.sup.iBU), and uridine (U), according to
standard solid phase oligonucleotide synthesis protocols as
previously described (1). The folate conjugated strands were
synthesized using folate containing solid support. (C, FIG. 2). The
introduction of cholesterol unit in the sequence was performed from
a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol was
tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage
to obtain a hydroxyprolinol-cholesterol moiety. 5'-end Cy-3 and
Cy-5.5 (fluorophore) labeled siRNAs were synthesized from the
corresponding Quasar-570 (Cy-3) phosphoramidite phosphoramidite
purchased from Biosearch Technologies). The disulfide linker was
introduced to the siRNA using C6-S--S--C6 phosphoramidite as
purchased from Glen Res. VA. An extended 15 min 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 tert-butyl
hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation
wait time afforded folate conjugated oligonucleotide.
[0516] Deprotection-I
[0517] After completeness of oligonucleotide synthesis, a solid
support was treated with 1 M aq. piperidine for 24 h at room
temperature. The solid support was washed with another portion of
deprotecting reagent and combined solutions were evaporated under
reduced pressure. Added a mixture of ethanolic ammonia [ammonia
(28-30%): ethanol (3:1) 1.0 mL] for 8 h at 55.degree. C. (2). The
vial was cooled briefly on ice and then the ethanolic ammonia
mixture was transferred to a new microfuge tube. The CPG was washed
with portions of deionized water (2.times.0.1 mL). The combined
filtrate was then put in dry ice for 10 min dried in a speed
vac.
[0518] Deprotection-II (Removal of 2'-O-TBDMS Group)
[0519] The white residue obtained was resuspended in a mixture of
triethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF)
and Dimethylsulfoxide (DMSO) (1:2:6)
[0520] (3.0 mL) and heated at 65.degree. C. for 2 h to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2'-position.
[0521] Analysis of Folate Conjugated siRNA:
[0522] The Folate conjugated sequences were analyzed by
high-performance liquid chromatography (HPLC) on Phenomenox C6
phenyl column. The buffer A was 50 mM TEAA, 10% Acetonitrile and
Buffer B was 50 mM Sodium Acetate, 70% Acetonitrile at 65.degree.
C. in gradient of 25-80% B in 35 min
[0523] HPLC Purification:
[0524] The Folate conjugated, Cy-3, Cy 5.5 and Cy-3-Folate hybrid
sequences were purified by high-performance liquid chromatography
(HPLC) on an in-house packed RPC-Source15 reverse-phase column. The
buffers were 20 mM NaOAc in 10% CH.sub.3CN (buffer A) and 20 mM
NaOAc in 70% CH.sub.3CN (buffer B). Fractions containing
full-length oligonucleotides were pooled, desalted and lyophilized.
Analytical HPLC, CGE and ES LC-MS established the integrity of the
compounds. 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. For duplex
generation, equimolar amounts of sense and antisense strand were
heated in 1.times.PBS at 95.degree. C. for 5 min and slowly cooled
to room temperature.
[0525] Duplex Preparation
[0526] For the preparation of duplexes, equimolar amounts of sense
and antisense strand were heated in 1.times.PBS at 95.degree. C.
for 5 min and slowly cooled to room temperature. Integrity of the
duplex was confirmed by HPLC analysis.
[0527] Synthesized oligonucleotides and duplexes are shown in Table
4-7.
TABLE-US-00004 TABLE 4 Oligonucleotides synthesized and evaluated
SEQ Duplex ID Target ID No S/AS Sequence 5'-3' Luc AD- 30 A-30678
5' CcAcAuGAAGcAGcACGACuUQ51sL37 3618 31 A-4381 5'
AAGUCGUGCUGCUUCAUGUGgsusC Luc AD- 32 A-30677 5'
AccGAAAGGucuuAccGGAdTdTQ51sL37 3614 33 A-30674 5'
UCCGGuAAGACCUUUCGGUdTdTsL10 Luc AD- 34 A-30659 5'
Q38cuuacgcugaguacuucgadTdTL37 3514 35 A-30660 5'
ucgaaguacucagcguaagdTdTL8 3' Luc AD- 36 A-30659 5'
Q38cuuacgcugaguacuucgadTdTL37 3515 37 A-30661 5'
ucgaaguacucagcguaagdTdT-L10 GFP AD- 38 A-30678 5'
CcAcAuGAAGcAGcACGACuUQ51sL37 3615 39 A-3389 5'
AAGUCGUGCUGCUUCAUGUGgusCs-L10 GFP AD- 40 A-30676 5'
cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3616 41 A-22826 5'
UGCUCUGAAAUUcAGCcAGdTsdT Luc AD- 42 A-30677 5'
AccGAAAGGucuuAccGGAdTdTQ51sL37 3617 43 A-24775 5'
UCCGGuAAGACCUUUCGGUdTsdT CD45 AD- 44 A-30676 5'
cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3613 45 A-30673 5'
UGCUCUGAAAUUcAGCcAGdTdTs L10 CD45 AD- 46 A-22825
5'-cuGGcuGAAuuucAGAGcAdTsdT 3637 47 A-30687 5'
Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD- 48 A-22825
5'-cuGGcuGAAuuucAGAGcAdTsdT 3638 49 A-30695 5'
Q39UGCUCUGAAAUUcAGCcAGdTdTsL10 CD45 AD- 50 A-30676 5'
cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3639 51 A-30852 5'
Q78UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD- 52 A-30676
cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3633 53 A-30821 5'
UGCUCUGAAAUUcAGCcAGdTsdTL10 CD45 AD- 54 A-22825
5'-cuGGcuGAAuuucAGAGcAdTsdT 3634 55 A-30821 5'
UGCUCUGAAAUUcAGCcAGdTsdTL10 CD45 AD- 56 A-30676
5'cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3635 57 A-30687 5'
Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD- 58 A-30676 5'
cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3636 59 A-30695 5'
Q39UGCUCUGAAAUUcAGCcAGdTdTsL10 CD45 AD- 60 A-30592 5'
cuGGcuGAAuuucAGAGcAdTdTQ51L37 3599 61 A-22826 5'
UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD- 62 A-31653 5'
cuGGcuGAAuuucAGAGcAdTsdTL37 18744 63 A-30687 5'
Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD- 64 A-31632 5'
cuGGcuGAAuuucAGAGcAUUZ32 18218 65 31633 and 5'
UGCUCUGAAAUUcAGCcAGUU 66 31634 5'Q67dAdAdCdCdGdTdGdGdTdCdAdTdGdCdT
dCdC GFP AD- 67 A-31635 5' CcAcAuGAAGcAGcACGACUUUUZ32 18219 68
31636 and 5' UGCUCUGAAAUUcAGCcAGUU 69 31634 5'
Q67dAdAdCdCdGdTdGdGdTdCdAdTdGdCdTd CdC CD45 AD- 70 A-31653
5'cuGGcuGAAuuucAGAGcAdTsdTL37 18367 71 A-31015
5'ugcucugaaauucagccagdTsdTL63 GFP AD- 72 A-33204 5'
AcAuGAAGcAGcACGACuUdTdTQ11L37 18860 73 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 74 A-33169 5'
AcAuGAAGcAGcACGACuUdTsdTL37 18866 75 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 76 A-33169 5'
AcAuGAAGcAGcACGACuUdTsdTL37 18866 77 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 78 A-32141
5'CcAcAuGAAGcAGcACGACusUL102 18576 79 A-4381 5'
AAGUCGUGCUGCUUCAUGUGgsusC GFP AD- 80 A-32142 5'
CcAcAuGAAGcAGcACGACusUL103 18578 81 A-4381 5'
AAGUCGUGCUGCUUCAUGUGgsusC GFP AD- 82 A-32142 5'
CcAcAuGAAGcAGcACGACusUL103 18579 83 A-32145 5'
AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD- 84 A-32143 5'
CcAcAuGAAGcAGcACGACusUL104 18580 85 A-4381 5'
AAGUCGUGCUGCUUCAUGUGgsusC GFP AD- 86 A-32143 5'
CcAcAuGAAGcAGcACGACusUL104 18581 87 A-32145 5'
AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD- 88 A-32593 5'
AcAuGAAGcAGcACGACuUdTsdT 18747 89 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 90 A-33199 5'
AcAuGAAGcAGcACGACuUdTdTL10 18858 91 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 92 A-33205 5'
Q11AcAuGAAGcAGcACGACuUdTsdT 18861 93 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 94 A-33199 5'
AcAuGAAGcAGcACGACuUdTdTL10 18862 95 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 96 A-33169 5'
AcAuGAAGcAGcACGACuUdTsdTL37 18867 97 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 98 A-33200 5'
AcAuGAAGcAGcACGACuUdTdTL103 19084 99 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 100 A-33201 5'
Q11AcAuGAAGcAGcACGACuUdTdTL103 19085 101 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 102 A-33203 5'
AcAuGAAGcAGcACGACuUdTdTQ11L103 19086 103 A-32592 5'
AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 104 A-33201 5'
Q11AcAuGAAGcAGcACGACuUdTdTL103 19087 105 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 106 A-33203 5'
AcAuGAAGcAGcACGACuUdTdTQ11L103 19088 107 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 108 A-33054 5'
CcAcAuGAAGcAGcACGACusUL113 19185 109 A-4381 5'
AAGUCGUGCUGCUUCAUGUGgsusC GFP AD- 110 A-32141 5'
CcAcAuGAAGcAGcACGACusUL102 18577 111 A-32145 5'
AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD- 112 A-33202 5'
Q11AcAuGAAGcAGcACGACuUdTsdT 18859 113 A-32594 5'
AAGUCGUGCUGCUUCAUGUdTsdT GFP 114 A-34122
AcAuGAAGcAGcACGACuUdTsdTL10 115 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT
GFP 116 A-34123 AcAuGAAGcAGcACGACuUdTsdTL103 117 A-32594
AAGUCGUGCUGCUUCAUGUdTsdT GFP 118 A-34124
Q11AcAuGAAGcAGcACGACuUdTsdTL103 119 A-32594
AAGUCGUGCUGCUUCAUGUdTsdT GFP 120 A-34125
Q11AcAuGAAGcAGcACGACuUdTsdTL37 121 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT
GFP 122 A-34126 AcAuGAAGcAGcACGACuUdTsdTQl1L103 123 A-32594
AAGUCGUGCUGCUUCAUGUdTsdT GFP 124 A-34127
AcAuGAAGcAGcACGACuUdTsdTQl1L37 125 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT
Note: Lower case = 2'-OMe, S = PS linkage, L37: = N-(folic
acidcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-folate), L63: =
Oregon Green 488-aminohexylcarboxamidocaproyl-prolinol-4-phosphate,
Q8: = N-(aminocaproyl)prolinol-4-phosphate, L8: =
N-(cholesterylcarboxamidocaproyl)-3-hydroxy-4-hydroxymethylpyrrolidine,
Q38: = Quasar 570 phosphate (BNS-5063, Biosearch Tech), L10: =
N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol),
Q39: = Quasar 705 phosphate (BNS-5067, Biosearch Tech), Q78: =
Atto-610-aminohexylphosphate, L102: = N-(folate
carboxamidohexadecanoylcarboxamidoethyl-dithio-1,1-dimethylbutyryl)-4-hyd-
roxyprolinol (Hyp-Me2S-S-C16-folate), L103: = N-(folate
carboxamidoethyl-dithio-butyryl)-4-hydroxyprolinol
(Hyp-S-S-folate), L104: = N-(folate
carboxamidohexadecanoyl)-4-hydroxyprolinol (Hyp-C16-folate), Q11: =
N-(cholesterylcarboxamidocaproyl)prolinol-4-phosphate, Q51: =
6-hydroxyhexyldithiohexylphosphate (Thiol-Modifier C6 S-S Glen Res.
10-1936), L48: = N-(Alexa647-carboxamidocaproyl)-4-hydroxyprolinol
(Hyp-C6-Alexa647), L113: = N-(folate
carboxamido-PEG12)-4-hydroxyprolinol (Hyp-PEG12-folate), Q67: =
Folate-gamma-carboxamidohexylphosphate (Folate C6), Z32: =
GGAGCAUGACCACGG (SEQ ID NO: 126).
TABLE-US-00005 TABLE 5 Oligonucleotides synthesized. SEQ ID Cal
Found No Sequence (5'-3') Mass Mass AL-3435 127 5' cuG AAG Acc uGA
AGA cAA uTT-s-Folate 7529.02 7530.8 AL-3436 128 5' AUu GUC uUc AGG
UCu UcA GTT-s-Folate 7385.76 7386.8 AL-3437 129 5' GAA CUG UGU GUG
AGA GGU CCU-s-Folate 7499.7 7498.7 AL-3482 130 5' GAA CUG UGU GUG
AGA GGU CCU chol s Folate 8203.7 8205.2 AL-3480 131 5' cuG AAG Acc
uGA AGA cAA uTT-Chol-s-Folate 8232.3 8234.4 AL-3481 132 5' GAA CUG
UGU GUG AGA GGU CCU-Chol-s-Folate 8203.7 8205.3 AL-3609 133 5' CUU
ACG CUG AGU ACU UCG AdTdT-Chol-C18-Folate 3' 8369.2 8371.70 AL-3610
134 5' Quasar CUU ACG CUG AGU ACU UCG AdTdT-Chol-C18- 8989.2
8990.15 Folate3' AL-3611 135 5' CUU ACG CUG AGU ACU UCG
AdTdT-C18-Chol-C18- 8715.96 8715.30 Folate 3' AL-3612 136 5' Quasar
CUU ACG CUG AGU ACU UCG AdTdT-C18-Chol- 9333.96 9334.36
C18-Folate3' AL-3639 137 5' CUU ACG CUG AGU ACU UCG
ATT-C12S-S-Folate3' 7650.05 7650.26 AL-3640 138 5' Quasar CUU ACG
CUG AGU ACU UCG ATT-C12S-S-Folate 8270.26 8270.18 3' AL-3664 139 5'
CUU ACG CUG AGU ACU UCG ATT-C12S-S-C18-Folate 3' 7994.35 7994.71
AL-3665 140 5' Quasar CUU ACG CUG AGU ACU UCG ATT-C12S-S-C18-
8614.67 8614.47 Folate3' AL-3668 141 5' cuG AAG Acc uGA AGA cAA uTT
C12S-S-Folate 3' 7841.35 7841.37 AL-3669 142 5' Quasar cuG AAG Acc
uGA AGA cAA uTT C12S-S- 8461.59 8461.47 Folate 3' AL-3670 143 5'
cuG AAG Acc uGA AGA cAA uTT C12S-S-C18-Folate3' 8185.68 8186.05
AL-3671 144 5' Quasar cuG AAG Acc uGA AGA cAA uTT C12S-S-C18-
8805.99 8805.83 Folate 3' Quasar = Cy3; C18 = Spacer C18 linker ,
C12 S-S = disulfide linker, Chol = Cholesterol, lower case =
2'-o-Me, s = phosphorothioate linkage, Folate = Folate. See FIG.
1-8 for a graphical representation of sequences with the associated
conjugates and structure of monomers used.
TABLE-US-00006 TABLE 6 Oligonucleotides synthesized. SEQ ID Cal
Found No Sequence (5'-3') Mass Mass Al-30658 145 5' Cy-3
cuuacgcugaguacuucgadTdT-Hyp-NH.sub.2 7786.5 7784.3 Al-30660 146 5'
ucgaaguacucagcguaagdTdT-Hyp-NH.sub.2 3' 7251.1 7250.6 Al-30659 147
5' Cy-3 cuuacgcugaguacuucgadTdT-Folate 3' 8209.9 8207.8 Al-30661
148 5' ucgaaguacucagcguaagdTdT-cholesterol 76634.4 7663.6 Al-30676
149 5' cuGGcuGAAuuucAGAGcAdTdT-C6-S-S-C6-s-Folate 7856.4 7855.4
Al-30677 150 5' AccGAAAGGucuuAccGGAdTdT- C6-S-S-C6-s- 7864.4 7863.4
Folate Al-30678 151 5' CcAcAuGAAGcAGcACGACuU- C6-S-S-C6-s- 7822.3
7821.2 Folate Al-30673 152 5' UGCUCUGAAAUUcAGCcAGdTdTs-cholesterol
7379.0 7377.1 Al-30674 153 5' UCCGGuAAGACCUUUCGGUdTdTs-cholesterol
7357.9 7357.1 Al-3389 154 5' AAGUCGUGCUGCUUCAUGUGgusCs-cholesterol
8082.4 8081.1 Al-24775 155 5' UCCGGuAAGACCUUUCGGUdTsdT 6653.1
6652.3 Al-4381 156 5' AAGUCGUGCUGCUUCAUGUGgsusC 7277.5 7376.6
Al-30687 157 5' Cy-5.5 UGCUCUGAAAUUcAGCcAGTsT 7421.02 7420.4
Al-30695 158 5' Cy-5.5 UGCUCUGAAAUUcAGCcAGTTs- 8125.9 8124.6
cholesterol Hyp-NH2 = Hydroxy prolinol linker; Cy3 = Cy3 dye; Cy5.5
= Cy-5.5 dye; C6 S-S-C6 = disulfide linker; Chol = Cholesterol;
lower case = 2'-O-Me; Folate = Folate; s = phosphorothioate
linkage.
TABLE-US-00007 TABLE 7 Some more folate-conjugated duplexes. Al-
SEQ Duplex ID Duplex # No Sequences Concentration AD-3513 159 5'
Cy-3 cuuacgcugaguacuucgadTdT-Hyp-NH.sub.2 10 mg/ml 160 5'
ucgaaguacucagcguaagdTdT-Hyp-NH.sub.2 3' AD-3514 161 5' Cy-3
cuuacgcugaguacuucgadTdT-Folate 3' 10 mg/ml 162 5'
ucgaaguacucagcguaagdTdT-Hyp-NH.sub.2 AD-3515 163 5' Cy-3
cuuacgcugaguacuucgadTdT-Folate 10 mg/ml 164 5'
ucgaaguacucagcguaagdTdT-Hyp-Cholesterol Hyp-NH2 = Hydroxy prolinol
linker; Cy3 = Cy3 dye; Chol = Cholesterol; lower case = 2'-O-Me;
Folate = Folate;
Example 14
Binding Competition Assays
[0528] Folate conjugated siRNAs were incubated with KB cells in
presence of FTIC-folate (PBS, on Ice). % competition for folate
receptor (FR) was plotted as a measure of activity. FIG. 10 shows
that different folate conjugates had different binding competition
but sequence of the oligonucleotide made no difference on binding
competition. The Kd were calculated from the binding assays and are
reported in table 8. The 3' sense folate with cleavable or
non-cleavable linkers shows lower affinity for FR than folic acid.
Introduction of 3' antisense cholesterol improves binding by
.about.50 fold. 5' folate attached to a DNA-oligo tether binds to
FR with an affinity similar to that of folic acid. FR ligands have
a slow off-rate--no significant competition is observed if
FITC-folate is added 1.sup.st.
TABLE-US-00008 TABLE 8 Folate receptor binding of folate conjugated
siRNAs duplex # sense antisense KD (uM) AD-18218 5' extended 5'
folate DNA oligo 0.3 AD-18219 5' extended 5' folate DNA oligo 0.2
AD-18576 3'-Hyp-Me2S-S-C2-C16-folate unconjugated 12.5 AD-18578
3'-Hyp-S-S-folate unconjugated 34.8 AD-18579 3'-Hyp-S-S-folate 3'
chol 0.3 AD-18580 3'-Hyp-C16-folate unconjugated 20.7 AD-18581
3'-Hyp-C16-folate sense 3' chol 0.3 AD-3614 3' C6-SS-C6-Hyp folate
3' chol 0.2 AD-3617 3' C6-SS-C6-Hyp folate unconjugated 13.7
Example 15
Folate GFP Duplexes, In Vitro Silencing with Transfection
Reagent
[0529] KB pEGFP clone 17 cells were used for all GFP silencing
experiments. For silencing experiments with transfection reagent
HiPerFect was used to transfect in the siRNAs tested and GFP
expression was analyzed 72 hrs later. As seen in FIG. 11, presence
of folate ligands (multiple linker designs) at the 3' of the sense
strand does not significantly impact silencing activity. 3'
antisense cholesterol decreased activity by 10.times. or greater
depending on design, similar trends were observed for CD45
duplexes. Addition of 3' sense folate to 3' or 5' sense cholesterol
duplex does not significantly alter silencing activity.
Example 16
Free Uptake Silencing Activity
[0530] Silencing by free uptake was evaluated in the KB-EGFP cells,
a cell line stably expressing the EGFP gene. KB-EGFP cells were
cultured continuously in folate free media with 10% FBS. For free
uptake silencing folate-siRNA conjugates were added to KB-EGFP
cells in 24-well plates in media with or without serum. For the "no
serum" wells complete media (with 10% FBS) was added 4 hrs later.
Cells were cultured for 72 hrs, removed from plates using Versene,
washed and GFP expression quantified by flow cytometry on a LSRII
instrument. Data was analyzed using the FlowJo software and median
fluorescence intensity of the GFP signal was plotted. As seen in
Table 9, various tethers and linker designs containing folate
conjugates can be accommodated at the 3'-end of the sense strand
without affecting RNAi activity. 25% silencing by free uptake is
observed for folate conjugated siRNA at 5 uM concentration
independent of serum. siRNAs comprising both cholesterol and folate
show 40% silencing by free uptake in absence of serum at 5 uM.
TABLE-US-00009 TABLE 9 Free uptake of folate conjugated siRNAs %
free uptake silencing KD IC.sub.50 (5 uM, no duplex # sense
antisense (uM) (nM) serum) singe overhang AD-5179 unconjugated
unconjugated ND 0.09 0 AD-3618 3' C6-SS-C6-Hyp folate unconjugated
ND 0.13 0 AD-3615 3' C6-SS-C6-Hyp folate 3' chol ND 0.69 0 AD-18576
3'-Hyp-Me2S-S-C2-C16-folate unconjugated 12.5 0.12 10 AD-18577
3'-Hyp-Me2S-S-C2-C16-folate 3' chol ND 4.09 ND AD-18578
3'-Hyp-S-S-folate unconjugated 34.8 0.09 0 AD-18579
3'-Hyp-S-S-folate 3' chol 0.3 0.80 20 AD-18580 3'-Hyp-C16-folate
unconjugated 20.7 0.14 0 AD-18581 3'-Hyp-C16-folate sense 3' chol
0.3 2.04 0 double overhang AD-18527 unconjugated unconjugated ND
0.03 0 AD-18526 unconjugated 3' chol ND 2.08 ND AD-18858 3'
cholesterol unconjugated ND 0.07 80 AD-18861 5' cholesterol
unconjugated ND 0.06 40 AD-18859 5' cholesterol, 3' folate
unconjugated ND 0.07 40 AD-18860 3' cholesterol, 3' folate
unconjugated ND 0.04 40 AD-18866 3' Hyp-C6 folate unconjugated ND
0.03 25 3-oligo duplex AD-18219 5' extended 5' folate DNA oligo 0.2
0.16 0
Example 17
Serum Stability of Folate Conjugated Oligonucleotides
[0531] siRNAs comprising folate conjugates with a cleavable linker
were incubated in mouse serum at 37.degree. C. for 0, 0.5, 1, 2, 6,
16 and 24 hours. After incubation, amount of individual strands
were quantified by IEX HPLC. Sense strand t.sub.1/2 decreased by
5-78.times. depending on the sequence. Table 10 summarizes the
t.sub.1/2 of individual strands of tested siRNAs. For a 3'sense
folate a gem-dimethyl linker duplex is more stable in serum then an
unhindered disulfide linked duplex. The 3' sense folate duplex with
a C16 non-cleavable linker is as stable as the unmodified parent
duplex. The 3' sense folate duplexes with a Glen research disulfide
linker show the lowest serum stability, however these molecules
also lack the PS group in the overhang.
TABLE-US-00010 TABLE 10 Serum stability of folate conjugated
duplexes in mouse serum (table discloses SEQ ID NOS 165-178,
respectively, in order of appearance) Single Strand Half-life
Duplex Strand Type 5-3 Full Strand (h) AD-3215 22825 s
5'-cuGGcuGAAuuucAGAGcAdTsdT 6.58 22826 as 5'
UGCUCUGAAAUUcAGCcAGdTsdT 1.83 AD-3616 30676 s
5'-cuGGcuGAAuuucAGAGcAdTdT-C6-S-S-C6-folate 1.22 22826 as
UGCUCUGAAAUUcAGCcAGdTsdT 2.90 AD-5179 4545-b1 s
CcAcAuGAAGcAGcACGACusU 15.00 4381-b11 as AAGUCGUGCUGCUUCAUGUGgsusC
11.50 AD-3618 30678 s 5' CcAcAuGAAGcAGcACGACuU-C6-S-S-C6-Folate
0.19 4381 as AAGUCGUGCUGCUUCAUGUGgsusC 1.59 AD-18576 32141 s
CcAcAuGAAGcAGcACGACusU-Hyp-Me2S-S-C16-folate 15.36 4381 as
AAGUCGUGCUGCUUCAUGUGgsusC 8.22 AD-18578 32142 s
CcAcAuGAAGcAGcACGACusU-Hyp-S-S-folate 6.39 4381 as
AAGUCGUGCUGCUUCAUGUGgsusC 4.69 AD-18580 32143 s
CcAcAuGAAGcAGcACGACusU-Hyp-C16-folate 15.72 4381 as
AAGUCGUGCUGCUUCAUGUGgsusC 10.44
Example 18
In Vivo Folate Conjugate Distribution in KB Xenograft Tumor
Model
[0532] KB cells were injected subcutaneously on both flanks of
Nu/Nu mice (3.times.10.sup.6 cells per site). Tumors were allowed
to develop for 2 weeks. Mice were injected with 60 nmol fluorophore
and folate conjugated siRNAs. Mice were saced at the desired time
point and tumors and kidneys harvested for imaging on the Maestro
imaging system and downstream processing. For lysate assays tissues
were frozen and ground to powder using a mortar and pestle. Tissue
powders (20 mg) were lysed using the Mammalian Cell Lysis Kit
(Sigma). Protein concentration was quantified using the BCA assay,
Oregon Green fluorescence in lysates was quantified using the
Victor plate reader. For tumor sections, animals were perfused with
4% PFA upon sacrifice, tumors were harvested and cryoprotected in
30% sucrose. Tumors were sectioned by cryostat. Sections were
post-fixed with PFA and stained for mouse CD11b or F.sub.4/80
macrophage markers. Sections were imaged on the Axiovision
fluorescence microscope and fluorescence normalized to protein
concentration. Conjugated siRNAs AD-18367 (3' non-cleavable folate
sense and 3' OregonGreen antisense) and AD-3716 (3' OregonGreen
antisense) were used. Figure x shows the accumulation of folate
conjugated siRNA (AD-18367) in the tumor and kidney at 4 hours.
FIG. 12 show tissue uptake of folate conjugated siRNA at 4 hour and
18 hours after injection. As can be seen in FIG. 13, folate
conjugated siRNA was present in significant amount in the tumor
after 18 hours. FIG. 14 shows the accumulation of Cy3 labeled
folate conjugated siRNAs in tumors. Accumulation was only seen when
the siRNA had a folate conjugate, thus confirming targeting by
folate conjugates.
[0533] Sections from a tumor treated with folate conjugated Cy3
labeled siRNA (AD-3514) were strained with an antibody to mouse
F4/80 or an isotype control. Results are shown in FIG. 15.
Example 19
KB EGFP Tumors in Nude Mice
[0534] Nu/Nu mice were SC implemented with KB or KB EGFP cells.
Tumors were imaged on the Maestro system. Tumor lysates were
prepared to measure GFP levels. FIG. 16 shows results of tumor
establishment and GFP levels in the tumors.
Sequence CWU 1
1
17814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Glu Ala Leu Ala 1 229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Ala
Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10
15 Leu Glu Ala Leu Ala Glu Ala Ala Ala Ala Gly Gly Cys 20 25
330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala 1 5 10 15 Glu Ala Leu Ala Glu Ala Leu Ala Ala
Ala Ala Gly Gly Cys 20 25 30 415PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 4Ala Leu Glu Ala Leu Ala
Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10 15 522PRTInfluenza A
virus 5Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu
Gly 1 5 10 15 Met Ile Trp Asp Tyr Gly 20 623PRTInfluenza A virus
6Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1
5 10 15 Met Ile Asp Gly Trp Tyr Gly 20 724PRTInfluenza A virus 7Gly
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10
15 Met Ile Asp Gly Trp Tyr Gly Cys 20 822PRTInfluenza A virus 8Gly
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10
15 Met Ile Asp Gly Gly Cys 20 935PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 9Gly Leu Phe Gly Ala Leu
Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala Glu
Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly Ser
Cys 35 1034PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn
Gly Trp Glu Gly 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu
Ala Leu Ala Ala Gly Gly 20 25 30 Ser Cys 1121PRTInfluenza A
virusMOD_RES(17)..(17)Norleucine 11Gly Leu Phe Glu Ala Ile Glu Gly
Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu Ile Asp Gly Lys 20
1220PRTInfluenza A virusMOD_RES(17)..(17)Norleucine 12Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu
Ile Asp Gly 20 1316PRTDrosophila sp. 13Arg Gln Ile Lys Ile Trp Phe
Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 1413PRTHuman
immunodeficiency virus 1 14Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln 1 5 10 1527PRTHuman immunodeficiency virus 1 15Gly Ala
Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15
Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 1618PRTMus sp.
16Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1
5 10 15 Ser Lys 1726PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Gly Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu Ala Lys
Lys Ile Leu 20 25 1818PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 18Lys Leu Ala Leu Lys Leu Ala
Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala
199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5
2010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys 1 5 10
2137PRTHomo sapiens 21Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu
Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys
Asp Phe Leu Arg Asn Leu Val 20 25 30 Pro Arg Thr Glu Ser 35
2231PRTAscaris suum 22Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu
Asn Ser Ala Lys Lys 1 5 10 15 Arg Ile Ser Glu Gly Ile Ala Ile Ala
Ile Gln Gly Gly Pro Arg 20 25 30 2330PRTHomo sapiens 23Ala Cys Tyr
Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly
Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30
2436PRTHomo sapiens 24Asp His Tyr Asn Cys Val Ser Ser Gly Gly Gln
Cys Leu Tyr Ser Ala 1 5 10 15 Cys Pro Ile Phe Thr Lys Ile Gln Gly
Thr Cys Tyr Arg Gly Lys Ala 20 25 30 Lys Cys Cys Lys 35
2512PRTUnknownDescription of Unknown Unknown bactenecin peptide
25Arg Lys Cys Arg Ile Val Val Ile Arg Val Cys Arg 1 5 10 2642PRTSus
scrofa 26Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Pro 1 5 10 15 Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro
Gly Phe Pro Pro 20 25 30 Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg 35
40 2713PRTUnknownDescription of Unknown Unknown indolicidin peptide
27Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10
2816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala
Leu Leu Ala Pro 1 5 10 15 2911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Ala Ala Leu Leu Pro Val Leu
Leu Ala Ala Pro 1 5 10 3021RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 30ccacaugaag
cagcacgacu u 213123RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 31aagucgugcu gcuucaugug guc
233221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 32accgaaaggu cuuaccggat t
213321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 33uccgguaaga ccuuucggut t
213421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 34cuuacgcuga guacuucgat t
213521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 35ucgaaguacu cagcguaagt t
213621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 36cuuacgcuga guacuucgat t
213721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 37ucgaaguacu cagcguaagt t
213821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 38ccacaugaag cagcacgacu u
213923RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 39aagucgugcu gcuucaugug guc
234021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 40cuggcugaau uucagagcat t
214121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 41ugcucugaaa uucagccagt t
214221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 42accgaaaggu cuuaccggat t
214321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 43uccgguaaga ccuuucggut t
214421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 44cuggcugaau uucagagcat t
214521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 45ugcucugaaa uucagccagt t
214621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 46cuggcugaau uucagagcat t
214721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 47ugcucugaaa uucagccagt t
214821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 48cuggcugaau uucagagcat t
214921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 49ugcucugaaa uucagccagt t
215021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 50cuggcugaau uucagagcat t
215121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 51ugcucugaaa uucagccagt t
215221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 52cuggcugaau uucagagcat t
215321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 53ugcucugaaa uucagccagt t
215421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 54cuggcugaau uucagagcat t
215521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 55ugcucugaaa uucagccagt t
215621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 56cuggcugaau uucagagcat t
215721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 57ugcucugaaa uucagccagt t
215821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 58cuggcugaau uucagagcat t
215921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 59ugcucugaaa uucagccagt t
216021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 60cuggcugaau uucagagcat t
216121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 61ugcucugaaa uucagccagt t
216221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 62cuggcugaau uucagagcat t
216321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 63ugcucugaaa uucagccagt t
216421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 64cuggcugaau uucagagcau u
216521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 65ugcucugaaa uucagccagu u
216617DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66aaccgtggtc atgctcc 176723RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67ccacaugaag cagcacgacu uuu 236821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 68ugcucugaaa uucagccagu u 216917DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 69aaccgtggtc atgctcc 177021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 70cuggcugaau uucagagcat t 217121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 71ugcucugaaa uucagccagt t 217221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 72acaugaagca gcacgacuut t 217321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 73aagucgugcu gcuucaugut t 217421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 74acaugaagca gcacgacuut t 217521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 75aagucgugcu gcuucaugut t 217621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 76acaugaagca gcacgacuut t 217721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 77aagucgugcu gcuucaugut t 217821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78ccacaugaag cagcacgacu u 217923RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79aagucgugcu gcuucaugug guc 238021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80ccacaugaag cagcacgacu u 218123RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81aagucgugcu gcuucaugug guc 238221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 82ccacaugaag cagcacgacu u 218323RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 83aagucgugcu gcuucaugug guc 238421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84ccacaugaag cagcacgacu u 218523RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85aagucgugcu gcuucaugug guc 238621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 86ccacaugaag cagcacgacu u 218723RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 87aagucgugcu gcuucaugug guc 238821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 88acaugaagca gcacgacuut t 218921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 89aagucgugcu gcuucaugut t 219021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 90acaugaagca gcacgacuut t 219121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 91aagucgugcu gcuucaugut t 219221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 92acaugaagca gcacgacuut t 219321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 93aagucgugcu gcuucaugut t 219421DNAArtificial
SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 94acaugaagca gcacgacuut t
219521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 95aagucgugcu gcuucaugut t
219621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 96acaugaagca gcacgacuut t
219721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 97aagucgugcu gcuucaugut t
219821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 98acaugaagca gcacgacuut t
219921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 99aagucgugcu gcuucaugut t
2110021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 100acaugaagca gcacgacuut t
2110121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 101aagucgugcu gcuucaugut t
2110221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 102acaugaagca gcacgacuut t
2110321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 103aagucgugcu gcuucaugut t
2110421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 104acaugaagca gcacgacuut t
2110521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 105aagucgugcu gcuucaugut t
2110621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 106acaugaagca gcacgacuut t
2110721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 107aagucgugcu gcuucaugut t
2110821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108ccacaugaag cagcacgacu u
2110923RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 109aagucgugcu gcuucaugug guc
2311021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110ccacaugaag cagcacgacu u
2111123RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111aagucgugcu gcuucaugug guc
2311221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 112acaugaagca gcacgacuut t
2111321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 113aagucgugcu gcuucaugut t
2111421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 114acaugaagca gcacgacuut t
2111521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 115aagucgugcu gcuucaugut t
2111621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 116acaugaagca gcacgacuut t
2111721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 117aagucgugcu gcuucaugut t
2111821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 118acaugaagca gcacgacuut t
2111921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 119aagucgugcu gcuucaugut t
2112021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 120acaugaagca gcacgacuut t
2112121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 121aagucgugcu gcuucaugut t
2112221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 122acaugaagca gcacgacuut t
2112321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 123aagucgugcu gcuucaugut t
2112421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 124acaugaagca gcacgacuut t
2112521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 125aagucgugcu gcuucaugut t
2112615RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126ggagcaugac cacgg 1512721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 127cugaagaccu gaagacaaut t 2112821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 128auugucuuca ggucuucagt t 2112921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 129gaacugugug ugagaggucc u 2113021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 130gaacugugug ugagaggucc u 2113121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 131cugaagaccu gaagacaaut t 2113221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 132gaacugugug ugagaggucc u 2113321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 133cuuacgcuga guacuucgat t 2113421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 134cuuacgcuga guacuucgat t 2113521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 135cuuacgcuga guacuucgat t 2113621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 136cuuacgcuga guacuucgat t 2113721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 137cuuacgcuga guacuucgat t 2113821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 138cuuacgcuga guacuucgat t 2113921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 139cuuacgcuga guacuucgat t 2114021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 140cuuacgcuga guacuucgat t 2114121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 141cugaagaccu gaagacaaut t 2114221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 142cugaagaccu gaagacaaut t 2114321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 143cugaagaccu gaagacaaut t 2114421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 144cugaagaccu gaagacaaut t 2114521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 145cuuacgcuga guacuucgat t 2114621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 146ucgaaguacu cagcguaagt t 2114721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 147cuuacgcuga guacuucgat t 2114821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 148ucgaaguacu cagcguaagt t 2114921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 149cuggcugaau uucagagcat t 2115021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 150accgaaaggu cuuaccggat t 2115121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 151ccacaugaag cagcacgacu u 2115221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 152ugcucugaaa uucagccagt t 2115321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 153uccgguaaga ccuuucggut t 2115423RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 154aagucgugcu gcuucaugug guc 2315521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 155uccgguaaga ccuuucggut t 2115623RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 156aagucgugcu gcuucaugug guc 2315721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 157ugcucugaaa uucagccagt t 2115821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 158ugcucugaaa uucagccagt t 2115921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 159cuuacgcuga guacuucgat t 2116021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 160ucgaaguacu cagcguaagt t 2116121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 161cuuacgcuga guacuucgat t 2116221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 162ucgaaguacu cagcguaagt t 2116321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 163cuuacgcuga guacuucgat t 2116421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 164ucgaaguacu cagcguaagt t 2116521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 165cuggcugaau uucagagcat t 2116621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 166ugcucugaaa uucagccagt t 2116721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 167cuggcugaau uucagagcat t 2116821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 168ugcucugaaa uucagccagt t 2116921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 169ccacaugaag cagcacgacu u 2117023RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 170aagucgugcu gcuucaugug guc 2317121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 171ccacaugaag cagcacgacu u 2117223RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 172aagucgugcu gcuucaugug guc 2317321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 173ccacaugaag cagcacgacu u 2117423RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 174aagucgugcu gcuucaugug guc 2317521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 175ccacaugaag cagcacgacu u 2117623RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 176aagucgugcu gcuucaugug guc 2317721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 177ccacaugaag cagcacgacu u 2117823RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 178aagucgugcu gcuucaugug guc 23
* * * * *