U.S. patent application number 12/891634 was filed with the patent office on 2011-05-26 for conjugates and compositions for cellular delivery.
Invention is credited to Tongqian CHEN, Peter Haeberli, Chandra Vargeese, Weimin Wang.
Application Number | 20110124853 12/891634 |
Document ID | / |
Family ID | 46204815 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110124853 |
Kind Code |
A1 |
CHEN; Tongqian ; et
al. |
May 26, 2011 |
Conjugates and Compositions for Cellular Delivery
Abstract
This invention features conjugates, degradable linkers,
compositions, methods of synthesis, and applications thereof,
including cholesterol, folate, galactose, galactosamine, N-acetyl
galactosamine, PEG, phospholipid, peptide and human serum albumin
(HSA) derived conjugates of biologically active compounds,
including antibodies, antivirals, chemotherapeutics, peptides,
proteins, hormones, nucleosides, nucleotides, non-nucleosides, and
nucleic acids including enzymatic nucleic acids, DNAzymes,
allozymes, antisense, dsRNA, siNA, siRNA, triplex oligonucleotides,
2,5-A chimeras, decoys and aptamers.
Inventors: |
CHEN; Tongqian; (Irvine,
CA) ; Haeberli; Peter; (San Francisco, CA) ;
Vargeese; Chandra; (Schwenksville, PA) ; Wang;
Weimin; (Churchville, PA) |
Family ID: |
46204815 |
Appl. No.: |
12/891634 |
Filed: |
September 27, 2010 |
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Application
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10427160 |
Apr 30, 2003 |
7833992 |
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12891634 |
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PCT/US02/15876 |
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10427160 |
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PCT/US03/05346 |
Feb 20, 2003 |
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10427160 |
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PCT/US03/05028 |
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PCT/US03/05346 |
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60292217 |
May 18, 2001 |
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60306883 |
Jul 20, 2001 |
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60311865 |
Aug 13, 2001 |
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60362016 |
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60358580 |
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60358580 |
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60363124 |
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60363124 |
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60386782 |
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60386782 |
Jun 6, 2002 |
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60406784 |
Aug 29, 2002 |
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60406784 |
Aug 29, 2002 |
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60408378 |
Sep 5, 2002 |
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60408378 |
Sep 5, 2002 |
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60409293 |
Sep 9, 2002 |
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60409293 |
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60440129 |
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60440129 |
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Current U.S.
Class: |
536/24.5 |
Current CPC
Class: |
A61K 47/544 20170801;
Y02A 50/465 20180101; A61K 38/00 20130101; Y02A 50/30 20180101;
Y02A 50/393 20180101 |
Class at
Publication: |
536/24.5 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Claims
1. A compound having Formula III: ##STR00202## wherein X comprises
a short interfering RNA (siRNA) comprising 2'-fluoro pyrimidines
and 2'-O-methyl purines, W comprises a linker molecule selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, and thiophosphate ester linkage, and n is an
integer from about 1 to about 20.
2. A compound having Formula 114: ##STR00203## wherein X comprises
a short interfering RNA (siRNA) comprising 2'-fluoro pyrimidines
and 2'-O-methyl purines, W comprises a linker molecule, and n is an
integer from about 1 to about 20.
3. The compound of claim 2, wherein W comprises a linker molecule
selected from the group consisting of amide, phosphate, phosphate
ester, phosphoramidate, and thiophosphate ester linkage.
4. The compound of claim 1, wherein said siRNA molecule comprises a
sense strand and an antisense strand, and wherein said sense strand
is conjugated with a compound comprising Formula 111.
5. The compound of claim 2, wherein said siRNA molecule comprises a
sense strand and an antisense strand, and wherein said sense strand
is conjugated with a compound comprising Formula 114.
Description
[0001] This patent application is a continuation-in-part of Adamic
et al., PCT/US02/15876, filed May 17, 2002, that claims the benefit
of Adamic et al., U.S. Ser. No. 60/292,217, filed May 18, 2001,
from Adamic et al., U.S. Ser. No. 60/362,016 filed Mar. 6, 2002,
both entitled `CONJUGATES AND COMPOSITIONS FOR CELLULAR DELIVERY`,
from Vargeese et al., U.S. Ser. No. 60/306,883, filed Jul. 20, 2001
entitled "CONJUGATES AND COMPOSITIONS FOR TRANSPORT ACROSS CELLULAR
MEMBRANES", and Vargeese et al., U.S. Ser. No. 60/311,865, filed
Aug. 13, 2001, entitled "CONJUGATES AND COMPOSITIONS FOR CELLULAR
DELIVERY"; and is also a continuation-in-part of Haeberli
PCT/US03/05346, filed Feb. 20, 2003, and McSwiggen PCT/US03/05028,
filed Feb. 20, 2003, both of which claim the benefit of Beigelman
U.S. Ser. No. 60/358,580 filed Feb. 20, 2002, of Beigelman U.S.
Ser. No. 60/363,124 filed Mar. 11, 2002, of Beigelman U.S. Ser. No.
60/386,782 filed Jun. 6, 2002, of Beigelman U.S. Ser. No.
60/406,784 filed Aug. 29, 2002, of Beigelman U.S. Ser. No.
60/408,378 filed Sep. 5, 2002, of Beigelman U.S. Ser. No.
60/409,293 filed Sep. 9, 2002, and of Beigelman U.S. Ser. No.
60/440,129 filed Jan. 15, 2003. These applications are hereby
incorporated by reference herein in their entirety including the
drawings.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to conjugates, compositions,
methods of synthesis, and applications thereof. The discussion is
provided only for understanding of the invention that follows. This
summary is not an admission that any of the work described below is
prior art to the claimed invention.
[0003] The cellular delivery of various therapeutic compounds, such
as antiviral and chemotherapeutic agents, is usually compromised by
two limitations. First the selectivity of therapeutic agents is
often low, resulting in high toxicity to normal tissues. Secondly,
the trafficking of many compounds into living cells is highly
restricted by the complex membrane systems of the cell. Specific
transporters allow the selective entry of nutrients or regulatory
molecules, while excluding most exogenous molecules such as nucleic
acids and proteins. Various strategies can be used to improve
transport of compounds into cells, including the use of lipid
carriers and various conjugate systems. Conjugates are often
selected based on the ability of certain molecules to be
selectively transported into specific cells, for example via
receptor mediated endocytosis. By attaching a compound of interest
to molecules that are actively transported across the cellular
membranes, the effective transfer of that compound into cells or
specific cellular organelles can be realized. Alternately,
molecules that are able to penetrate cellular membranes without
active transport mechanisms, for example, various lipophilic
molecules, can be used to deliver compounds of interest. Examples
of molecules that can be utilized as conjugates include but are not
limited to peptides, hormones, fatty acids, vitamins, flavonoids,
sugars, reporter molecules, reporter enzymes, chelators,
porphyrins, intercalcators, and other molecules that are capable of
penetrating cellular membranes, either by active transport or
passive transport.
[0004] The delivery of compounds to specific cell types, for
example, cancer cells or cells specific to particular tissues and
organs, can be accomplished by utilizing receptors associated with
specific cell types. Particular receptors are overexpressed in
certain cancerous cells, including the high affinity folic acid
receptor. For example, the high affinity folate receptor is a tumor
marker that is overexpressed in a variety of neoplastic tissues,
including breast, ovarian, cervical, colorectal, renal, and
nasoparyngeal tumors, but is expressed to a very limited extent in
normal tissues. The use of folic acid based conjugates to transport
exogenous compounds across cell membranes can provide a targeted
delivery approach to the treatment and diagnosis of disease and can
provide a reduction in the required dose of therapeutic compounds.
Furthermore, therapeutic bioavialability, pharmacodynamics, and
pharmacokinetic parameters can be modulated through the use of
bioconjugates, including folate bioconjugates. Godwin et al., 1972,
J. Biol. Chem., 247, 2266-2271, report the synthesis of
biologically active pteroyloligo-L-glutamates. Habus et al., 1998,
Bioconjugate Chem., 9, 283-291, describe a method for the solid
phase synthesis of certain oligonucleotide-folate conjugates. Cook,
U.S. Pat. No. 6,721,208, describes certain oligonucleotides
modified with specific conjugate groups. The use of biotin and
folate conjugates to enhance transmembrane transport of exogenous
molecules, including specific oligonucleotides has been reported by
Low et al., U.S. Pat. Nos. 5,416,016, 5,108,921, and International
PCT publication No. WO 90/12096. Manoharan et al., International
PCT publication No. WO 99/66063 describe certain folate conjugates,
including specific nucleic acid folate conjugates with a
phosphoramidite moiety attached to the nucleic acid component of
the conjugate, and methods for the synthesis of these folate
conjugates. Nomura et al., 2000, Org. Chem., 65, 5016-5021,
describe the synthesis of an intermediate,
alpha-[2-(trimethylsilyl)ethoxycarbonl]folic acid, useful in the
synthesis of ceratin types of folate-nucleoside conjugates. Guzaev
et al., U.S. Pat. No. 6,335,434, describes the synthesis of certain
folate oligonucleotide conjugates.
[0005] The delivery of compounds to other cell types can be
accomplished by utilizing receptors associated with a certain type
of cell, such as hepatocytes. For example, drug delivery systems
utilizing receptor-mediated endocytosis have been employed to
achieve drug targeting as well as drug-uptake enhancement. The
asialoglycoprotein receptor (ASGPr) (see for example Wu and Wu,
1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and
binds branched galactose-terminal glycoproteins, such as
asialoorosomucoid (ASOR). Binding of such glycoproteins or
synthetic glycoconjugates to the receptor takes place with an
affinity that strongly depends on the degree of branching of the
oligosaccharide chain, for example, triatennary structures are
bound with greater affinity than biatenarry or monoatennary chains
(Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al.,
1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,
Glycoconjugate J., 4, 317-328, obtained this high specificity
through the use of N-acetyl-D-galactosamine as the carbohydrate
moiety, which has higher affinity for the receptor, compared to
galactose. This "clustering effect" has also been described for the
binding and uptake of mannosyl-terminating glycoproteins or
glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24,
1388-1395). The use of galactose and galactosamine based conjugates
to transport exogenous compounds across cell membranes can provide
a targeted delivery approach to the treatment of liver disease such
as HBV and HCV infection or hepatocellular carcinoma. The use of
bioconjugates can also provide a reduction in the required dose of
therapeutic compounds required for treatment. Furthermore,
therapeutic bioavialability, pharmacodynamics, and pharmacokinetic
parameters can be modulated through the use of bioconjugates.
[0006] A number of peptide based cellular transporters have been
developed by several research groups. These peptides are capable of
crossing cellular membranes in vitro and in vivo with high
efficiency. Examples of such fusogenic peptides include a 16-amino
acid fragment of the homeodomain of ANTENNAPEDIA, a Drosophila
transcription factor (Wang et al., 1995, PNAS USA., 92, 3318-3322);
a 17-mer fragment representing the hydrophobic region of the signal
sequence of Kaposi fibroblast growth factor with or without NLS
domain (Antopolsky et al., 1999, Bioconj. Chem., 10, 598-606); a
17-mer signal peptide sequence of caiman crocodylus Ig(5) light
chain (Chaloin et al., 1997, Biochem. Biophys. Res. Comm., 243,
601-608); a 17-amino acid fusion sequence of HIV envelope
glycoprotein gp4114, (Morris et al., 1997, Nucleic Acids Res., 25,
2730-2736); the HIV-1 Tat49-57 fragment (Schwarze et al., 1999,
Science, 285, 1569-1572); a transportan A-achimeric 27-mer
consisting of N-terminal fragment of neuropeptide galanine and
membrane interacting wasp venom peptide mastoporan (Lindgren et
al., 2000, Bioconjugate Chem., 11, 619-626); and a 24-mer derived
from influenza virus hemagglutinin envelop glycoprotein (Bongartz
et al., 1994, Nucleic Acids Res., 22, 4681-4688).
[0007] These peptides were successfully used as part of an
antisense oligonucleotide-peptide conjugate for cell culture
transfection without lipids. In a number of cases, such conjugates
demonstrated better cell culture efficacy then parent
oligonucleotides transfected using lipid delivery. In addition, use
of phage display techniques has identified several organ targeting
and tumor targeting peptides in vivo (Ruoslahti, 1996, Ann. Rev.
Cell Dev. Biol., 12, 697-715). Conjugation of tumor targeting
peptides to doxorubicin has been shown to significantly improve the
toxicity profile and has demonstrated enhanced efficacy of
doxorubicin in the in vivo murine cancer model MDA-MB-435 breast
carcinoma (Arap et al., 1998, Science, 279, 377-380).
[0008] Hudson et al., 1999, Int. J. Pharm., 182, 49-58, describes
the cellular delivery of specific hammerhead ribozymes conjugated
to a transferrin receptor antibody. Janjic et al., U.S. Pat. No.
6,168,778, describes specific VEGF nucleic acid ligand complexes
for targeted drug delivery. Bonora et al., 1999, Nucleosides
Nucleotides, 18, 1723-1725, describes the biological properties of
specific antisense oligonucleotides conjugated to certain
polyethylene glycols. Davis and Bishop, International PCT
publication No. WO 99/17120 and Jaeschke et al., 1993, Tetrahedron
Lett., 34, 301-4 describe specific methods of preparing
polyethylene glycol conjugates. Tullis, International PCT
Publication No. WO 88/09810; Jaschke, 1997, ACS Sympl Ser., 680,
265-283; Jaschke et al., 1994, Nucleic Acids Res., 22, 4810-17;
Efimov et al., 1993, Bioorg. Khim., 19, 800-4; and Bonora et al.,
1997, Bioconjugate Chem., 8, 793-797, describe specific
oligonucleotide polyethylene glycol conjugates. Manoharan,
International PCT Publication No. WO 00/76554, describes the
preparation of specific ligand-conjugated oligodeoxyribonucleotides
with certain cellular, serum, or vascular proteins. Defrancq and
Lhomme, 2001, Bioorg Med Chem Lett., 11, 931-933; Cebon et al.,
2000, Aust. J. Chem., 53, 333-339; and Salo et al., 1999,
Bioconjugate Chem., 10, 815-823 describe specific aminooxy peptide
oligonucleotide conjugates.
SUMMARY OF THE INVENTION
[0009] The present invention features compositions and conjugates
to facilitate delivery of molecules into a biological system, such
as cells. The conjugates provided by the instant invention can
impart therapeutic activity by transferring therapeutic compounds
across cellular membranes. The present invention encompasses the
design and synthesis of novel agents for the delivery of molecules,
including but not limited to small molecules, lipids, nucleosides,
nucleotides, nucleic acids, polynucleotides, oligonucleotides,
antibodies, toxins, negatively charged polymers and other polymers,
for example proteins, peptides, hormones, carbohydrates, or
polyamines, across cellular membranes. In general, the transporters
described are designed to be used either individually or as part of
a multi-component system, with or without degradable linkers. The
compounds of the invention generally shown in the Formulae below
are expected to improve delivery of molecules into a number of cell
types originating from different tissues, in the presence or
absence of serum.
[0010] The present invention features a compound having the Formula
1:
##STR00001##
[0011] wherein each R.sub.1, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 is independently hydrogen, alkyl, substituted
alkyl, aryl, substituted aryl, or a protecting group, each "n" is
independently an integer from 0 to about 200, R.sub.12 is a
straight or branched chain alkyl, substituted alkyl, aryl, or
substituted aryl, and R.sub.2 is a phosphorus containing group,
nucleoside, nucleotide, small molecule, nucleic acid,
polynucleotide, or oligonucleotide such as an enzymatic nucleic
acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, siNA or a portion
thereof, or a solid support comprising a linker.
[0012] The present invention features a compound having the Formula
2:
##STR00002##
[0013] wherein each R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7
is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently
an integer from 0 to about 200, R.sub.12 is a straight or branched
chain alkyl, substituted alkyl, aryl, or substituted aryl, and
R.sub.2 is a phosphorus containing group, nucleoside, nucleotide,
small molecule, nucleic acid, polynucleotide, or oligonucleotide
such as an enzymatic nucleic acid, allozyme, antisense nucleic
acid, 2,5-A chimera, decoy, aptamer or triplex forming
oligonucleotide, siNA or a portion thereof, or a solid support
comprising a linker.
[0014] The present invention features a compound having the Formula
3:
##STR00003##
[0015] wherein each R.sub.1, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently
an integer from 0 to about 200, R.sub.12 is a straight or branched
chain alkyl, substituted alkyl, aryl, or substituted aryl, and
R.sub.2 is a phosphorus containing group, nucleoside, nucleotide,
small molecule, or nucleic acid, polynucleotide, or oligonucleotide
such as an enzymatic nucleic acid, allozyme, antisense nucleic
acid, 2,5-A chimera, decoy, aptamer or triplex forming
oligonucleotide, siNA or a portion thereof.
[0016] The present invention features a compound having the Formula
4:
##STR00004##
[0017] wherein each R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7
is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently
an integer from 0 to about 200, R.sub.2 is a phosphorus containing
group, nucleoside, nucleotide, small molecule, nucleic acid,
polynucleotide, or oligonucleotide such as an enzymatic nucleic
acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, siNA or a portion
thereof, or a solid support comprising a linker, and R.sub.13 is an
amino acid side chain.
[0018] The present invention features a compound having the Formula
5:
##STR00005##
[0019] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 is independently hydrogen, alkyl or nitrogen
protecting group, each "n" is independently an integer from 0 to
about 200, R.sub.12 is a straight or branched chain alkyl,
substituted alkyl, aryl, or substituted aryl, and each R.sub.9 and
R.sub.10 is independently a nitrogen containing group, cyanoalkoxy,
alkoxy, aryloxy, or alkyl group.
[0020] The present invention features a compound having the Formula
6:
##STR00006##
[0021] wherein each R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is
independently hydrogen, alkyl, substituted alkyl, aryl, substituted
aryl, or a protecting group, R.sub.2 is a phosphorus containing
group, nucleoside, nucleotide, small molecule, nucleic acid,
polynucleotide, or oligonucleotide such as an enzymatic nucleic
acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, siNA or a portion
thereof, or a solid support comprising a linker, each "n" is
independently an integer from 0 to about 200, and L is a degradable
linker.
[0022] The present invention features a compound having the Formula
7:
##STR00007##
[0023] wherein each R.sub.1, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently
an integer from 0 to about 200, R.sub.12 is a straight or branched
chain alkyl, substituted alkyl, aryl, or substituted aryl, and
R.sub.2 is a phosphorus containing group, nucleoside, nucleotide,
small molecule, nucleic acid, polynucleotide, or oligonucleotide
such as an enzymatic nucleic acid, allozyme, antisense nucleic
acid, 2,5-A chimera, decoy, aptamer or triplex forming
oligonucleotide, siNA or a portion thereof, or a solid support
comprising a linker.
[0024] The present invention features a compound having the Formula
8:
##STR00008##
[0025] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl or nitrogen protecting
group, each "n" is independently an integer from 0 to about 200,
R.sub.12 is a straight or branched chain alkyl, substituted alkyl,
aryl, or substituted aryl, and each R.sub.9 and R.sub.10 is
independently a nitrogen containing group, cyanoalkoxy, alkoxy,
aryloxy, or alkyl group.
[0026] The present invention features a method for synthesizing a
compound having Formula 5:
##STR00009##
[0027] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl or nitrogen protecting
group, each "n" is independently an integer from 0 to about 200,
R.sub.12 is a straight or branched chain alkyl, substituted alkyl,
aryl, or substituted aryl, and each R.sub.9 and R.sub.10 is
independently a nitrogen containing group, cyanoalkoxy, alkoxy,
aryloxy, or alkyl group, comprising: coupling a bis-hydroxy
aminoalkyl derivative, for example D-threoninol, with a N-protected
aminoalkanoic acid to yield a compound of Formula 9;
##STR00010##
[0028] wherein R.sub.11 is an amino protecting group, R.sub.12 is a
straight or branched chain alkyl, substituted alkyl, aryl, or
substituted aryl, and each "n" is independently an integer from 0
to about 200; introducing primary hydroxy protection R.sub.1
followed by amino deprotection of R.sub.11 to yield a compound of
Formula 10;
##STR00011##
[0029] wherein R.sub.1 is a protecting group, R.sub.12 is a
straight or branched chain alkyl, substituted alkyl, aryl, or
substituted aryl, and each "n" is independently an integer from 0
to about 200; coupling the deprotected amine of Formula 10 with a
protected amino acid, for example glutamic acid, to yield a
compound of Formula 11;
##STR00012##
[0030] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each "n" is independently an integer
from 0 to about 200, R.sub.11 is an amino protecting group, and
R.sub.12 is a straight or branched chain alkyl, substituted alkyl,
aryl, or substituted aryl; deprotecting the amine R.sub.11 of the
conjugated glutamic acid of Formula XI to yield a compound of
Formula 12;
##STR00013##
[0031] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each "n" is independently an integer
from 0 to about 200, R.sub.11 is an amino protecting group, and
R.sub.12 is a straight or branched chain alkyl, substituted alkyl,
aryl, or substituted aryl; coupling the deprotected amine of
Formula 12 with an amino protected pteroic acid to yield a compound
of Formula 13;
##STR00014##
[0032] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl or nitrogen protecting
group, R.sub.12 is a straight or branched chain alkyl, substituted
alkyl, aryl, or substituted aryl, and each "n" is independently an
integer from 0 to about 200; and introducing a phosphorus
containing group at the secondary hydroxyl of Formula 13 to yield a
compound of Formula 5.
[0033] The present invention features a method for synthesizing a
compound having Formula 8:
##STR00015##
[0034] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl or nitrogen protecting
group, each "n" is independently an integer from 0 to about 200,
each R.sub.9 and R.sub.10 is independently a nitrogen containing
group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group, and R.sub.12
is a straight or branched chain alkyl, substituted alkyl, aryl, or
substituted aryl, comprising; coupling a bis-hydroxy aminoalkyl
derivative, for example D-threoninol, with a protected amino acid,
for example glutamic acid, to yield a compound of Formula 14;
##STR00016##
[0035] wherein R.sub.11 is an amino protecting group, each "n" is
independently an integer from 0 to about 200, R.sub.4 is
independently a protecting group, and R.sub.12 is a straight or
branched chain alkyl, substituted alkyl, aryl, or substituted aryl;
introducing primary hydroxy protection R.sub.1 followed by amino
deprotection of R.sub.11 of Formula 14 to yield a compound of
Formula 15;
##STR00017##
[0036] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, R.sub.12 is a straight or branched
chain alkyl, substituted alkyl, aryl, or substituted aryl, and each
"n" is independently an integer from 0 to about 200; coupling the
deprotected amine of Formula 15 with an amino protected pteroic
acid to yield a compound of Formula 16;
##STR00018##
[0037] wherein each R.sub.1 and R.sub.4 is independently a
protecting group or hydrogen, each R.sub.3, R.sub.5, R.sub.6 and
R.sub.7 is independently hydrogen, alkyl or nitrogen protecting
group, R.sub.12 is a straight or branched chain alkyl, substituted
alkyl, aryl, or substituted aryl, and each "n" is independently an
integer from 0 to about 200; and introducing a phosphorus
containing group at the secondary hydroxyl of Formula 16 to yield a
compound of Formula 8.
[0038] In one embodiment, R.sub.2 of a compound of the invention
comprises a phosphorus containing group.
[0039] In another embodiment, R.sub.2 of a compound of the
invention comprises a nucleoside, for example, a nucleoside with
beneficial activity such as anticancer or antiviral activity.
[0040] In yet another embodiment, R.sub.2 of a compound of the
invention comprises a nucleotide, for example, a nucleotide with
beneficial activity such as anticancer or antiviral activity.
[0041] In a further embodiment, R.sub.2 of a compound of the
invention comprises a small molecule, for example, a small molecule
with beneficial activity such as anticancer or antiviral
activity.
[0042] In another embodiment, R.sub.2 of a compound of the
invention comprises a nucleic acid, polynucleotide, or
oligonucleotide, for example, a nucleic acid, polynucleotide, or
oligonucleotide with beneficial activity such as anticancer or
antiviral activity. Non-limiting examples of nucleic acid,
polynucleotide, and oligonucleotide compounds include enzymatic
nucleic acid molecules, antisense molecules, aptamers, triplex
forming oligonucleotides, decoys, 2,5-A chimera molecules, and siNA
or a portion thereof.
[0043] In one embodiment, R.sub.2 of a compound of the invention
comprises a solid support comprising a linker.
[0044] In another embodiment, a nucleoside (R.sub.2) of the
invention comprises a nucleoside with anticancer activity.
[0045] In another embodiment, a nucleoside (R.sub.2) of the
invention comprises a nucleoside with antiviral activity.
[0046] In another embodiment, the nucleoside (R.sub.2) of the
invention comprises fludarabine, lamivudine (3TC), 5-fluoro
uridine, AZT, ara-adenosine, ara-adenosine monophosphate, a dideoxy
nucleoside analog, carbodeoxyguanosine, ribavirin, fialuridine,
lobucavir, a pyrophosphate nucleoside analog, an acyclic nucleoside
analog, acyclovir, gangciclovir, penciclovir, famciclovir, an
L-nucleoside analog, FTC, L-FMAU, L-ddC, L-FddC, L-d4C, L-Fd4C, an
L-dideoxypurine nucleoside analog, cytallene, bis-POM PMEA
(GS-840), BMS-200,475, carbovir or abacavir.
[0047] In one embodiment, R.sub.13 of a compound of the invention
comprises an alkylamino or an alkoxy group, for example,
--CH.sub.2O-- or --CH(CH.sub.2)CH.sub.2O--.
[0048] In another embodiment, R.sub.12 of a compound of the
invention is an alkylhyrdroxyl, for example, --(CH.sub.2).sub.nOH,
where n comprises an integer from about 1 to about 10.
[0049] In another embodiment, L of Formula 6 of the invention
comprises serine, threonine, or a photolabile linkage.
[0050] In one embodiment, R.sub.9 of a compound of the invention
comprises a phosphorus protecting group, for example
--OCH.sub.2CH.sub.2CN (oxyethylcyano).
[0051] In one embodiment, R.sub.10 of a compound of the invention
comprises a nitrogen containing group, for example, --N(R.sub.14)
wherein R.sub.14 is a straight or branched chain alkyl having from
about 1 to about 10 carbons.
[0052] In another embodiment, R.sub.10 of a compound of the
invention comprises a heterocycloalkyl or heterocycloalkenyl ring
containing from about 4 to about 7 atoms, and having from about 1
to about 3 heteroatoms comprising oxygen, nitrogen, or sulfur.
[0053] In another embodiment, R.sub.1 of a compound of the
invention comprises an acid labile protecting group, such as a
trityl or substituted trityl group, for example, a dimethoxytrityl
or mono-methoxytrityl group.
[0054] In another embodiment, R.sub.4 of a compound of the
invention comprises a tert-butyl, Fm (fluorenyl-methoxy), or allyl
group.
[0055] In one embodiment, R.sub.6 of a compound of the invention
comprises a TFA (trifluoracetyl) group.
[0056] In another embodiment, R.sub.3, R.sub.5, R.sub.7 and R.sub.8
of a compound of the invention are independently hydrogen.
[0057] In one embodiment, R.sub.7 of a compound of the invention is
independently isobutyryl, dimethylformamide, or hydrogen.
[0058] In another embodiment, R.sub.12 of a compound of the
invention comprises a methyl group or ethyl group.
[0059] In one embodiment, a nucleic acid of the invention comprises
a siNA molecule or a portion thereof.
[0060] In one embodiment, a nucleic acid of the invention comprises
an enzymatic nucleic acid, for example a hammerhead, Inozyme,
DNAzyme, G-cleaver, Zinzyme, Amberzyme, or allozyme or a portion
thereof.
[0061] In another embodiment, a nucleic acid of the invention
comprises an antisense nucleic acid, 2-5A nucleic acid chimera, or
decoy nucleic acid or a portion thereof.
[0062] In another embodiment, the solid support having a linker of
the invention comprises a structure of Formula 17:
##STR00019##
[0063] wherein SS is a solid support, and each "n" is independently
an integer from about 1 to about 200.
[0064] In another embodiment, the solid support of the instant
invention is controlled pore glass (CPG) or polystyrene, and can be
used in the synthesis of a nucleic acid, polynucleotide, or
oligonucleotide or the invention, such as an enzymatic nucleic
acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,
aptamer, triplex forming oligonucleotide, siNA or a portion
thereof.
[0065] In one embodiment, the invention features a pharmaceutical
composition comprising a compound of the invention and a
pharmaceutically acceptable carrier.
[0066] In another embodiment, the invention features a method of
treating a cancer patient, comprising contacting cells of the
patient with a pharmaceutical composition of the invention under
conditions suitable for the treatment. This treatment can comprise
the use of one or more other drug therapies under conditions
suitable for the treatment. The cancers contemplated by the instant
invention include but are not limited to breast cancer, lung
cancer, colorectal cancer, brain cancer, esophageal cancer, stomach
cancer, bladder cancer, pancreatic cancer, cervical cancer, head
and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or
multidrug resistant cancers.
[0067] In one embodiment, the invention features a method of
treating a patient infected with a virus, comprising contacting
cells of the patient with a pharmaceutical composition of the
invention, under conditions suitable for the treatment. This
treatment can comprise the use of one or more other drug therapies
under conditions suitable for the treatment. The viruses
contemplated by the instant invention include but are not limited
to HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus,
west nile virus, Ebola virus, foot and mouth virus, and papilloma
virus.
[0068] In one embodiment, the invention features a kit for
detecting the presence of a nucleic acid molecule or other target
molecule in a sample, for example, a gene in a cancer cell,
comprising a compound of the instant invention.
[0069] In one embodiment, the invention features a kit for
detecting the presence of a nucleic acid molecule, or other target
molecule in a sample, for example, a gene in a virus-infected cell,
comprising a compound of the instant invention.
[0070] In another embodiment, the invention features a compound of
the instant invention comprising a modified phosphate group, for
example, a phosphoramidite, phosphodiester, phosphoramidate,
phosphorothioate, phosphorodithioate, alkylphosphonate,
arylphosphonate, monophosphate, diphosphate, triphosphate, or
pyrophosphate.
[0071] In one embodiment, the invention features a method for
synthesizing a compound having Formula 18:
##STR00020##
[0072] wherein each R.sub.6 and R.sub.7 is independently hydrogen,
alkyl or nitrogen protecting group, comprising: reacting folic acid
with a carboxypeptidase to yield a compound of Formula 19;
##STR00021##
[0073] introducing a protecting group R.sub.6 on the secondary
amine of Formula 19 to yield a compound of Formula 20;
##STR00022##
[0074] wherein R.sub.6 is a nitrogen protecting group; and
introducing a protecting group R.sub.7 on the primary amine of
Formula 20 to yield a compound of Formula 18.
[0075] In another embodiment, the amino protected pteroic acid of
the invention is a compound of Formula 18.
[0076] In one embodiment, the invention encompasses a compound of
Formula 1 having Formula 21:
##STR00023##
[0077] wherein each "n" is independently an integer from 0 to about
200.
[0078] In another embodiment, the invention encompasses a compound
of Formula 7 having Formula 22:
##STR00024##
[0079] wherein each "n" is independently an integer from 0 to about
200.
[0080] In another embodiment, the invention encompasses a compound
of Formula 4 having Formula 23:
##STR00025##
[0081] wherein "n" is an integer from 0 to about 200.
[0082] In another embodiment, the invention encompasses a compound
of Formula 4 having Formula 24:
##STR00026##
[0083] wherein "n" is an integer from 0 to about 200.
[0084] In another embodiment, the invention features a compound
having Formula 25:
##STR00027##
[0085] wherein each R.sub.5 and R.sub.7 is independently hydrogen,
alkyl or a nitrogen protecting group, each R.sub.15, R.sub.16,
R.sub.17, and R.sub.18 is independently O, S, alkyl, substituted
alkyl, aryl, substituted aryl, or halogen, X.sub.1 is --CH(X.sub.10
or a group of Formula 38:
##STR00028##
[0086] wherein R.sub.4 is a protecting group and "n" is an integer
from 0 to about 200;
[0087] X.sub.1' is the protected or unprotected side chain of a
naturally occurring or non-naturally-occurring amino acid, X.sub.2
is amide, alkyl, or carbonyl containing linker or a bond, and
X.sub.3 is a degradable linker which is optionally absent.
[0088] In another embodiment, the X.sub.3 group of Formula 25
comprises a group of Formula 26:
##STR00029##
[0089] wherein R.sub.4 is hydrogen or a protecting group, "n" is an
integer from 0 to about 200 and R.sub.12 is a straight or branched
chain alkyl, substituted alkyl, aryl, or substituted aryl.
[0090] In yet another embodiment, R.sub.4 of Formula 26 is hydrogen
and R.sub.12 is methyl or hyrdogen.
[0091] In still another embodiment, the invention features a
compound having Formula 27:
##STR00030##
[0092] wherein "n" is an integer from about 0 to about 20, R.sub.4
is H or a cationic salt, and R.sub.24 is a sulfur containing
leaving group, for example a group comprising:
##STR00031##
[0093] In another embodiment, the invention features a method for
synthesizing a compound having Formula 27 comprising:
[0094] (a) selective tritylation of the thiol of cysteamine under
conditions suitable to yield a compound having Formula 28:
##STR00032##
[0095] wherein "n" is an integer from about 0 to about 20 and
R.sub.19 is a thiol protecting group;
[0096] (b) peptide coupling of the product of (a) with a compound
having Formula 29:
##STR00033##
[0097] wherein R.sub.20 is a carboxylic acid protecting group and
R.sub.21 is an amino protecting group, under conditions suitable to
yield a compound having Formula 30:
##STR00034##
[0098] wherein "n" is an integer from about 0 to about 20, R.sub.19
is a thiol protecting group, R.sub.20 is a carboxylic acid
protecting group and R.sub.21 is an amino protecting group;
[0099] (c) removing the amino protecting group R.sub.21 of the
product of (b) under conditions suitable to yield a compound having
Formula 31:
##STR00035##
[0100] wherein "n" is an integer from about 0 to about 20 and
R.sub.19 and R.sub.20 are as described in (b);
[0101] (d) condensation of the product of (c) with a compound
having Formula 32:
##STR00036##
[0102] wherein R.sub.22 is an amino protecting group, under
conditions suitable to yield a compound having Formula 33:
##STR00037##
[0103] wherein "n" is an integer from about 0 to about 20 and
R.sub.19 and R.sub.20 are as described in (b) and R.sub.22 is as
described in (d);
[0104] (e) selective cleavage of R.sub.22 from the product of (d)
under conditions suitable to yield a compound having Formula
34:
##STR00038##
[0105] wherein "n" is an integer from about 0 to about 20 and
R.sub.19 and R.sub.20 are as described in (b);
[0106] (f) coupling the product of (e) with a compound having
Formula 35:
##STR00039##
[0107] wherein R.sub.23 is an amino protecting group under
conditions suitable to yield a compound having Formula 36:
##STR00040##
[0108] wherein R.sub.23 is an amino protecting group, "n" is an
integer from about 0 to about 20 and R.sub.19 and R.sub.20 are as
described in (b);
[0109] (g) deprotecting the product of (f) under conditions
suitable to yield a compound having Formula 37.
##STR00041##
[0110] wherein "n" is an integer from about 0 to about 20; and
[0111] (h) introducing a disulphide-based leaving group to the
product of (g) under conditions suitable to yield a compound having
Formula 27.
[0112] In one embodiment, the invention features a compound having
Formula 39:
##STR00042##
[0113] wherein "n" is an integer from about 0 to about 20, X is a
nucleic acid, polynucleotide, or oligonucleotide such as an
enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,
2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide,
and P is a phosphorus containing group. In another embodiment, X
comprises a siNA molecule or a portion thereof.
[0114] In another embodiment, the invention features a method for
synthesizing a compound having Formula 39, comprising:
[0115] (a) Coupling a thiol containing linker to a nucleic acid,
polynucleotide or oligonucleotide under conditions suitable to
yield a compound having Formula 40:
##STR00043##
[0116] wherein "n" is an integer from about 0 to about 20, X is a
nucleic acid, polynucleotide, or oligonucleotide, and P is a
phosphorus containing group; and
[0117] (b) coupling the product of (a) with a compound having
Formula 37 under conditions suitable to yield a compound having
Formula 39.
[0118] In another embodiment, the thiol containing linker of the
invention is a compound having Formula 41:
##STR00044##
[0119] wherein "n" is an integer from about 0 to about 20, P is a
phosphorus containing group, for example a phosphine, phosphite, or
phosphate, and R.sub.24 is any alkyl, substituted alkyl, alkoxy,
aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, or
substituted alkynyl group with or without additional protecting
groups.
[0120] In another embodiment, the conditions suitable to yield a
compound having Formula 40 comprises reduction, for example using
dithiothreitol (DTT) or any equivalent disulphide reducing agent,
of the disulfide bond of a compound having Formula 42:
##STR00045##
[0121] wherein "n" is an integer from about 0 to about 20, X is a
nucleic acid, polynucleotide, or oligonucleotide, P is a phosphorus
containing group, and R.sub.24 is any alkyl, substituted alkyl,
alkoxy, aryl, substituted aryl, alkenyl, substituted alkenyl,
alkynyl, or substituted alkynyl group with or without additional
protecting groups. In another embodiment, X comprises a siNA
molecule or a portion thereof.
[0122] In one embodiment, the invention features a compound having
Formula 43:
##STR00046##
[0123] wherein X comprises a biologically active molecule; W
comprises a degradable nucleic acid linker; Y comprises a linker
molecule or amino acid that can be present or absent; Z comprises
H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,
substituted aryl, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,
phospholipid, or label; n is an integer from about 1 to about 100;
and N' is an integer from about 1 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0124] In another embodiment, the invention features a compound
having Formula 44:
##STR00047##
[0125] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent; n is an integer from about 1 to about 50, and PEG
represents a compound having Formula 45:
##STR00048##
[0126] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; and n is an
integer from about 1 to about 100. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0127] In another embodiment, the invention features a compound
having Formula 46:
##STR00049##
[0128] wherein X comprises a biologically active molecule; each W
independently comprises linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule or chemical
linkage that can be present or absent; and PEG represents a
compound having Formula 45:
##STR00050##
[0129] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; and n is an
integer from about 1 to about 100. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0130] In one embodiment, the invention features a compound having
Formula 47:
##STR00051##
[0131] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be the same or different and can be present or absent, Y
comprises a linker molecule that can be present or absent; each Q
independently comprises a hydrophobic group or phospholipid; each
R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, and n is an integer from about 1 to about 10. In
another embodiment, X comprises a siNA molecule or a portion
thereof. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.
[0132] In another embodiment, the invention features a compound
having Formula 48:
##STR00052##
[0133] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and B represents a lipophilic
group, for example a saturated or unsaturated linear, branched, or
cyclic alkyl group, cholesterol, or a derivative thereof. In
another embodiment, X comprises a siNA molecule or a portion
thereof. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.
[0134] In another embodiment, the invention features a compound
having Formula 49:
##STR00053##
[0135] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, and B represents a lipophilic group, for
example a saturated or unsaturated linear, branched, or cyclic
alkyl group, cholesterol, or a derivative thereof. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0136] In another embodiment, the invention features a compound
having Formula 50:
##STR00054##
[0137] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule or chemical linkage that
can be present or absent; and each Q independently comprises a
hydrophobic group or phospholipid. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0138] In one embodiment, the invention features a compound having
Formula 51:
##STR00055##
[0139] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent; Y comprises a linker molecule or amino acid that can be
present or absent; Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; SG comprises
a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers, and n is an integer
from about 1 to about 20. In another embodiment, X comprises a siNA
molecule or a portion thereof. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0140] In another embodiment, the invention features a compound
having Formula 52:
##STR00056##
[0141] wherein X comprises a biologically active molecule; Y
comprises a linker molecule or chemical linkage that can be present
or absent; each R1, R2, R3, R4, and R5 independently comprises O,
OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N; Z comprises H, OH, O-alkyl, SH,
S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,
or label; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose,
or fucose and the respective D or L, alpha or beta isomers, n is an
integer from about 1 to about 20; and N' is an integer from about 1
to about 20. In another embodiment, X comprises a siNA molecule or
a portion thereof. In another embodiment, Y is selected from the
group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0142] In another embodiment, the invention features a compound
having Formula 53:
##STR00057##
[0143] wherein B comprises H, a nucleoside base, or a
non-nucleosidic base with or without protecting groups; each R1
independently comprises O, N, S, alkyl, or substituted N; each R2
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, or a phosphorus containing group;
each R3 independently comprises N or O--N, each R4 independently
comprises O, CH2, S, sulfone, or sulfoxy; X comprises H, a
removable protecting group, amino, substituted amino, nucleotide,
nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid,
siNA or a portion thereof, amino acid, peptide, protein, lipid,
phospholipid, or label; W comprises a linker molecule or chemical
linkage that can be present or absent; SG comprises a sugar, for
example galactose, galactosamine, N-acetyl-galactosamine, glucose,
mannose, fructose, or fucose and the respective D or L, alpha or
beta isomers, each n is independently an integer from about 1 to
about 50; and N' is an integer from about 1 to about 10. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0144] In another embodiment, the invention features a compound
having Formula 54:
##STR00058##
[0145] wherein B comprises H, a nucleoside base, or a
non-nucleosidic base with or without protecting groups; each R1
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, or a phosphorus containing group;
X comprises H, a removable protecting group, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide,
enzymatic nucleic acid, siNA or a portion thereof, amino acid,
peptide, protein, lipid, phospholipid, or label; W comprises a
linker molecule or chemical linkage that can be present or absent;
and SG comprises a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0146] In one embodiment, the invention features a compound having
Formula 55:
##STR00059##
[0147] wherein each R1 independently comprises O, N, S, alkyl, or
substituted N; each R2 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a
phosphorus containing group; each R3 independently comprises H, OH,
alkyl, substituted alkyl, or halo; X comprises H, a removable
protecting group, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA or a portion thereof, 2,5-A
chimera, decoy, aptamer or triplex forming oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, biologically active
molecule or label; W comprises a linker molecule or chemical
linkage that can be present or absent; SG comprises a sugar, for
example galactose, galactosamine, N-acetyl-galactosamine, glucose,
mannose, fructose, or fucose and the respective D or L, alpha or
beta isomers, each n is independently an integer from about 1 to
about 50; and N' is an integer from about 1 to about 100. In
another embodiment, X comprises a siNA molecule or a portion
thereof. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.
[0148] In another embodiment, the invention features a compound
having Formula 56:
##STR00060##
[0149] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N,
or a phosphorus containing group; R2 comprises H, O, OH, alkyl,
alkylhalo, halo, S, N, substituted N, or a phosphorus containing
group; X comprises H, a removable protecting group, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide such as an enzymatic nucleic acid, allozyme,
antisense nucleic acid, 2,5-A chimera, decoy, aptamer or triplex
forming oligonucleotide, siNA or a portion thereof, amino acid,
peptide, protein, lipid, phospholipid, biologically active molecule
or label; W comprises a linker molecule or chemical linkage that
can be present or absent; SG comprises a sugar, for example
galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,
fructose, or fucose and the respective D or L, alpha or beta
isomers, and each n is independently an integer from about 0 to
about 20. In another embodiment, X comprises a siNA molecule or a
portion thereof. In another embodiment, W is selected from the
group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0150] In another embodiment, the invention features a compound
having Formula 57:
##STR00061##
[0151] wherein R1 can include the groups:
##STR00062##
[0152] and wherein R2 can include the groups:
##STR00063##
[0153] and wherein Tr is a removable protecting group, for example
a trityl, monomethoxytrityl, or dimethoxytrityl; SG comprises a
sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers, and n is an integer
from about 1 to about 20.
[0154] In one embodiment, compounds having Formula 52, 53, 54, 55,
56, and 57 are featured wherein each nitrogen adjacent to a
carbonyl can independently be substituted for a carbonyl adjacent
to a nitrogen or each carbonyl adjacent to a nitrogen can be
substituted for a nitrogen adjacent to a carbonyl.
[0155] In another embodiment, the invention features a compound
having Formula 58:
##STR00064##
[0156] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent; Y comprises a linker molecule or amino acid that can be
present or absent; V comprises a signal protein or peptide, for
example Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide; each n is independently an integer from
about 1 to about 50; and N' is an integer from about 1 to about
100. In another embodiment, X comprises a siNA molecule or a
portion thereof. In another embodiment, W is selected from the
group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0157] In another embodiment, the invention features a compound
having Formula 59:
##STR00065##
[0158] wherein each R1 independently comprises O, S, N, substituted
N, or a phosphorus containing group; each R2 independently
comprises O, S, or N; X comprises H, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, or enzymatic
nucleic acid or other biologically active molecule; n is an integer
from about 1 to about 50, Q comprises H or a removable protecting
group which can be optionally absent, each W independently
comprises a linker molecule or chemical linkage that can be present
or absent, and V comprises a signal protein or peptide, for example
Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide, or a compound having Formula 45
##STR00066##
[0159] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide such
as an enzymatic nucleic acid, allozyme, antisense nucleic acid,
siNA, 2,5-A chimera, decoy, aptamer or triplex forming
oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,
or label; and n is an integer from about 1 to about 100. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0160] In another embodiment, the invention features a compound
having Formula 60:
##STR00067##
[0161] wherein R1 can include the groups:
##STR00068##
[0162] and wherein R2 can include the groups:
##STR00069##
[0163] and wherein Tr is a removable protecting group, for example
a trityl, monomethoxytrityl, or dimethoxytrityl; n is an integer
from about 1 to about 50; and R8 is a nitrogen protecting group,
for example a phthaloyl, trifluoroacetyl, FMOC, or
monomethoxytrityl group.
[0164] In another embodiment, the invention features a compound
having Formula 61:
##STR00070##
[0165] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be the same or different and can be present or absent, Y
comprises a linker molecule that can be present or absent; each 5
independently comprises a signal protein or peptide, for example
Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide; each R1, R2, R3, and R4 independently
comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S,
S-alkyl, S-alkylcyano, N or substituted N, and n is an integer from
about 1 to about 10. In another embodiment, X comprises a siNA
molecule or a portion thereof. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0166] In another embodiment, the invention features a compound
having Formula 62:
##STR00071##
[0167] wherein X comprises a biologically active molecule; each 5
independently comprises a signal protein or peptide, for example
Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide; W comprises a linker molecule or chemical
linkage that can be present or absent; each R1, R2, and R3
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and
each n is independently an integer from about 1 to about 10. In
another embodiment, X comprises a siNA molecule or a portion
thereof. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.
[0168] In another embodiment, the invention features a compound
having Formula 63:
##STR00072##
[0169] wherein X comprises a biologically active molecule; V
comprises a signal protein or peptide, for example Human serum
albumin protein, Antennapedia peptide, Kaposi fibroblast growth
factor peptide, Caiman crocodylus Ig(5) light chain peptide, HIV
envelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenza
hemagglutinin envelope glycoprotein peptide, or transportan A
peptide; W comprises a linker molecule or chemical linkage that can
be present or absent; each R1, R2, R3 independently comprises O,
OH, II, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, R4 represents an ester, amide, or
protecting group, and each n is independently an integer from about
1 to about 10. In another embodiment, X comprises a siNA molecule
or a portion thereof. In another embodiment, W is selected from the
group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0170] In another embodiment, the invention features a compound
having Formula 64:
##STR00073##
[0171] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, A comprises a nitrogen containing
group, and B comprises a lipophilic group. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0172] In another embodiment, the invention features a compound
having Formula 65:
##STR00074##
[0173] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, RV comprises the lipid or
phospholipid component of any of Formulae 47-50, and R6 comprises a
nitrogen containing group. In another embodiment, X comprises a
siNA molecule or a portion thereof. In another embodiment, W is
selected from the group consisting of amide, phosphate, phosphate
ester, phosphoramidate, or thiophosphate ester linkage.
[0174] In another embodiment, the invention features a compound
having Formula 92:
##STR00075##
[0175] wherein B comprises H, a nucleoside base, or a
non-nucleosidic base with or without protecting groups; each R1
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, or a phosphorus containing group;
X comprises H, a removable protecting group, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide,
enzymatic nucleic acid, amino acid, peptide, protein, lipid,
phospholipid, biologically active molecule or label; W comprises a
linker molecule or chemical linkage that can be present or absent;
R2 comprises O, NH, S, CO, COO, ON.dbd.C, or alkyl; R3 comprises
alkyl, akloxy, or an aminoacyl side chain; and SG comprises a
sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0176] In another embodiment, the invention features a compound
having Formula 86:
##STR00076##
[0177] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N,
or a phosphorus containing group; R2 comprises H, O, OH, alkyl,
alkylhalo, halo, S, N, substituted N, or a phosphorus containing
group; X comprises H, a removable protecting group, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide, enzymatic nucleic acid, amino acid, peptide,
protein, lipid, phospholipid, biologically active molecule or
label; W comprises a linker molecule or chemical linkage that can
be present or absent; R3 comprises O, NH, S, CO, COO, ON.dbd.C, or
alkyl; R4 comprises alkyl, akloxy, or an aminoacyl side chain; and
SG comprises a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers, and each n is
independently an integer from about 0 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0178] In another embodiment, the invention features a compound
having Formula 87:
##STR00077##
[0179] wherein X comprises a protein, peptide, antibody, lipid,
phospholipid, oligosaccharide, label, biologically active molecule,
for example a vitamin such as folate, vitamin A, E, B6, B12,
coenzyme, antibiotic, antiviral, nucleic acid, nucleotide,
nucleoside, or oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, or polymers such as
polyethylene glycol; W comprises a linker molecule or chemical
linkage that can be present or absent; and Y comprises a
biologically active molecule, for example an enzymatic nucleic
acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, peptide, protein, or
antibody; R1 comprises H, alkyl, or substituted alkyl. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0180] In another embodiment, the invention features a compound
having Formula 88:
##STR00078##
[0181] wherein X comprises a protein, peptide, antibody, lipid,
phospholipid, oligosaccharide, label, biologically active molecule,
for example a vitamin such as folate, vitamin A, E, B6, B12,
coenzyme, antibiotic, antiviral, nucleic acid, nucleotide,
nucleoside, or oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, or polymers such as
polyethylene glycol; W comprises a linker molecule or chemical
linkage that can be present or absent, and Y comprises a
biologically active molecule, for example an enzymatic nucleic
acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, peptide, protein, or
antibody. In another embodiment, X comprises a siNA molecule or a
portion thereof. In another embodiment, W is selected from the
group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0182] In another embodiment, the invention features a compound
having Formula 99:
##STR00079##
[0183] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and SG comprises a sugar, for
example galactose, galactosamine, N-acetyl-galactosamine or
branched derivative thereof, glucose, mannose, fructose, or fucose
and the respective D or L, alpha or beta isomers. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0184] In another embodiment, the invention features a compound
having Formula 100:
##STR00080##
[0185] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, and SG comprises a sugar, for example
galactose, galactosamine, N-acetyl-galactosamine or branched
derivative thereof, glucose, mannose, fructose, or fucose and the
respective D or L, alpha or beta isomers. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0186] In one embodiment, the SG component of any compound having
Formulae 99 or 100 comprises a compound having Formula 101:
##STR00081##
[0187] wherein Y comprises a linker molecule or chemical linkage
that can be present or absent and each R7 independently is hydrogen
or an acyl group, for example an acetyl group.
[0188] In one embodiment, the W-SG component of a compound having
Formulae 99 comprises a compound having Formula 102:
##STR00082##
[0189] wherein R2 comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, a protecting group, or another
compound having Formula 102; R1 independently H, OH, alkyl,
substituted alkyl, or halo and each R7 independently is hydrogen or
an acyl group, for example an acetyl group, and R3 comprises O or
R3 in Formula 99, and n is an integer from about 1 to about 20.
[0190] In one embodiment, the W-SG component of a compound having
Formulae 99 comprises a compound having Formula 103:
##STR00083##
[0191] wherein R1 comprises H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, a protecting group, or another
compound having Formula 103; each R7 independently is hydrogen or
an acyl group, for example an acetyl group, and R3 comprises H or
R3 in Formula 99, and each n is independently an integer from about
1 to about 20.
[0192] In one embodiment, the invention features a compound having
Formula 104:
##STR00084##
[0193] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each R7 independently is hydrogen or an acyl
group, for example an acetyl group, and each n is independently an
integer from about 1 to about 20, and
[0194] wherein R1 can include the groups:
##STR00085##
[0195] and wherein R2 can include the groups:
##STR00086##
[0196] In one embodiment, the invention features a compound having
Formula 105:
##STR00087##
[0197] wherein X comprises a nucleotide, polynucleotide, or
oligonucleotide or a portion thereof, R2 comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, a protecting
group, or a nucleotide, polynucleotide, or oligonucleotide or a
portion thereof; R1 independently H, OH, alkyl, substituted alkyl,
or halo and each R7 independently is hydrogen or an acyl group, for
example an acetyl group, and n is an integer from about 1 to about
20. In another embodiment, X comprises a siNA molecule or a portion
thereof.
[0198] In one embodiment, the invention features a compound having
Formula 106:
##STR00088##
[0199] wherein X comprises a nucleotide, polynucleotide, or
oligonucleotide or a portion thereof, R1 comprises H, OH, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,
label, or a portion thereof, or OR5 where R5 a removable protecting
group, each R7 independently is hydrogen or an acyl group, for
example an acetyl group, and each n is independently an integer
from about 1 to about 20. In another embodiment, X comprises a siNA
molecule or a portion thereof.
[0200] In another embodiment, the invention features a compound
having Formula 107:
##STR00089##
[0201] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and Cholesterol comprises
cholesterol or an analog, derivative, or metabolite thereof. In
another embodiment, X comprises a siNA molecule or a portion
thereof. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.
[0202] In another embodiment, the invention features a compound
having Formula 108:
##STR00090##
[0203] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, and Cholesterol comprises cholesterol or an
analog, derivative, or metabolite thereof. In another embodiment, X
comprises a siNA molecule or a portion thereof. In another
embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.
[0204] In one embodiment, the W-Cholesterol component of a compound
having Formula 107 comprises a compound having Formula 109:
##STR00091##
[0205] wherein R3 comprises R3 as described in Formula 107, and n
is independently an integer from about 1 to about 20.
[0206] In one embodiment, the invention features a compound having
Formula 110:
##STR00092##
[0207] wherein R4 comprises O, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S. S-alkyl, S-alkylcyano, N or substituted N, each n
is independently an integer from about 1 to about 20, and
[0208] wherein R1 can include the groups:
##STR00093##
[0209] and wherein R2 can include the groups:
##STR00094##
[0210] In one embodiment, the invention features a compound having
Formula 111:
##STR00095##
[0211] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, and n is an integer from about 1 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0212] In one embodiment, the invention features a compound having
Formula 112:
##STR00096##
[0213] wherein n is an integer from about 1 to about 20. In another
embodiment, a compound having Formula 112 is used to generate a
compound having Formula III via NHS ester mediated coupling with a
biologically active molecule, such as a siNA molecule or a portion
thereof. In a non-limiting example, the NHS ester coupling can be
effectuated via attachment to a free amine present in the siNA
molecule, such as an amino linker molecule present on a nucleic
acid sugar (e.g. 2'-amino linker) or base (e.g., C5 alkyl amine
linker) component of the siNA molecule.
[0214] In one embodiment, the invention features a compound having
Formula 113:
##STR00097##
[0215] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each n is independently an integer from about 1
to about 20, and
[0216] wherein R1 can include the groups:
##STR00098##
[0217] and wherein R2 can include the groups:
##STR00099##
[0218] In another embodiment, a compound having Formula 113 is used
to generate a compound having Formula 111 via phosphoramidite
mediated coupling with a biologically active molecule, such as a
siNA molecule or a portion thereof.
[0219] In one embodiment, the invention features a compound having
Formula 114:
##STR00100##
[0220] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, and n is an integer from about 1 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0221] In one embodiment, the invention features a compound having
Formula 115:
##STR00101##
[0222] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, and each n is
independently an integer from about 1 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0223] In one embodiment, the invention features a compound having
Formula 116:
##STR00102##
[0224] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each n is independently an integer from about 1
to about 20, and
[0225] wherein R1 can include the groups:
##STR00103##
[0226] and wherein R2 can include the groups:
##STR00104##
[0227] In another embodiment, a compound having Formula 116 is used
to generate a compound having Formula 114 or 115 via
phosphoramidite mediated coupling with a biologically active
molecule, such as a siNA molecule or a portion thereof.
[0228] In one embodiment, the invention features a compound having
Formula 117:
##STR00105##
[0229] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each R7 independently is hydrogen or an acyl
group, for example an acetyl group, each n is independently an
integer from about 1 to about 20, and
[0230] wherein R1 can include the groups:
##STR00106##
[0231] and wherein R2 can include the groups:
##STR00107##
[0232] In another embodiment, a compound having Formula 117 is used
to generate a compound having Formula 105 via phosphoramidite
mediated coupling with a biologically active molecule, such as a
siNA molecule or a portion thereof.
[0233] In one embodiment, the invention features a compound having
Formula 118:
##STR00108##
[0234] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, each R7 independently
is hydrogen or an acyl group, for example an acetyl group, and each
n is independently an integer from about 1 to about 20. In another
embodiment, X comprises a siNA molecule or a portion thereof. In
another embodiment, W is selected from the group consisting of
amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.
[0235] In one embodiment, the invention features a compound having
Formula 119:
##STR00109##
[0236] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, each R7 independently is hydrogen or an acyl group, for
example an acetyl group, and each n is independently an integer
from about 1 to about 20. In another embodiment, X comprises a siNA
molecule or a portion thereof. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0237] In one embodiment, the invention features a compound having
Formula 120:
##STR00110##
[0238] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each R7 independently is hydrogen or an acyl
group, for example an acetyl group, each n is independently an
integer from about 1 to about 20, and
[0239] wherein R1 can include the groups:
##STR00111##
[0240] and wherein R2 can include the groups:
##STR00112##
[0241] In another embodiment, a compound having Formula 120 is used
to generate a compound having Formula 118 or 119 via
phosphoramidite mediated coupling with a biologically active
molecule, such as a siNA molecule or a portion thereof.
[0242] In one embodiment, the invention features a compound having
Formula 121:
##STR00113##
[0243] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, each R7 independently is hydrogen or an acyl group, for
example an acetyl group, and each n is independently an integer
from about 1 to about 20. In another embodiment, X comprises a siNA
molecule or a portion thereof. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.
[0244] In one embodiment, the invention features a compound having
Formula 122:
##STR00114##
[0245] wherein R3 comprises H, OH, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,
peptide, protein, lipid, phospholipid, label, or a portion thereof,
or OR5 where R5 a removable protecting group, R4 comprises O,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N, each R7 independently is hydrogen or an acyl
group, for example an acetyl group, each n is independently an
integer from about 1 to about 20, and
[0246] wherein R1 can include the groups:
##STR00115##
[0247] and wherein R2 can include the groups:
##STR00116##
[0248] In another embodiment, a compound having Formula 122 is used
to generate a compound having Formula 121 via phosphoramidite
mediated coupling with a biologically active molecule, such as a
siNA molecule or a portion thereof.
[0249] In one embodiment, the invention features a method for the
synthesis of a compound having Formula 48:
##STR00117##
[0250] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N; and each B independently
represents a lipophilic group, for example a saturated or
unsaturated linear, branched, or cyclic alkyl group, comprising:
(a) introducing a compound having Formula 66:
##STR00118##
[0251] wherein R1 is defined as in Formula 48 and can include the
groups:
##STR00119##
[0252] and wherein R2 is defined as in Formula 48 and can include
the groups:
##STR00120##
[0253] and wherein each R5 independently comprises O, N, or S and
each R6 independently comprises a removable protecting group, for
example a trityl, monomethoxytrityl, or dimethoxytrityl group, to a
compound having Formula 67:
X--W--Y 67
[0254] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, and Y comprises a linker molecule that can be present or
absent, under conditions suitable for the formation of a compound
having Formula 68:
##STR00121##
[0255] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; and each R1, R2, R3, and R4 independently comprises O, OH,
H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N comprising, each R5 independently
comprises O, S, or N; and each R6 is independently a removable
protecting group, for example a trityl, monomethoxytrityl, or
dimethoxytrityl group; (b) removing R6 from the compound having
Formula 26 and (c) introducing a compound having Formula 69:
##STR00122##
[0256] wherein R1 is defined as in Formula 48 and can include the
groups:
##STR00123##
[0257] and wherein R2 is defined as in Formula 48 and can include
the groups:
##STR00124##
[0258] and wherein W and B are defined as in Formula 48, to the
compound having Formula 68 under conditions suitable for the
formation of a compound having Formula 48.
[0259] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 49:
##STR00125##
[0260] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H,
alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano,
N or substituted N; each R5 independently comprises O, S, or N; and
each B independently comprises a lipophilic group, for example a
saturated or unsaturated linear, branched, or cyclic alkyl group,
comprising: (a) coupling a compound having Formula 70:
##STR00126##
[0261] wherein R1 is defined as in Formula 49 and can include the
groups:
##STR00127##
[0262] and wherein R2 is defined as in Formula 49 and can include
the groups:
##STR00128##
[0263] and wherein each R5 independently comprises O, S, or N, and
wherein each B independently comprises a lipophilic group, for
example a saturated or unsaturated linear, branched, or cyclic
alkyl group, with a compound having Formula 67:
X--W--Y 67
[0264] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, and Y comprises a linker molecule that can be present or
absent, under conditions suitable for the formation of a compound
having Formula 49.
[0265] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 52:
##STR00129##
[0266] wherein X comprises a biologically active molecule; Y
comprises a linker molecule or chemical linkage that can be present
or absent; each R1, R2, R3, and R4 independently comprises O, OH,
H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N; Z comprises H, OH, O-alkyl, SH,
S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,
or label; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose,
or fucose and the respective D or L, alpha or beta isomers, n is an
integer from about 1 to about 20; and N' is an integer from about 1
to about 20, comprising: (a) coupling a compound having Formula
71:
##STR00130##
[0267] wherein R1, R2, R3, R5, SG, and n is as defined in Formula
52, and wherein R1 can include the groups:
##STR00131##
[0268] and wherein R2 can include the groups:
##STR00132##
[0269] and R6 comprises a removable protecting group, for example a
trityl, monomethoxytrityl, or dimethoxytrityl group; with a
compound having Formula 72:
X--Y
[0270] wherein X comprises a biologically active molecule and Y
comprises a linker molecule that can be present or absent, under
conditions suitable for the formation of a compound having Formula
95:
##STR00133##
[0271] (b) removing R6 from the compound having Formula 95 and (c)
optionally coupling a nucleotide, nucleoside, nucleic acid,
oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,
or label, or optionally; coupling a compound having Formula 71
under and optionally repeating (b) and (c) under conditions
suitable for the formation of a compound having Formula 52.
[0272] In another embodiment, the invention features a method for
synthesizing a compound having Formula 53:
##STR00134##
[0273] wherein B comprises H, a nucleoside base, or a
non-nucleosidic base with or without protecting groups; each R1
independently comprises O, N, S, alkyl, or substituted N; each R2
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, or a phosphorus containing group;
each R3 independently comprises N or O--N, each R4 independently
comprises O, CH2, S, sulfone, or sulfoxy; X comprises H, a
removable protecting group, amino, substituted amino, nucleotide,
nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid,
amino acid, peptide, protein, lipid, phospholipid, or label; W
comprises a linker molecule or chemical linkage that can be present
or absent; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose,
or fucose and the respective D or L, alpha or beta isomers, each n
is independently an integer from about 1 to about 50; and N' is an
integer from about 1 to about 10, comprising: coupling a compound
having Formula 73:
##STR00135##
[0274] wherein R1, R2, R3, R4, X, W, B, N' and n are as defined in
Formula 53, with a sugar, for example a compound having Formula
74:
##STR00136##
[0275] wherein Y comprises a linker molecule or chemical linkage
that can be present or absent; L represents a reactive chemical
group, for example a NHS ester, and each R7 independently is
hydrogen or an acyl group, for example an acetyl group; under
conditions suitable for the formation of a compound having Formula
53.
[0276] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 54:
##STR00137##
[0277] wherein B comprises H, a nucleoside base, or a
non-nucleosidic base with or without protecting groups; each R1
independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, or a phosphorus containing group;
X comprises H, a removable protecting group, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide,
enzymatic nucleic acid, amino acid, peptide, protein, lipid,
phospholipid, biologically active molecule or label; W comprises a
linker molecule or chemical linkage that can be present or absent;
SG comprises a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers, comprising (a)
coupling a compound having Formula 75:
##STR00138##
[0278] wherein R1, R2, R3, R4, X, W, and B are as defined in
Formula 53, with a sugar, for example a compound having Formula
74.
##STR00139##
[0279] wherein Y comprises a C11 alkyl linker molecule; L
represents a reactive chemical group, for example a NHS ester, and
each R7 independently is hydrogen or an acyl group, for example an
acetyl group; under conditions suitable for the formation of a
compound having Formula 54.
[0280] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 55:
##STR00140##
[0281] wherein each R1 independently comprises O, N, S, alkyl, or
substituted N; each R2 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a
phosphorus containing group; each R3 independently comprises H, OH,
alkyl, substituted alkyl, or halo; X comprises H, a removable
protecting group, nucleotide, nucleoside, nucleic acid,
oligonucleotide, or enzymatic nucleic acid or biologically active
molecule; W comprises a linker molecule or chemical linkage that
can be present or absent; SG comprises a sugar, for example
galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,
fructose, or fucose and the respective D or L, alpha or beta
isomers, each n is independently an integer from about 1 to about
50; and N' is an integer from about 1 to about 100, comprising: (a)
coupling a compound having Formula 76:
##STR00141##
[0282] wherein R1 can include the groups:
##STR00142##
[0283] and wherein R2 can include the groups:
##STR00143##
[0284] and wherein each R3 independently comprises H, OH, alkyl,
substituted alkyl, or halo; SG comprises a sugar, for example
galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,
fructose, or fucose and the respective D or L, alpha or beta
isomers, and n is an integer from about 1 to about 20, to a
compound X--W, wherein X comprises a nucleotide, nucleoside,
nucleic acid, oligonucleotide, enzymatic nucleic acid, amino acid,
peptide, protein, lipid, phospholipid, biologically active molecule
or label, and W comprises a linker molecule or chemical linkage
that can be present or absent; and (b) optionally repeating step
(a) under conditions suitable for the formation of a compound
having Formula 55.
[0285] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 56:
##STR00144##
[0286] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N,
or a phosphorus containing group; R2 comprises H, O, OH, alkyl,
alkylhalo, halo, S, N, substituted N, or a phosphorus containing
group; X comprises H, a removable protecting group, amino,
substituted amino, nucleotide, nucleoside, nucleic acid,
oligonucleotide, enzymatic nucleic acid, amino acid, peptide,
protein, lipid, phospholipid, biologically active molecule or
label; W comprises a linker molecule or chemical linkage that can
be present or absent; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose,
or fucose and the respective D or L, alpha or beta isomers, and
each n is independently an integer from about 0 to about 20,
comprising: (a) coupling a compound having Formula 77:
##STR00145##
[0287] wherein each R1, X, W, and n are as defined in Formula 56,
to a sugar, for example a compound having Formula 74:
##STR00146##
[0288] wherein Y comprises an alkyl linker molecule of length n,
where n is an integer from about 1 to about 20; L represents a
reactive chemical group, for example a NHS ester, and each R7
independently is hydrogen or an acyl group, for example an acetyl
group; and (b) optionally coupling X--W, wherein X comprises a
removable protecting group, amino, substituted amino, nucleotide,
nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid,
amino acid, peptide, protein, lipid, phospholipid, or label and W
comprises a linker molecule or chemical linkage that can be present
or absent, under conditions suitable for the formation of a
compound having Formula 56.
[0289] In another embodiment, the invention features method for
synthesizing a compound having Formula 57:
##STR00147##
[0290] wherein R1 can include the groups:
##STR00148##
[0291] and wherein R2 can include the groups:
##STR00149##
[0292] and wherein Tr is a removable protecting group, for example
a trityl, monomethoxytrityl, or dimethoxytrityl; SG comprises a
sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and
the respective D or L, alpha or beta isomers, and n is an integer
from about 1 to about 20, comprising: (a) coupling a compound
having Formula 77:
##STR00150##
[0293] wherein R1 and X comprise H, to a sugar, for example a
compound having Formula 74:
##STR00151##
[0294] wherein Y comprises an alkyl linker molecule of length n,
where n is an integer from about 1 to about 20; L represents a
reactive chemical group, for example a NHS ester, and each R7
independently is hydrogen or an acyl group, for example an acetyl
group; and (b) introducing a trityl group, for example a
dimethoxytrityl, monomethoxytrityl, or trityl group to the primary
hydroxyl of the product of (a) and (c) introducing a phosphorus
containing group having Formula 78:
##STR00152##
[0295] wherein R1 can include the groups:
##STR00153##
[0296] and wherein each R2 and R3 independently can include the
groups:
##STR00154##
[0297] to the secondary hydroxyl of the product of (b) under
conditions suitable for the formation of a compound having Formula
57.
[0298] In another embodiment, the invention features a method for
synthesizing a compound having Formula 60:
##STR00155##
[0299] wherein R1 can include the groups:
##STR00156##
[0300] and wherein R2 can include the groups:
##STR00157##
[0301] and wherein Tr is a removable protecting group, for example
a trityl, monomethoxytrityl, or dimethoxytrityl; n is an integer
from about 1 to about 50; and R8 is a nitrogen protecting group,
for example a phthaloyl, trifluoroacetyl, FMOC, or
monomethoxytrityl group, comprising: (a) introducing carboxy
protection to a compound having Formula 79:
##STR00158##
[0302] wherein n is an integer from about 1 to about 50, under
conditions suitable for the formation of a compound having Formula
80:
##STR00159##
[0303] wherein n is an integer from about 1 to about 50 and R7 is a
carboxylic acid protecting group, for example a benzyl group; (b)
introducing a nitrogen containing group to the product of (a) under
conditions suitable for the formation of a compound having Formula
81:
##STR00160##
[0304] wherein n and R7 are as defined in Formula 80 and R8 is a
nitrogen protecting group, for example a phthaloyl,
trifluoroacetyl, FMOC, or monomethoxytrityl group; (c) removing the
carboxylic acid protecting group from the product of (b) and
introducing aminopropanediol under conditions suitable for the
formation of a compound having Formula 82:
##STR00161##
[0305] wherein n and R8 are as defined in Formula 81; (d)
introducing a removable protecting group, for example a trityl,
monomethoxytrityl, or dimethoxytrityl to the product of (c) under
conditions suitable for the formation of a compound having Formula
83:
##STR00162##
[0306] wherein Tr, n and R8 are as defined in Formula 60; and (e)
introducing a phosphorus containing group having Formula 78:
##STR00163##
[0307] wherein R1 can include the groups:
##STR00164##
[0308] and wherein each R2 and R3 independently can include the
groups:
##STR00165##
[0309] to the product of (d) under conditions suitable for the
formation of a compound having Formula 60.
[0310] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 59:
##STR00166##
[0311] wherein each R1 independently comprises O, S, N, substituted
N, or a phosphorus containing group; each R2 independently
comprises O, S, or N; X comprises H, amino, substituted amino,
nucleotide, nucleoside, nucleic acid, oligonucleotide, such as an
enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,
2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide or
other biologically active molecule or a portion thereof; n is an
integer from about 1 to about 50, Q comprises H or a removable
protecting group which can be optionally absent, each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, and V comprises a protein or peptide, for
example Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide, or a compound having Formula 45:
##STR00167##
[0312] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; and n is an
integer from about 1 to about 100, comprising: (a) removing R8 from
a compound having Formula 84:
##STR00168##
[0313] wherein Q, X, W, R1, R2, and n are as defined in Formula 59
and R8 is a nitrogen protecting group, for example a phthaloyl,
trifluoroacetyl, FMOC, or monomethoxytrityl group, under conditions
suitable for the formation of a compound having Formula 85:
##STR00169##
[0314] wherein Q, X, W, R1, R2, and n are as defined in Formula 59;
(b) introducing a group V to the product of (a) via the formation
of an oxime linkage, wherein V comprises a protein or peptide, for
example Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide, or a compound having Formula 45:
##STR00170##
[0315] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; and n is an
integer from about 1 to about 100, under conditions suitable for
the formation of a compound having Formula 59.
[0316] In another embodiment, the invention features a method for
synthesizing a compound having Formula 64:
##STR00171##
[0317] wherein X comprises a biologically active molecule; each W
independently comprises a linker molecule or chemical linkage that
can be present or absent, Y comprises a linker molecule that can be
present or absent; each R1, R2, R3, and R4 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, A comprises a nitrogen containing
group, and B comprises a lipophilic group, comprising: (a)
introducing a compound having Formula 66:
##STR00172##
[0318] wherein R1 is defined as in Formula 64 and can include the
groups:
##STR00173##
[0319] and wherein R2 is defined as in Formula 64 and can include
the groups:
##STR00174##
[0320] and wherein each R5 independently comprises O, N, or S and
each R6 independently comprises a removable protecting group, for
example a trityl, monomethoxytrityl, or dimethoxytrityl group, to a
compound having Formula 67:
X--W--Y 67
[0321] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, and Y comprises a linker molecule that can be present or
absent, under conditions suitable for the formation of a compound
having Formula 68:
##STR00175##
[0322] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent, Y comprises a linker molecule that can be present or
absent; and each R1, R2, R3, and R4 independently comprises O, OH,
H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N comprising, each R5 independently
comprises O, S, or N; and each R6 is independently a removable
protecting group, for example a trityl, monomethoxytrityl, or
dimethoxytrityl group; (b) removing R6 from the compound having
Formula 68 and (c) introducing a compound having Formula 69:
##STR00176##
[0323] wherein R1 is defined as in Formula 64 and can include the
groups:
##STR00177##
[0324] and wherein R2 is defined as in Formula 64 and can include
the groups:
##STR00178##
[0325] and wherein R3, W and B are defined as in Formula 64; and
introducing a compound having Formula 69':
##STR00179##
[0326] wherein R1 is defined as in Formula 64 and can include the
groups:
##STR00180##
[0327] and wherein R2 is defined as in Formula 48 and can include
the groups:
##STR00181##
[0328] and wherein R3, W and A are defined as in Formula 64; to the
compound having Formula 68 under conditions suitable for the
formation of a compound having Formula 64.
[0329] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 62:
##STR00182##
[0330] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent; each 5 independently comprises a protein or peptide, for
example Human serum albumin protein, Antennapedia peptide, Kaposi
fibroblast growth factor peptide, Caiman crocodylus Ig(5) light
chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat
peptide, Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide; each R1, R2, and R3 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and each n is independently an
integer from about 1 to about 10, comprising: (a) introducing a
compound having Formula 93:
##STR00183##
[0331] wherein V and n are as defined in Formula 62, to a compound
having Formula 86:
##STR00184##
[0332] wherein X, W, R1, R2, R3, and n are as defined in Formula
62, under conditions suitable for the formation of a compound
having Formula 62.
[0333] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 63:
##STR00185##
[0334] wherein X comprises a biologically active molecule; W
comprises a linker molecule or chemical linkage that can be present
or absent; V comprises a protein or peptide, for example Human
serum albumin protein, Antennapedia peptide, Kaposi fibroblast
growth factor peptide, Caiman crocodylus Ig(5) light chain peptide,
HIV envelope glycoprotein gp41 peptide, HIV-1 Tat peptide,
Influenza hemagglutinin envelope glycoprotein peptide, or
transportan A peptide; each R1, R2, and R3 independently comprises
O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, R4 represents an ester, amide, or
protecting group, and each n is independently an integer from about
1 to about 10, comprising: (a) introducing a compound having
Formula 96:
##STR00186##
[0335] wherein V and R4 are as defined in Formula 63, to a compound
having Formula 86:
##STR00187##
[0336] wherein X, W, R1, R2, R3, and n are as defined in Formula
63, under conditions suitable for the formation of a compound
having Formula 63.
[0337] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 87:
##STR00188##
[0338] wherein X comprises a protein, peptide, antibody, lipid,
phospholipid, oligosaccharide, label, biologically active molecule,
for example a vitamin such as folate, vitamin A, E, B6, B12,
coenzyme, antibiotic, antiviral, nucleic acid, nucleotide,
nucleoside, or oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, or polymers such as
polyethylene glycol; W comprises a linker molecule or chemical
linkage that can be present or absent; and Y comprises a
biologically active molecule, for example an enzymatic nucleic
acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, peptide, protein, or
antibody; R1 comprises H, alkyl, or substituted alkyl, comprising
(a) coupling a compound having Formula 89:
##STR00189##
[0339] wherein Y, W and R are as defined in Formula 87, with a
compound having Formula 90:
H.sub.2N--O--X 90
[0340] wherein X is as defined in Formula 87, under conditions
suitable for the formation of a compound having Formula 87, for
example by post-synthetic conjugation of a compound having Formula
89 with a compound having Formula 90, wherein X of compound 90
comprises an enzymatic nucleic acid molecule and Y of Formula 89
comprises a peptide.
[0341] In another embodiment, the invention features a method for
the synthesis of a compound having Formula 88:
##STR00190##
[0342] wherein X comprises a protein, peptide, antibody, lipid,
phospholipid, oligosaccharide, label, biologically active molecule,
for example a vitamin such as folate, vitamin A, E, B6, B12,
coenzyme, antibiotic, antiviral, nucleic acid, nucleotide,
nucleoside, or oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, or polymers such as
polyethylene glycol; W comprises a linker molecule or chemical
linkage that can be present or absent, and Y comprises a
biologically active molecule, for example an enzymatic nucleic
acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, peptide, protein, or
antibody, comprising (a) coupling a compound having Formula 91:
##STR00191##
[0343] wherein Y and W are as defined in Formula 88, with a
compound having Formula 90:
H.sub.2N--O--X 90
[0344] wherein X is as defined in Formula 88, under conditions
suitable for the formation of a compound having Formula 88, for
example by post-synthetic conjugation of a compound having Formula
91 with a compound having Formula 90, wherein X of compound 90
comprises an enzymatic nucleic acid molecule and Y of Formula 91
comprises a peptide.
[0345] In one embodiment, the invention features a compound having
Formula 94,
X--Y--W--Y--Z 94
[0346] wherein X comprises a protein, peptide, antibody, lipid,
phospholipid, oligosaccharide, label, biologically active molecule,
for example a vitamin such as folate, vitamin A, E, B6, B12,
coenzyme, antibiotic, antiviral, nucleic acid, nucleotide,
nucleoside, or oligonucleotide such as an enzymatic nucleic acid,
allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, or polymers such as
polyethylene glycol; each Y independently comprises a linker or
chemical linkage that can be present or absent, W comprises a
biodegradable nucleic acid linker molecule, and Z comprises a
biologically active molecule, for example an enzymatic nucleic
acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,
aptamer or triplex forming oligonucleotide, peptide, protein, or
antibody.
[0347] In another embodiment, W of a compound having Formula 94 of
the invention comprises
5'-cytidine-deoxythymidine-3',5'-deoxythymidine-cytidine-3',5'-cytidine-d-
eoxyuridine-3',5'-deoxyuridine-cytidine-3',5'-uridine-deoxythymidine-3',
or 5'-deoxythymidine-uridine-3'.
[0348] In yet another embodiment, W of a compound having Formula 94
of the invention comprises
5'-adenosine-deoxythymidine-3',5'-deoxythymidine-adenosine-3',5'-adenosin-
e-deoxyuridine-3', or 5'-deoxyuridine-adenosine-3'.
[0349] In another embodiment, Y of a compound having Formula 94 of
the invention comprises a phosphorus containing linkage,
phoshoramidate linkage, phosphodiester linkage, phosphorothioate
linkage, amide linkage, ester linkage, carbamate linkage, disulfide
linkage, oxime linkage, or morpholino linkage.
[0350] In another embodiment, compounds having Formula 89 and 91 of
the invention are synthesized by periodate oxidation of an
N-terminal Serine or Threonine residue of a peptide or protein.
[0351] In one embodiment, X of compounds having Formulae 43, 44,
46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114,
115, 118, 119, or 121 of the invention comprises a siNA molecule or
a portion thereof. In one embodiment, the siNA molecule can be
conjugated at the 5' end, 3'-end, or both 5' and 3' ends of the
sense strand or region of the siNA. In one embodiment, the siNA
molecule can be conjugated at the 3'-end of the antisense strand or
region of the siNA with a compound of the invention. In one
embodiment, both the sense strand and antisense strands or regions
of the siNA molecule are conjugated with a compound of the
invention. In one embodiment, only the sense strand or region of
the siNA is conjugated with a compound of the invention. In one
embodiment, only the antisense strand or region of the siNA is
conjugated with a compound of the invention.
[0352] In one embodiment, X of compounds having Formulae 43, 44,
46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114,
115, 118, 119, or 121 of the invention comprises an enzymatic
nucleic acid.
[0353] In another embodiment, X of compounds having Formulae 43,
44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111,
114, 115, 118, 119, or 121 of the invention comprises an antibody.
In yet another embodiment, X of compounds having Formulae 43, 44,
46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114,
115, 118, 119, or 121 of the invention comprises an interferon.
[0354] In another embodiment, X of compounds having Formulae 43,
44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111,
114, 115, 118, 119, or 121 of the invention comprises an antisense
nucleic acid, dsRNA, ssRNA, decoy, triplex oligonucleotide,
aptamer, or 2,5-A chimera.
[0355] In one embodiment, W and/or Y of compounds having Formulae
43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107,
108, 111, 114, 115, 118, 119, or 121 of the invention comprises a
degradable or cleavable linker, for example a nucleic acid sequence
comprising ribonucleotides and/or deoxynucleotides, such as a
dimer, trimer, or tetramer. A non limiting example of a nucleic
acid cleavable linker is an adenosine-deoxythymidine (A-dT) dimer
or a cytidine-deoxythymidine (C-dT) dimer. In yet another
embodiment, W and/or V of compounds having Formulae 43, 44, 48-51,
58, 63-65, 96, 99, 100, 107, 108, 111, 114, 115, 118, 119, or 121
of the invention comprises a N-hydroxy succinimide (NHS) ester
linkage, oxime linkage, disulfide linkage, phosphoramidate,
phosphorothioate, phosphorodithioate, phosphodiester linkage, or
NHC(O), CH.sub.3NC(O), CONH, C(O)NCH.sub.3, S, SO, SO.sub.2, O, NH,
NCH.sub.3 group. In another embodiment, the degradable linker, W
and/or Y, of compounds having Formulae Formulae 43, 44, 46-52, 58,
61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115,
118, 119, or 121 of the invention comprises a linker that is
susceptible to cleavage by carboxypeptidase activity.
[0356] In another embodiment, W and/or Y of Formulae 43, 44, 46-52,
58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114,
115, 118, 119, or 121 comprises a polyethylene glycol linker having
Formula 45:
##STR00192##
[0357] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,
substituted alkyl, aryl, substituted aryl, amino, substituted
amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino
acid, peptide, protein, lipid, phospholipid, or label; and n is an
integer from about 1 to about 100.
[0358] In one embodiment, the nucleic acid conjugates of the
instant invention are assembled by solid phase synthesis, for
example on an automated peptide synthesizer, for example a Miligen
9050 synthesizer and/or an automated oligonucleotide synthesizer
such as an ABI 394, 390Z, or Pharmacia OligoProcess, OligoPilot,
OligoMax, or AKTA synthesizer. In another embodiment, the nucleic
acid conjugates of the invention are assembled post synthetically,
for example, following solid phase oligonucleotide synthesis (see
for example FIG. 15).
[0359] In another embodiment, V of compounds having Formula 58-63
and 96 comprise peptides having SEQ ID NOS: 14-23 (Table 3).
[0360] In one embodiment, the nucleic acid conjugates of the
instant invention are assembled post synthetically, for example,
following solid phase oligonucleotide synthesis.
[0361] The present invention provides compositions and conjugates
comprising nucleosidic and non-nucleosidic derivatives. The present
invention also provides nucleic acid, polynucleotide and
oligonucleotide derivatives including RNA, DNA, and PNA based
conjugates. The attachment of compounds of the invention to
nucleosides, nucleotides, non-nucleosides, and nucleic acid
molecules is provided at any position within the molecule, for
example, at internucleotide linkages, nucleosidic sugar hydroxyl
groups such as 5', 3', and 2'-hydroxyls, and/or at nucleobase
positions such as amino and carbonyl groups.
[0362] The exemplary conjugates of the invention are described as
compounds of the formulae herein, however, other peptide, protein,
phospholipid, and poly-alkyl glycol derivatives are provided by the
invention, including various analogs of the compounds of formulae
1-122, including but not limited to different isomers of the
compounds described herein.
[0363] In one embodiment, the present invention features molecules,
compositions and conjugates of molecules, for example,
non-nucleosidic small molecules, nucleosides, nucleotides, and
nucleic acids, such as enzymatic nucleic acid molecules, antisense
nucleic acids, 2-5A antisense chimeras, triplex oligonucleotides,
decoys, siNA, allozymes, aptamers, and antisense nucleic acids
containing RNA cleaving chemical groups.
[0364] The exemplary folate conjugates of the invention are
described as compounds shown by formulae herein, however, other
folate and antifolate derivatives are provided by the invention,
including various folate analogs of the formulae of the invention,
including dihydrofloates, tetrahydrofolates, tetrahydorpterins,
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 acids. As used
herein, the term "folate" is meant to refer to folate and folate
derivatives, including pteroic acid derivatives and analogs.
[0365] The present invention features compositions and conjugates
to facilitate delivery of molecules into a biological system such
as cells. The conjugates provided by the instant invention can
impart therapeutic activity by transferring therapeutic compounds
across cellular membranes. The present invention encompasses the
design and synthesis of novel agents for the delivery of molecules,
including but not limited to small molecules, lipids, nucleosides,
nucleotides, nucleic acids, negatively charged polymers and other
polymers, for example proteins, peptides, carbohydrates, or
polyamines. In general, the transporters described are designed to
be used either individually or as part of a multi-component system.
The compounds of the invention generally shown in Formulae herein
are expected to improve delivery of molecules into a number of cell
types originating from different tissues, in the presence or
absence of serum.
[0366] In another embodiment, the present invention features
methods to modulate gene expression, for example, genes involved in
the progression and/or maintenance of cancer or in a viral
infection. For example, in one embodiment, the invention features
the use of one or more of the nucleic acid-based molecules and
methods independently or in combination to inhibit the expression
of the gene(s) encoding proteins associated with cancerous
conditions, for example breast cancer, lung cancer, colorectal
cancer, brain cancer, esophageal cancer, stomach cancer, bladder
cancer, pancreatic cancer, cervical cancer, head and neck cancer,
ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant
cancer associated genes.
[0367] In another embodiment, the invention features the use of one
or more of the nucleic acid-based molecules and methods
independently or in combination to inhibit the expression of the
gene(s) encoding viral proteins, for example HIV, HBV, HCV, CMV,
RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus, Ebola
virus, foot and mouth virus, and papilloma virus associated
genes.
[0368] In one embodiment, the invention features the use of an
enzymatic nucleic acid molecule conjugate comprising compounds of
formulae 1-122, preferably in the hammerhead, NCH, G-cleaver,
amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression
of cancer and virus associated genes.
[0369] In another embodiment, the invention features the use of an
enzymatic nucleic acid molecule as a conjugate. These enzymatic
nucleic acids can catalyze the hydrolysis of RNA phosphodiester
bonds in trans (and thus can cleave other RNA molecules) under
physiological conditions. Table I summarizes some of the
characteristics of these enzymatic nucleic acids. Without being
bound by any particular theory, in general, enzymatic nucleic acids
act by first binding to a target RNA. Such binding occurs through
the target binding portion of an enzymatic nucleic acid which is
held in close proximity to an enzymatic portion of the molecule
that acts to cleave the target RNA. Thus, the enzymatic nucleic
acid first recognizes and then binds a target RNA through
complementary base-pairing, and once bound to the correct site,
acts enzymatically to cut the target RNA. Strategic cleavage of
such a target RNA destroys its ability to direct synthesis of an
encoded protein. After an enzymatic nucleic acid has bound and
cleaved its RNA target, it is released from that RNA to search for
another target and can repeatedly bind and cleave new targets.
Thus, a single enzymatic nucleic acid molecule is able to cleave
many molecules of target RNA. In addition, the enzymatic nucleic
acid is a highly specific inhibitor of gene expression, with the
specificity of inhibition depending not only on the base-pairing
mechanism of binding to the target RNA, but also on the mechanism
of target RNA cleavage. Single mismatches, or base-substitutions,
near the site of cleavage can completely eliminate catalytic
activity of an enzymatic nucleic acid.
[0370] In one embodiment of the invention described herein, the
enzymatic nucleic acid molecule component of the conjugate is
formed in a hammerhead or hairpin motif, but can also be formed in
the motif of a hepatitis delta virus, group I intron, group II
intron or RNase P RNA (in association with an RNA guide sequence),
Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.
Examples of such hammerhead motifs are described by Dreyfus, supra,
Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of
hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989
Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53,
Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990
Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, U.S. Pat. No.
5,631,359; of the hepatitis delta virus motif is described by
Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif
by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman,
1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24,
835; Neurospora VS RNA ribozyme motif is described by Collins
(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins,
1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive,
1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J.
14, 363); Group II introns are described by Griffin et al., 1995,
Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965;
Pyle et al., International PCT Publication No. WO 96/22689; of the
Group I intron by Cech et al., U.S. Pat. No. 4,987,071 and of
DNAzymes by Usman et al., International PCT Publication No. WO
95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al.,
1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262, and
Beigelman et al., International PCT publication No. WO 99/55857.
NCH cleaving motifs are described in Ludwig & Sproat,
International PCT Publication No. WO 98/58058; and G-cleavers are
described in Kore et al., 1998, Nucleic Acids Research 26,
4116-4120 and Eckstein et al., International PCT Publication No. WO
99/16871. Additional motifs such as the Aptazyme (Breaker et al.,
WO 98/43993), Amberzyme (Class I motif; FIG. 3; Beigelman et al.,
U.S. Ser. No. 09/301,511) and Zinzyme (FIG. 4) (Beigelman et al.,
U.S. Ser. No. 09/301,511), all incorporated by reference herein
including drawings, can also be used in the present invention.
These specific motifs are not limiting in the invention and those
skilled in the art will recognize that all that is important in an
enzymatic nucleic acid molecule of this invention is that it has a
specific substrate binding site which is complementary to one or
more of the target gene RNA regions, and that it have nucleotide
sequences within or surrounding that substrate binding site which
impart an RNA cleaving activity to the molecule (Cech et al., U.S.
Pat. No. 4,987,071).
[0371] In one embodiment of the present invention, a nucleic acid
molecule component of a conjugate of the instant invention can be
about 12 to about 100 nucleotides in length. For example, enzymatic
nucleic acid molecules of the invention are preferably about 15 to
about 50 nucleotides in length, more preferably about 25 to about
40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length
(for example see Jarvis et al., 1996, J. Biol. Chem., 271,
29107-29112). Exemplary DNAzymes of the invention are preferably
about 15 to about 40 nucleotides in length, more preferably about
25 to about 35 nucleotides in length, e.g., 29, 30, 31, or 32
nucleotides in length (see for example Santoro et al., 1998,
Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic
Acids Research, 23, 4092-4096). Exemplary antisense molecules of
the invention are preferably about 15 to about 75 nucleotides in
length, more preferably about 20 to about 35 nucleotides in length,
e.g., 25, 26, 27, or 28 nucleotides in length (see, for example,
Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997,
Nature Biotechnology, 15, 537-541). Exemplary triplex forming
oligonucleotide molecules of the invention are preferably about 10
to about 40 nucleotides in length, more preferably about 12 to
about 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides
in length (see for example Maher et al., 1990, Biochemistry, 29,
8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75).
Exemplary double stranded siNA molecules of the invention comprise
about 19 to about 25 nucleotides in length, e.g., about 19, 20, 21,
22, 23, 24, or 25 nucleotides in length, for each strand of the
siNA molecule. Exemplary single stranded siNA molecules of the
invention are about 38 to about 50 nucleotides in length, e.g.,
about 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
nucleotides in length. The length of the nucleic acid molecules
described and exemplified herein are not limiting within the
general size ranges stated.
[0372] The conjugates of the invention are added directly, or can
be complexed with cationic lipids, packaged within liposomes, or
otherwise delivered to target cells or tissues. The conjugates
and/or conjugate complexes can be locally administered to relevant
tissues ex vivo, or in vivo through injection or infusion pump,
with or without their incorporation in biopolymers. The
compositions and conjugates of the instant invention, individually,
or in combination or in conjunction with other drugs, can be used
to treat diseases or conditions discussed above. For example, to
treat a disease or condition associated with the levels of a
pathogenic protein, the patient can be treated, or other
appropriate cells can be treated, as is evident to those skilled in
the art, individually or in combination with one or more drugs
under conditions suitable for the treatment.
[0373] In a further embodiment, the described molecules can be used
in combination with other known treatments to treat conditions or
diseases discussed above. For example, the described molecules can
be used in combination with one or more known therapeutic agents to
treat breast, lung, prostate, colorectal, brain, esophageal,
bladder, pancreatic, cervical, head and neck, and ovarian cancer,
melanoma, lymphoma, glioma, multidrug resistant cancers, and/or
HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus,
west nile virus, Ebola virus, foot and mouth virus, and papilloma
virus infection.
[0374] Included in another embodiment are a series of multi-domain
cellular transport vehicles (MCTV) including one or more compounds
of Formulae 1-122 herein that enhance the cellular uptake and
transmembrane permeability of negatively charged molecules in a
variety of cell types. The compounds of the invention are used
either alone or in combination with other compounds with a neutral
or a negative charge including but not limited to neutral lipid
and/or targeting components, to improve the effectiveness of the
formulation or conjugate in delivering and targeting the
predetermined compound or molecule to cells. Another embodiment of
the invention encompasses the utility of these compounds for
increasing the transport of other impermeable and/or lipophilic
compounds into cells. Targeting components include ligands for cell
surface receptors including, peptides and proteins, glycolipids,
lipids, carbohydrates, and their synthetic variants, for example
folate receptors.
[0375] In another embodiment, the compounds of the invention are
provided as a surface component of a lipid aggregate, such as a
liposome encapsulated with the predetermined molecule to be
delivered. Liposomes, which can be unilamellar or multilamellar,
can introduce encapsulated material into a cell by different
mechanisms. For example, the liposome can directly introduce its
encapsulated material into the cell cytoplasm by fusing with the
cell membrane. Alternatively, the liposome can be compartmentalized
into an acidic vacuole (i.e., an endosome) and its contents
released from the liposome and out of the acidic vacuole into the
cellular cytoplasm.
[0376] In one embodiment the invention features a lipid aggregate
formulation of the compounds described herein, including
phosphatidylcholine (of varying chain length; e.g., egg yolk
phosphatidylcholine), cholesterol, a cationic lipid, and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000
(DSPE-PEG2000). The cationic lipid component of this lipid
aggregate can be any cationic lipid known in the art such as
dioleoyl 1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In
another embodiment this cationic lipid aggregate comprises a
covalently bound compound described in any of the Formulae
herein.
[0377] In another embodiment, polyethylene glycol (PEG) is
covalently attached to the compounds of the present invention. The
attached PEG can be any molecular weight but is preferably between
2000-50,000 daltons.
[0378] The compounds and methods of the present invention are
useful for introducing nucleotides, nucleosides, nucleic acid
molecules, lipids, peptides, proteins, and/or non-nucleosidic small
molecules into a cell. For example, the invention can be used for
nucleotide, nucleoside, nucleic acid, lipids, peptides, proteins,
and/or non-nucleosidic small molecule delivery where the
corresponding target site of action exists intracellularly.
[0379] In one embodiment, the compounds of the instant invention
provide conjugates of molecules that can interact with cellular
receptors, such as high affinity folate receptors and ASGPr
receptors, and provide a number of features that allow the
efficient delivery and subsequent release of conjugated compounds
across biological membranes. The compounds utilize chemical
linkages between the receptor ligand and the compound to be
delivered of length that can interact preferentially with cellular
receptors. Furthermore, the chemical linkages between the ligand
and the compound to be delivered can be designed as degradable
linkages, for example by utilizing a phosphate linkage that is
proximal to a nucleophile, such as a hydroxyl group. Deprotonation
of the hydroxyl group or an equivalent group, as a result of pH or
interaction with a nuclease, can result in nucleophilic attack of
the phosphate resulting in a cyclic phosphate intermediate that can
be hydrolyzed. This cleavage mechanism is analogous RNA cleavage in
the presence of a base or RNA nuclease. Alternately, other
degradable linkages can be selected that respond to various factors
such as UV irradiation, cellular nucleases, pH, temperature etc.
The use of degradable linkages allows the delivered compound to be
released in a predetermined system, for example in the cytoplasm of
a cell, or in a particular cellular organelle.
[0380] The present invention also provides ligand derived
phosphoramidites that are readily conjugated to compounds and
molecules of interest. Phosphoramidite compounds of the invention
permit the direct attachment of conjugates to molecules of interest
without the need for using nucleic acid phosphoramidite species as
scaffolds. As such, the used of phosphoramidite chemistry can be
used directly in coupling the compounds of the invention to a
compound of interest, without the need for other condensation
reactions, such as condensation of the ligand to an amino group on
the nucleic acid, for example at the N6 position of adenosine or a
2'-deoxy-2'-amino function. Additionally, compounds of the
invention can be used to introduce non-nucleic acid based
conjugated linkages into oligonucleotides that can provide more
efficient coupling during oligonucleotide synthesis than the use of
nucleic acid-based phosphoramidites. This improved coupling can
take into account improved steric considerations of abasic or
non-nucleosidic scaffolds bearing pendant alkyl linkages.
[0381] Compounds of the invention utilizing triphosphate groups can
be utilized in the enzymatic incorporation of conjugate molecules
into oligonucleotides. Such enzymatic incorporation is useful when
conjugates are used in post-synthetic enzymatic conjugation or
selection reactions, (see for example Matulic-Adamic et al., 2000,
Bioorg. Med. Chem. Lett., 10, 1299-1302; Lee et al., 2001, NAR.,
29, 1565-1573; Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992,
Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97;
Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science
261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995,
FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442;
Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et
al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long
& Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish
et al., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr.
Opin. Chem. Biol., 4, 669).
[0382] Compounds of the invention can be used to detect the
presence of a target molecule in a biological system, such as
tissue, cell or cell lysate. Examples of target molecules include
nucleic acids, proteins, peptides, antibodies, polysaccharides,
lipids, hormones, sugars, metals, microbial or cellular
metabolites, analytes, pharmaceuticals, and other organic and
inorganic molecules or other biomolecules in a sample. The
compounds of the instant invention can be conjugated to a
predetermined compound or molecule that is capable of interacting
with the target molecule in the system and providing a detectable
signal or response. Various compounds and molecules known in the
art that can be used in these applications include but are not
limited to antibodies, labeled antibodies, allozymes, aptamers,
labeled nucleic acid probes, molecular beacons, fluorescent
molecules, radioisotopes, polysaccharides, and any other compound
capable of interacting with the target molecule and generating a
detectable signal upon target interaction. For example, such
compounds are described in application entitled "NUCLEIC ACID
SENSOR MOLECULES", U.S. Ser. No. 09/800,594 filed on Mar. 6, 2001
(Not yet assigned; Attorney Docket No. MBHB00-816-A 700.001) with
inventors Nassim Usman and James A. McSwiggen, which is
incorporated by reference in its entirety, including the
drawings.
[0383] The term "biodegradable nucleic acid linker molecule" as
used herein, refers to a nucleic acid molecule that is designed as
a biodegradable linker to connect one molecule to another molecule,
for example, a biologically active molecule. The stability of the
biodegradable nucleic acid linker molecule can be modulated by
using various combinations of ribonucleotides,
deoxyribonucleotides, and chemically modified nucleotides, for
example 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl,
2'-O-allyl, and other 2'-modified or base modified nucleotides. The
biodegradable nucleic acid linker molecule can be a dimer, trimer,
tetramer or longer nucleic acid molecule, for example an
oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can
comprise a single nucleotide with a phosphorus based linkage, for
example a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise
nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
[0384] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example enzymatic
degradation or chemical degradation.
[0385] The term "biologically active molecule" as used herein,
refers to compounds or molecules that are capable of eliciting or
modifying a biological response in a system. Non-limiting examples
of biologically active molecules contemplated by the instant
invention include therapeutically active molecules such as
antibodies, hormones, antivirals, peptides, proteins,
chemotherapeutics, small molecules, vitamins, co-factors,
nucleosides, nucleotides, oligonucleotides, enzymatic nucleic
acids, antisense nucleic acids, triplex forming oligonucleotides,
2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and
analogs thereof. Biologically active molecules of the invention
also include molecules capable of modulating the pharmacokinetics
and/or pharmacodynamics of other biologically active molecules, for
example lipids and polymers such as polyamines, polyamides,
polyethylene glycol and other polyethers.
[0386] The term "phospholipid" as used herein, refers to a
hydrophobic molecule comprising at least one phosphorus group. For
example, a phospholipid can comprise a phosphorus containing group
and saturated or unsaturated alkyl group, optionally substituted
with OH, COOH, oxo, amine, or substituted or unsubstituted aryl
groups.
[0387] The term "nitrogen containing group" as used herein refers
to any chemical group or moiety comprising a nitrogen or
substituted nitrogen. Non-limiting examples of nitrogen containing
groups include amines, substituted amines, amides, alkylamines,
amino acids such as arginine or lysine, polyamines such as spermine
or spermidine, cyclic amines such as pyridines, pyrimidines
including uracil, thymine, and cytosine, morpholines, phthalimides,
and heterocyclic amines such as purines, including guanine and
adenine.
[0388] The term "target molecule" as used herein, refers to nucleic
acid molecules, proteins, peptides, antibodies, polysaccharides,
lipids, sugars, metals, microbial or cellular metabolites,
analytes, pharmaceuticals, and other organic and inorganic
molecules that are present in a system.
[0389] By "inhibit" or "down-regulate" it is meant that the
expression of the gene, or level of RNAs or equivalent RNAs
encoding one or more protein subunits, or activity of one or more
protein subunits, such as pathogenic protein, viral protein or
cancer related protein subunit(s), is reduced below that observed
in the absence of the compounds or combination of compounds of the
invention. In one embodiment, inhibition or down-regulation with an
enzymatic nucleic acid molecule preferably is below that level
observed in the presence of an enzymatically inactive or attenuated
molecule that is able to bind to the same site on the target RNA,
but is unable to cleave that RNA. In another embodiment, inhibition
or down-regulation with antisense oligonucleotides is preferably
below that level observed in the presence of, for example, an
oligonucleotide with scrambled sequence or with mismatches. In
another embodiment, inhibition or down-regulation of viral or
oncogenic RNA, protein, or protein subunits with a compound of the
instant invention is greater in the presence of the compound than
in its absence.
[0390] By "up-regulate" is meant that the expression of the gene,
or level of RNAs or equivalent RNAs encoding one or more protein
subunits, or activity of one or more protein subunits, such as
viral or oncogenic protein subunit(s), is greater than that
observed in the absence of the compounds or combination of
compounds of the invention. For example, the expression of a gene,
such as a viral or cancer related gene, can be increased in order
to treat, prevent, ameliorate, or modulate a pathological condition
caused or exacerbated by an absence or low level of gene
expression.
[0391] By "modulate" is meant that the expression of the gene, or
level of RNAs or equivalent RNAs encoding one or more protein
subunits, or activity of one or more protein subunit(s) of a
protein, for example a viral or cancer related protein is
up-regulated or down-regulated, such that the expression, level, or
activity is greater than or less than that observed in the absence
of the compounds or combination of compounds of the invention.
[0392] The term "enzymatic nucleic acid molecule" as used herein
refers to a nucleic acid molecule which has complementarity in a
substrate binding region to a specified gene target, and also has
an enzymatic activity which is active to specifically cleave target
RNA. That is, the enzymatic nucleic acid molecule is able to
intermolecularly cleave RNA and thereby inactivate a target RNA
molecule. These complementary regions allow sufficient
hybridization of the enzymatic nucleic acid molecule to the target
RNA and thus permit cleavage. One hundred percent complementarity
is preferred, but complementarity as low as 50-75% can also be
useful in this invention (see for example Werner and Uhlenbeck,
1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,
Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids
can be modified at the base, sugar, and/or phosphate groups. The
term enzymatic nucleic acid is used interchangeably with phrases
such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA,
aptazyme or aptamer-binding ribozyme, regulatable ribozyme,
catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme,
endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or
DNA enzyme. All of these terminologies describe nucleic acid
molecules with enzymatic activity. The specific enzymatic nucleic
acid molecules described in the instant application are not
limiting in the invention and those skilled in the art will
recognize that all that is important in an enzymatic nucleic acid
molecule of this invention is that it has a specific substrate
binding site which is complementary to one or more of the target
nucleic acid regions, and that it have nucleotide sequences within
or surrounding that substrate binding site which impart a nucleic
acid cleaving and/or ligation activity to the molecule (Cech et
al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA
3030).
[0393] The term "nucleic acid molecule" as used herein, refers to a
molecule having nucleotides. The nucleic acid can be single,
double, or multiple stranded and can comprise modified or
unmodified nucleotides or non-nucleotides or various mixtures and
combinations thereof.
[0394] The term "enzymatic portion" or "catalytic domain" as used
herein refers to that portion/region of the enzymatic nucleic acid
molecule essential for cleavage of a nucleic acid substrate (for
example see FIG. 1).
[0395] The term "substrate binding arm" or "substrate binding
domain" as used herein refers to that portion/region of a enzymatic
nucleic acid which is able to interact, for example via
complementarity (i.e., able to base-pair with), with a portion of
its substrate. Preferably, such complementarity is 100%, but can be
less if desired. For example, as few as 10 bases out of 14 can be
base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic
Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and
Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown
generally in FIGS. 1-4. That is, these arms contain sequences
within a enzymatic nucleic acid which are intended to bring
enzymatic nucleic acid and target RNA together through
complementary base-pairing interactions. The enzymatic nucleic acid
of the invention can have binding arms that are contiguous or
non-contiguous and can be of varying lengths. The length of the
binding arm(s) are preferably greater than or equal to four
nucleotides and of sufficient length to stably interact with the
target RNA; preferably 12-100 nucleotides; more preferably 14-24
nucleotides long (see for example Werner and Uhlenbcck, supra;
Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herrance et
al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen,
the design is such that the length of the binding arms are
symmetrical (i.e., each of the binding arms is of the same length;
e.g., five and five nucleotides, or six and six nucleotides, or
seven and seven nucleotides long) or asymmetrical (i.e., the
binding arms are of different length; e.g., six and three
nucleotides; three and six nucleotides long; four and five
nucleotides long; four and six nucleotides long; four and seven
nucleotides long; and the like).
[0396] The term "Inozyme" or "NCH" motif as used herein, refers to
an enzymatic nucleic acid molecule comprising a motif as is
generally described as NCH Rz in FIG. 1. Inozymes possess
endonuclease activity to cleave RNA substrates having a cleavage
triplet NCH/, where N is a nucleotide, C is cytidine and H is
adenosine, uridine or cytidine, and/represents the cleavage site. H
is used interchangeably with X. Inozymes can also possess
endonuclease activity to cleave RNA substrates having a cleavage
triplet NCN/, where N is a nucleotide, C is cytidine,
and/represents the cleavage site. "I" in FIG. 2 represents an
Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine
nucleoside.
[0397] The term "G-cleaver" motif as used herein, refers to an
enzymatic nucleic acid molecule comprising a motif as is generally
described as G-cleaver Rz in FIG. 1. G-cleavers possess
endonuclease activity to cleave RNA substrates having a cleavage
triplet NYN/, where N is a nucleotide, Y is uridine or cytidine
and/represents the cleavage site. G-cleavers can be chemically
modified as is generally shown in FIG. 2.
[0398] The term "amberzyme" motif as used herein, refers to an
enzymatic nucleic acid molecule comprising a motif as is generally
described in FIG. 2. Amberzymes possess endonuclease activity to
cleave RNA substrates having a cleavage triplet NG/N, where N is a
nucleotide, G is guanosine, and/represents the cleavage site.
Amberzymes can be chemically modified to increase nuclease
stability through substitutions as are generally shown in FIG. 3.
In addition, differing nucleoside and/or non-nucleoside linkers can
be used to substitute the 5'-gaaa-3' loops shown in the figure.
Amberzymes represent a non-limiting example of an enzymatic nucleic
acid molecule that does not require a ribonucleotide (2'-OH) group
within its own nucleic acid sequence for activity.
[0399] The term "zinzyme" motif as used herein, refers to an
enzymatic nucleic acid molecule comprising a motif as is generally
described in FIG. 3. Zinzymes possess endonuclease activity to
cleave RNA substrates having a cleavage triplet including but not
limited to YG/Y, where Y is uridine or cytidine, and G is guanosine
and/represents the cleavage site. Zinzymes can be chemically
modified to increase nuclease stability through substitutions as
are generally shown in FIG. 3, including substituting 2'-O-methyl
guanosine nucleotides for guanosine nucleotides. In addition,
differing nucleotide and/or non-nucleotide linkers can be used to
substitute the 5'-gaaa-2' loop shown in the figure Zinzymes
represent a non-limiting example of an enzymatic nucleic acid
molecule that does not require a ribonucleotide (2'-OH) group
within its own nucleic acid sequence for activity.
[0400] The term `DNAzyme` as used herein, refers to an enzymatic
nucleic acid molecule that does not require the presence of a 2'-OH
group for its activity. In particular embodiments the enzymatic
nucleic acid molecule can have an attached linker(s) or other
attached or associated groups, moieties, or chains containing one
or more nucleotides with 2'-OH groups. DNAzymes can be synthesized
chemically or expressed endogenously in vivo, by means of a single
stranded DNA vector or equivalent thereof. An example of a DNAzyme
is shown in FIG. 4 and is generally reviewed in Usman et al.,
International PCT Publication No. WO 95/11304; Chartrand et al.,
1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655;
Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature
Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem.
Soc., 122, 2433-39. Additional DNAzyme motifs can be selected for
using techniques similar to those described in these references,
and hence, are within the scope of the present invention.
[0401] The term "sufficient length" as used herein, refers to an
oligonucleotide of length great enough to provide the intended
function under the expected condition, i.e., greater than or equal
to 3 nucleotides. For example, for binding arms of enzymatic
nucleic acid "sufficient length" means that the binding arm
sequence is long enough to provide stable binding to a target site
under the expected binding conditions. Preferably, the binding arms
are not so long as to prevent useful turnover of the nucleic acid
molecule.
[0402] The term "stably interact" as used herein, refers to
interaction of the oligonucleotides with target nucleic acid (e.g.,
by forming hydrogen bonds with complementary nucleotides in the
target under physiological conditions) that is sufficient to the
intended purpose (e.g., cleavage of target RNA by an enzyme).
[0403] The term "homology" as used herein, refers to the nucleotide
sequence of two or more nucleic acid molecules is partially or
completely identical.
[0404] The term "antisense nucleic acid", as used herein, refers to
a non-enzymatic nucleic acid molecule that binds to target RNA by
means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid;
Egholm et al., 1993 Nature 365, 566) interactions and alters the
activity of the target RNA (for a review, see Stein and Cheng, 1993
Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902).
Typically, antisense molecules are complementary to a target
sequence along a single contiguous sequence of the antisense
molecule. However, in certain embodiments, an antisense molecule
can bind to substrate such that the substrate molecule forms a
loop, and/or an antisense molecule can bind such that the antisense
molecule forms a loop. Thus, the antisense molecule can be
complementary to two (or even more) non-contiguous substrate
sequences or two (or even more) non-contiguous sequence portions of
an antisense molecule can be complementary to a target sequence or
both. For a review of current antisense strategies, see Schmajuk et
al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997,
Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev.,
7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998,
Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad.
Pharmacol., 40, 1-49. In addition, antisense DNA can be used to
target RNA by means of DNA-RNA interactions, thereby activating
RNase H, which digests the target RNA in the duplex. The antisense
oligonucleotides can comprise one or more RNAse H activating
region, which is capable of activating RNAse H cleavage of a target
RNA. Antisense DNA can be synthesized chemically or expressed via
the use of a single stranded DNA expression vector or equivalent
thereof.
[0405] The term "RNase H activating region" as used herein, refers
to a region (generally greater than or equal to 4-25 nucleotides in
length, preferably from 5-11 nucleotides in length) of a nucleic
acid molecule capable of binding to a target RNA to form a
non-covalent complex that is recognized by cellular RNase H enzyme
(see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et
al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the
nucleic acid molecule-target RNA complex and cleaves the target RNA
sequence. The RNase H activating region comprises, for example,
phosphodiester, phosphorothioate (preferably at least four of the
nucleotides are phosphorothiote substitutions; more specifically,
4-11 of the nucleotides are phosphorothiote substitutions);
phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone
chemistry or a combination thereof. In addition to one or more
backbone chemistries described above, the RNase H activating region
can also comprise a variety of sugar chemistries. For example, the
RNase H activating region can comprise deoxyribose, arabino,
fluoroarabino or a combination thereof, nucleotide sugar chemistry.
Those skilled in the art will recognize that the foregoing are
non-limiting examples and that any combination of phosphate, sugar
and base chemistry of a nucleic acid that supports the activity of
RNase H enzyme is within the scope of the definition of the RNase H
activating region and the instant invention.
[0406] The term "2-5A antisense chimera" as used herein, refers to
an antisense oligonucleotide containing a 5'-phosphorylated
2'-5'-linked adenylate residue. These chimeras bind to target RNA
in a sequence-specific manner and activate a cellular
2-5A-dependent ribonuclease which, in turn, cleaves the target RNA
(Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300;
Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and
Torrence, 1998, Pharmacol. Ther., 78, 55-113).
[0407] The term "triplex forming oligonucleotides" as used herein,
refers to an oligonucleotide that can bind to a double-stranded DNA
in a sequence-specific manner to form a triple-strand helix.
Formation of such triple helix structure has been shown to inhibit
transcription of the targeted gene (Duval-Valentin et al., 1992
Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7,
17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489,
181-206).
[0408] The term "gene" it as used herein, refers to a nucleic acid
that encodes an RNA, for example, nucleic acid sequences including
but not limited to structural genes encoding a polypeptide.
[0409] The term "pathogenic protein" as used herein, refers to
endogenous or exongenous proteins that are associated with a
disease state or condition, for example a particular cancer or
viral infection.
[0410] The term "complementarity" refers to the ability of a
nucleic acid to form hydrogen bond(s) with another RNA sequence by
either traditional Watson-Crick or other non-traditional types. In
reference to the nucleic molecules of the present invention, the
binding free energy for a nucleic acid molecule with its target or
complementary sequence is sufficient to allow the relevant function
of the nucleic acid to proceed, e.g., enzymatic nucleic acid
cleavage, antisense or triple helix inhibition. Determination of
binding free energies for nucleic acid molecules is well known in
the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII
pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785). A percent complementarity indicates the percentage
of contiguous residues in a nucleic acid molecule which can form
hydrogen bonds (e.g., Watson-Crick base pairing) with a second
nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%,
60%, 70%, 80%, 90%, and 100% complementary). "Perfectly
complementary" means that all the contiguous residues of a nucleic
acid sequence will hydrogen bond with the same number of contiguous
residues in a second nucleic acid sequence.
[0411] The term "RNA" as used herein, refers to a molecule
comprising at least one ribonucleotide residue. By "ribonucleotide"
or "2'-OH" is meant a nucleotide with a hydroxyl group at the 2'
position of a .beta.-D-ribo-furanose moiety.
[0412] The term "decoy RNA" as used herein, refers to a RNA
molecule or aptamer that is designed to preferentially bind to a
predetermined ligand. Such binding can result in the inhibition or
activation of a target molecule. The decoy RNA or aptamer can
compete with a naturally occurring binding target for the binding
of a specific ligand. For example, it has been shown that
over-expression of HIV trans-activation response (TAR) RNA can act
as a "decoy" and efficiently binds HIV tat protein, thereby
preventing it from binding to TAR sequences encoded in the HIV RNA
(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific
example and those in the art will recognize that other embodiments
can be readily generated using techniques generally known in the
art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64,
763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr.
Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27;
Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999,
Clinical Chemistry, 45, 1628. Similarly, a decoy RNA can be
designed to bind to a receptor and block the binding of an effector
molecule or a decoy RNA can be designed to bind to receptor of
interest and prevent interaction with the receptor.
[0413] The term "single stranded RNA" (ssRNA) as used herein refers
to a naturally occurring or synthetic ribonucleic acid molecule
comprising a linear single strand, for example a ssRNA can be a
messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA)
etc. of a gene.
[0414] The term "single stranded DNA" (ssDNA) as used herein refers
to a naturally occurring or synthetic deoxyribonucleic acid
molecule comprising a linear single strand, for example, a ssDNA
can be a sense or antisense gene sequence or EST (Expressed
Sequence Tag).
[0415] The term "double stranded RNA" or "dsRNA" as used herein
refers to a double stranded RNA molecule capable of RNA
interference, including short interfering RNA (siNA).
[0416] The term "short interfering nucleic acid", "siNA", "short
interfering RNA", "siRNA", "short interfering nucleic acid
molecule", "short interfering oligonucleotide molecule", or
"chemically-modified short interfering nucleic acid molecule" as
used herein refers to any nucleic acid molecule capable of
inhibiting or down regulating gene expression or viral replication,
for example by mediating RNA interference "RNAi" or gene silencing
in a sequence-specific manner; see for example Bass, 2001, Nature,
411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and
Kreutzer et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;
Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al.,
2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,
1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
Non limiting examples of siNA molecules of the invention are
described in Haeberli et al., PCT/US03/05346 and McSwiggen et al.,
PCT/US03/05028, both incorporated by reference herein in their
entirety including the drawings, and in FIGS. 34-42 herein.
Chemical modifications described in Haeberli et al., PCT/US03/05346
and McSwiggen et al., PCT/US03/05028 and/or shown in Table 4 can be
applied to any siNA sequence of the invention. For example the siNA
can be a double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be assembled from two
separate oligonucleotides, where one strand is the sense strand and
the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e. each strand comprises
nucleotide sequence that is complementary to nucleotide sequence in
the other strand; such as where the antisense strand and sense
strand form a duplex or double stranded structure, for example
wherein the double stranded region is about 19 base pairs); the
antisense strand comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. Alternatively, the siNA is assembled
from a single oligonucleotide, where the self-complementary sense
and antisense regions of the siNA are linked by means of a nucleic
acid based or non-nucleic acid-based linker(s). The siNA can be a
polynucleotide with a hairpin secondary structure, having
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a separate target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be a circular
single-stranded polynucleotide having two or more loop structures
and a stem comprising self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof, and wherein the circular
polynucleotide can be processed either in vivo or in vitro to
generate an active siNA molecule capable of mediating RNAi. The
siNA can also comprise a single stranded polynucleotide having
nucleotide sequence complementary to nucleotide sequence in a
target nucleic acid molecule or a portion thereof (for example,
where such siNA molecule does not require the presence within the
siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single
stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,
2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell,
10, 537-568), or 5',3'-diphosphate. In certain embodiment, the siNA
molecule of the invention comprises separate sense and antisense
sequences or regions, wherein the sense and antisense regions are
covalently linked by nucleotide or non-nucleotide linkers molecules
as is known in the art, or are alternately non-covalently linked by
ionic interactions, hydrogen bonding, van der waals interactions,
hydrophobic interactions, and/or stacking interactions. In certain
embodiments, the siNA molecules of the invention comprise
nucleotide sequence that is complementary to nucleotide sequence of
a target gene. In another embodiment, the siNA molecule of the
invention interacts with nucleotide sequence of a target gene in a
manner that causes inhibition of expression of the target gene. As
used herein, siNA molecules need not be limited to those molecules
containing only RNA, but further encompasses chemically-modified
nucleotides and non-nucleotides. In certain embodiments, the short
interfering nucleic acid molecules of the invention lack 2'-hydroxy
(2'-OH) containing nucleotides. Applicant describes in certain
embodiments short interfering nucleic acids that do not require the
presence of nucleotides having a 2'-hydroxy group for mediating
RNAi and as such, short interfering nucleic acid molecules of the
invention optionally do not include any ribonucleotides (e.g.,
nucleotides having a 2'-OH group). Such siNA molecules that do not
require the presence of ribonucleotides within the siNA molecule to
support RNAi can however have an attached linker or linkers or
other attached or associated groups, moieties, or chains containing
one or more nucleotides with 2'-OH groups. Optionally, siNA
molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40,
or 50% of the nucleotide positions. The modified short interfering
nucleic acid molecules of the invention can also be referred to as
short interfering modified oligonucleotides "siMON." As used
herein, the term siNA is meant to be equivalent to other terms used
to describe nucleic acid molecules that are capable of mediating
sequence specific RNAi, for example short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide,
chemically-modified siRNA, post-transcriptional gene silencing RNA
(ptgsRNA), translational silencing, and others. In addition, as
used herein, the term RNAi is meant to be equivalent to other terms
used to describe sequence specific RNA interference, such as post
transcriptional gene silencing, or epigenetics. For example, siNA
molecules of the invention can be used to epigenetically silence
genes at both the post-transcriptional level or the
pre-transcriptional level. In a non-limiting example, epigenetic
regulation of gene expression by siNA molecules of the invention
can result from siNA mediated modification of chromatin structure
to alter gene expression (see, for example, Allshire, 2002,
Science, 297, 1818-1819; Volpe et al., 2002, Science, 297,
1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et
al., 2002, Science, 297, 2232-2237).
[0417] The term "allozyme" as used herein refers to an allosteric
enzymatic nucleic acid molecule, see for example see for example
George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al.,
U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914,
Nathan and Ellington, International PCT publication No. WO
00/24931, Breaker et al., International PCT Publication Nos. WO
00/26226 and 98/27104, and Sullenger et al., International PCT
publication No. WO 99/29842.
[0418] The term "cell" as used herein, refers to its usual
biological sense, and does not refer to an entire multicellular
organism. The cell can, for example, be in vitro, e.g., in cell
culture, or present in a multicellular organism, including, e.g.,
birds, plants and mammals such as humans, cows, sheep, apes,
monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,
bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
[0419] The term "highly conserved sequence region" as used herein,
refers to a nucleotide sequence of one or more regions in a target
gene docs not vary significantly from one generation to the other
or from one biological system to the other.
[0420] The term "non-nucleotide" as used herein, refers to any
group or compound which can be incorporated into a nucleic acid
chain in the place of one or more nucleotide units, including
either sugar and/or phosphate substitutions, and allows the
remaining bases to exhibit their enzymatic activity. The group or
compound is abasic in that it does not contain a commonly
recognized nucleotide base, such as adenosine, guanine, cytosine,
uracil or thymine.
[0421] The term "nucleotide" as used herein, refers to a
heterocyclic nitrogenous base in N-glycosidic linkage with a
phosphorylated sugar. Nucleotides are recognized in the art to
include natural bases (standard), and modified bases well known in
the art. Such bases are generally located at the 1' position of a
nucleotide sugar moiety. Nucleotides generally comprise a base,
sugar and a phosphate group. The nucleotides can be unmodified or
modified at the sugar, phosphate and/or base moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides,
non-natural nucleotides, non-standard nucleotides and other; see
for example, Usman and McSwiggen, supra; Eckstein et al.,
International PCT Publication No. WO 92/07065; Usman et al.,
International PCT Publication No. WO 93/15187; Uhlman & Peyman,
supra all are hereby incorporated by reference herein). There are
several examples of modified nucleic acid bases known in the art as
summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
Some of the non-limiting examples of chemically modified and other
natural nucleic acid bases that can be introduced into nucleic
acids include, for example, inosine, purine, pyridin-4-one,
pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene,
3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,
5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,
ribothymidine), 5-halouridine (e.g., 5-bromouridine) or
6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine),
propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine,
wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,
5'-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonylmethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,
beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,
threonine derivatives and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine,
guanine, cytosine and uracil at 1' position or their equivalents;
such bases can be used at any position, for example, within the
catalytic core of an enzymatic nucleic acid molecule and/or in the
substrate-binding regions of the nucleic acid molecule.
[0422] The term "nucleoside" as used herein, refers to a
heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.
Nucleosides are recognized in the art to include natural bases
(standard), and modified bases well known in the art. Such bases
are generally located at the 1' position of a nucleoside sugar
moiety. Nucleosides generally comprise a base and sugar group. The
nucleosides can be unmodified or modified at the sugar, and/or base
moiety, (also referred to interchangeably as nucleoside analogs,
modified nucleosides, non-natural nucleosides, non-standard
nucleosides and other; see for example, Usman and McSwiggen, supra;
Eckstein et al., International PCT Publication No. WO 92/07065;
Usman et al., International PCT Publication No. WO 93/15187; Uhlman
& Peyman, supra all are hereby incorporated by reference
herein). There are several examples of modified nucleic acid bases
known in the art as summarized by Limbach et al., 1994, Nucleic
Acids Res. 22, 2183. Some of the non-limiting examples of
chemically modified and other natural nucleic acid bases that can
be introduced into nucleic acids include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,
wybutosine, wybutoxosine, 4-acetylcytidine,
5-(carboxyhydroxymethyl)uridine,
5'-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonylmethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,
beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,
threonine derivatives and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleoside bases other than adenine,
guanine, cytosine and uracil at 1' position or their equivalents;
such bases can be used at any position, for example, within the
catalytic core of an enzymatic nucleic acid molecule and/or in the
substrate-binding regions of the nucleic acid molecule.
[0423] The term "cap structure" as used herein, refers to chemical
modifications, which have been incorporated at either terminus of
the oligonucleotide (see for example Wincott et al., WO 97/26270,
incorporated by reference herein). These terminal modifications
protect the nucleic acid molecule from exonuclease degradation, and
can help in delivery and/or localization within a cell. The cap can
be present at the 5'-terminus (5'-cap) or at the 3'-terminus
(3'-cap) or can be present on both terminus. In non-limiting
examples, the 5'-cap includes inverted abasic residue (moiety),
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,
4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol
nucleotide; L-nucleotides; alpha-nucleotides; modified base
nucleotide; phosphorodithioate linkage; threo-pentofuranosyl
nucleotide; acyclic 3',4'-seco nucleotide; acyclic
3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic
moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic
moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate;
3'-phosphorothioate; phosphorodithioate; or bridging or
non-bridging methylphosphonate moiety (for more details see Wincott
et al., International PCT publication No. WO 97/26270, incorporated
by reference herein).
[0424] The term "abasic" as used herein, refers to sugar moieties
lacking a base or having other chemical groups in place of a base
at the 1' position, for example a 3',3'-linked or 5',5'-linked
deoxyabasic ribose derivative (for more details see Wincott et al.,
International PCT publication No. WO 97/26270).
[0425] The term "unmodified nucleoside" as used herein, refers to
one of the bases adenine, cytosine, guanine, thymine, uracil joined
to the 1' carbon of 13-D-ribo-furanose.
[0426] The term "modified nucleoside" as used herein, refers to any
nucleotide base which contains a modification in the chemical
structure of an unmodified nucleotide base, sugar and/or
phosphate.
[0427] The term "consists essentially of" as used herein, is meant
that the active nucleic acid molecule of the invention, for
example, an enzymatic nucleic acid molecule, contains an enzymatic
center or core equivalent to those in the examples, and binding
arms able to bind RNA such that cleavage at the target site occurs.
Other sequences can be present which do not interfere with such
cleavage. Thus, a core region can, for example, include one or more
loop, stem-loop structure, or linker which does not prevent
enzymatic activity. For example, a core sequence for a hammerhead
enzymatic nucleic acid can comprise a conserved sequence, such as
5'-CUGAUGAG-3' and 5'-CGAA-3' connected by "X", where X is
5'-GCCGUUAGGC-3' (SEQ ID NO 1), or any other Stem II region known
in the art, or a nucleotide and/or non-nucleotide linker.
Similarly, for other nucleic acid molecules of the instant
invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme,
antisense, 2-5A antisense, triplex forming nucleic acid, and decoy
nucleic acids, other sequences or non-nucleotide linkers can be
present that do not interfere with the function of the nucleic acid
molecule.
[0428] Sequence X can be a linker of .gtoreq.2 nucleotides in
length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where
the nucleotides can preferably be internally base-paired to form a
stem of preferably 2 base pairs. In yet another embodiment, the
nucleotide linker X can be a nucleic acid aptamer, such as an ATP
aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others
(for a review see Gold et al., 1995, Anna. Rev. Biochem., 64, 763;
and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland
and Atkins, pp. 511, CSH Laboratory Press). A "nucleic acid
aptamer" as used herein is meant to indicate a nucleic acid
sequence capable of interacting with a ligand. The ligand can be
any natural or a synthetic molecule, including but not limited to a
resin, metabolites, nucleosides, nucleotides, drugs, toxins,
transition state analogs, peptides, lipids, proteins, amino acids,
nucleic acid molecules, hormones, carbohydrates, receptors, cells,
viruses, bacteria and others.
[0429] Alternatively or in addition, sequence X can be a
non-nucleotide linker. Non-nucleotides can include abasic
nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,
lipid, or polyhydrocarbon compounds. Specific examples include
those described by Seela and Kaiser, Nucleic Acids Res. 1990,
18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz,
J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am.
Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,
21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic
Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993,
34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al.,
International Publication No. WO 89/02439; Usman et al.,
International Publication No. WO 95/06731; Dudycz et al.,
International Publication No. WO 95/11910 and Ferentz and Verdine,
J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein. A "non-nucleotide" further means any group or
compound which can be incorporated into a nucleic acid chain in the
place of one or more nucleotide units, including either sugar
and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic activity. The group or compound can be
abasic in that it does not contain a commonly recognized nucleotide
base, such as adenosine, guanine, cytosine, uracil or thymine.
Thus, in a preferred embodiment, the invention features an
enzymatic nucleic acid molecule having one or more non-nucleotide
moieties, and having enzymatic activity to cleave an RNA or DNA
molecule.
[0430] The term "patient" as used herein, refers to an organism,
which is a donor or recipient of explanted cells or the cells
themselves. "Patient" also refers to an organism to which the
nucleic acid molecules of the invention can be administered.
Preferably, a patient is a mammal or mammalian cells. More
preferably, a patient is a human or human cells.
[0431] The term "enhanced enzymatic activity" as used herein,
includes activity measured in cells and/or in vivo where the
activity is a reflection of both the catalytic activity and the
stability of the nucleic acid molecules of the invention. In this
invention, the product of these properties can be increased in vivo
compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In
some cases, the activity or stability of the nucleic acid molecule
can be decreased (i.e., less than ten-fold), but the overall
activity of the nucleic acid molecule is enhanced, in vivo.
[0432] By "comprising" is meant including, but not limited to,
whatever follows the word "comprising". Thus, use of the term
"comprising" indicates that the listed elements are required or
mandatory, but that other elements are optional and can or can not
be present. By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of". Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements can be present.
[0433] The term "negatively charged molecules" as used herein,
refers to molecules such as nucleic acid molecules (e.g., RNA, DNA,
oligonucleotides, mixed polymers, peptide nucleic acid, and the
like), peptides (e.g., polyaminoacids, polypeptides, proteins and
the like), nucleotides, pharmaceutical and biological compositions,
that have negatively charged groups that can ion-pair with the
positively charged head group of the cationic lipids of the
invention.
[0434] The term "coupling" as used herein, refers to a reaction,
either chemical or enzymatic, in which one atom, moiety, group,
compound or molecule is joined to another atom, moiety, group,
compound or molecule.
[0435] The terms "deprotection" or "deprotecting" as used herein,
refers to the removal of a protecting group.
[0436] The term "alkyl" as used herein refers to a saturated
aliphatic hydrocarbon, including straight-chain, branched-chain
"isoalkyl", and cyclic alkyl groups. The term "alkyl" also
comprises alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl,
alkylamino, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl,
cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl,
aryl or substituted aryl groups. Preferably, the alkyl group has 1
to 12 carbons. More preferably it is a lower alkyl of from about 1
to about 7 carbons, more preferably about 1 to about 4 carbons. The
alkyl group can be substituted or unsubstituted. When substituted
the substituted group(s) preferably comprise hydroxy, oxy, thio,
amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,
alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy,
cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl,
heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The
term "alkyl" also includes alkenyl groups containing at least one
carbon-carbon double bond, including straight-chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group
has about 2 to about 12 carbons. More preferably it is a lower
alkenyl of from about 2 to about 7 carbons, more preferably about 2
to about 4 carbons. The alkenyl group can be substituted or
unsubstituted. When substituted the substituted group(s) preferably
comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,
alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl,
alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl,
cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl,
aryl or substituted aryl groups. The term "alkyl" also includes
alkynyl groups containing at least one carbon-carbon triple bond,
including straight-chain, branched-chain, and cyclic groups.
Preferably, the alkynyl group has about 2 to about 12 carbons. More
preferably it is a lower alkynyl of from about 2 to about 7
carbons, more preferably about 2 to about 4 carbons. The alkynyl
group can be substituted or unsubstituted. When substituted the
substituted group(s) preferably comprise hydroxy, oxy, thio, amino,
nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl,
alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,
cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6
hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or
moieties of the invention can also include aryl, alkylaryl,
carbocyclic aryl, heterocyclic aryl, amide and ester groups. The
preferred substituent(s) of aryl groups are halogen, trihalomethyl,
hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino
groups. An "alkylaryl" group refers to an alkyl group (as described
above) covalently joined to an aryl group (as described above).
Carbocyclic aryl groups are groups wherein the ring atoms on the
aromatic ring are all carbon atoms. The carbon atoms are optionally
substituted. Heterocyclic aryl groups are groups having from about
1 to about 3 heteroatoms as ring atoms in the aromatic ring and the
remainder of the ring atoms are carbon atoms. Suitable heteroatoms
include oxygen, sulfur, and nitrogen, and include furanyl, thienyl,
pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl,
imidazolyl and the like, all optionally substituted. An "amide"
refers to an --C(O)--NH--R, where R is either alkyl, aryl,
alkylaryl or hydrogen. An "ester" refers to an --C(O)--OR', where R
is either alkyl, aryl, alkylaryl or hydrogen.
[0437] The term "alkoxyalkyl" as used herein refers to an
alkyl-O-alkyl ether, for example, methoxyethyl or ethoxymethyl.
[0438] The term "alkyl-thio-alkyl" as used herein refers to an
alkyl-5-alkyl thioether, for example, methylthiomethyl or
methylthioethyl.
[0439] The term "amino" as used herein refers to a nitrogen
containing group as is known in the art derived from ammonia by the
replacement of one or more hydrogen radicals by organic radicals.
For example, the terms "aminoacyl" and "aminoalkyl" refer to
specific N-substituted organic radicals with acyl and alkyl
substituent groups respectively.
[0440] The term "amination" as used herein refers to a process in
which an amino group or substituted amine is introduced into an
organic molecule.
[0441] The term "exocyclic amine protecting moiety" as used herein
refers to a nucleobase amino protecting group compatible with
oligonucleotide synthesis, for example, an acyl or amide group.
[0442] The term "alkenyl" as used herein refers to a straight or
branched hydrocarbon of a designed number of carbon atoms
containing at least one carbon-carbon double bond. Examples of
"alkenyl" include vinyl, allyl, and 2-methyl-3-heptene.
[0443] The term "alkoxy" as used herein refers to an alkyl group of
indicated number of carbon atoms attached to the parent molecular
moiety through an oxygen bridge. Examples of alkoxy groups include,
for example, methoxy, ethoxy, propoxy and isopropoxy.
[0444] The term "alkynyl" as used herein refers to a straight or
branched hydrocarbon of a designed number of carbon atoms
containing at least one carbon-carbon triple bond. Examples of
"alkynyl" include propargyl, propyne, and 3-hexyne.
[0445] The term "aryl" as used herein refers to an aromatic
hydrocarbon ring system containing at least one aromatic ring. The
aromatic ring can optionally be fused or otherwise attached to
other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings.
Examples of aryl groups include, for example, phenyl, naphthyl,
1,2,3,4-tetrahydronaphthalene and biphenyl. Preferred examples of
aryl groups include phenyl and naphthyl.
[0446] The term "cycloalkenyl" as used herein refers to a C3-C8
cyclic hydrocarbon containing at least one carbon-carbon double
bond. Examples of cycloalkenyl include cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene,
cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
[0447] The term "cycloalkyl" as used herein refers to a C3-C8
cyclic hydrocarbon. Examples of cycloalkyl include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl.
[0448] The term "cycloalkylalkyl," as used herein, refers to a
C3-C7 cycloalkyl group attached to the parent molecular moiety
through an alkyl group, as defined above. Examples of
cycloalkylalkyl groups include cyclopropylmethyl and
cyclopentylethyl.
[0449] The terms "halogen" or "halo" as used herein refers to
indicate fluorine, chlorine, bromine, and iodine.
[0450] The term "heterocycloalkyl," as used herein refers to a
non-aromatic ring system containing at least one heteroatom
selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl
ring can be optionally fused to or otherwise attached to other
heterocycloalkyl rings and/or non-aromatic hydrocarbon rings.
Preferred heterocycloalkyl groups have from 3 to 7 members.
Examples of heterocycloalkyl groups include, for example,
piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine,
and pyrazole. Preferred heterocycloalkyl groups include
piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.
[0451] The term "heteroaryl" as used herein refers to an aromatic
ring system containing at least one heteroatom selected from
nitrogen, oxygen, and sulfur. The heteroaryl ring can be fused or
otherwise attached to one or more heteroaryl rings, aromatic or
non-aromatic hydrocarbon rings or heterocycloalkyl rings. Examples
of heteroaryl groups include, for example, pyridine, furan,
thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred
examples of heteroaryl groups include thienyl, benzothienyl,
pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,
benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,
isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,
tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.
[0452] The term "C1-C6 hydrocarbyl" as used herein refers to
straight, branched, or cyclic alkyl groups having 1-6 carbon atoms,
optionally containing one or more carbon-carbon double or triple
bonds. Examples of hydrocarbyl groups include, for example, methyl,
ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl,
2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl,
3-methylpentyl, vinyl, 2-pentene, cyclopropylmethyl, cyclopropyl,
cyclohexylmethyl, cyclohexyl and propargyl. When reference is made
herein to C1-C6 hydrocarbyl containing one or two double or triple
bonds it is understood that at least two carbons are present in the
alkyl for one double or triple bond, and at least four carbons for
two double or triple bonds.
[0453] The term "protecting group" as used herein, refers to groups
known in the art that are readily introduced and removed from an
atom, for example O, N, P, or S. Protecting groups are used to
prevent undesirable reactions from taking place that can compete
with the formation of a specific compound or intermediate of
interest. See also "Protective Groups in Organic Synthesis", 3rd
Ed., 1999, Greene, T. W. and related publications.
[0454] The term "nitrogen protecting group," as used herein, refers
to groups known in the art that are readily introduced on to and
removed from a nitrogen. Examples of nitrogen protecting groups
include Boc, Cbz, benzoyl, and benzyl. See also "Protective Groups
in Organic Synthesis", 3rd Ed., 1999, Greene, T. W. and related
publications.
[0455] The term "hydroxy protecting group," or "hydroxy protection"
as used herein, refers to groups known in the art that are readily
introduced on to and removed from an oxygen, specifically an --OH
group. Examples of hyroxy protecting groups include trityl or
substituted trityl groups, such as monomethoxytrityl and
dimethoxytrityl, or substituted silyl groups, such as
tert-butyldimethyl, trimethylsilyl, or tert-butyldiphenyl silyl
groups. See also "Protective Groups in Organic Synthesis", 3rd Ed.,
1999, Greene, T. W. and related publications.
[0456] The term "acyl" as used herein refers to --C(O)R groups,
wherein R is an alkyl or aryl.
[0457] The term "phosphorus containing group" as used herein,
refers to a chemical group containing a phosphorus atom. The
phosphorus atom can be trivalent or pentavalent, and can be
substituted with O, H, N, S, C or halogen atoms. Examples of
phosphorus containing groups of the instant invention include but
are not limited to phosphorus atoms substituted with O, H, N, S, C
or halogen atoms, comprising phosphonate, alkylphosphonate,
phosphate, diphosphate, triphosphate, pyrophosphate,
phosphorothioate, phosphorodithioate, phosphoramidate,
phosphoramidite groups, nucleotides and nucleic acid molecules.
[0458] The term "phosphine" or "phosphite" as used herein refers to
a trivalent phosphorus species, for example compounds having
Formula 97:
##STR00193##
[0459] wherein R can include the groups:
##STR00194##
[0460] and wherein S and T independently include the groups:
##STR00195##
[0461] The term "phosphate" as used herein refers to a pentavalent
phosphorus species, for example a compound having Formula 98:
##STR00196##
[0462] wherein R includes the groups:
##STR00197##
[0463] and wherein S and T each independently can be a sulfur or
oxygen atom or a group which can include:
##STR00198##
[0464] and wherein M comprises a sulfur or oxygen atom. The
phosphate of the invention can comprise a nucleotide phosphate,
wherein any R, S, or T in Formula 98 comprises a linkage to a
nucleic acid or nucleoside.
[0465] The term "cationic salt" as used herein refers to any
organic or inorganic salt having a net positive charge, for example
a triethylammonium (TEA) salt.
[0466] The term "degradable linker" as used herein, refers to
linker moieties that are capable of cleavage under various
conditions. Conditions suitable for cleavage can include but are
not limited to pH, UV irradiation, enzymatic activity, temperature,
hydrolysis, elimination, and substitution reactions, and
thermodynamic properties of the linkage.
[0467] The term "photolabile linker" as used herein, refers to
linker moieties as are known in the art, that are selectively
cleaved under particular UV wavelengths. Compounds of the invention
containing photolabile linkers can be used to deliver compounds to
a target cell or tissue of interest, and can be subsequently
released in the presence of a UV source.
[0468] The term "nucleic acid conjugates" as used herein, refers to
nucleoside, nucleotide and oligonucleotide conjugates.
[0469] The term "lipid" as used herein, refers to any lipophilic
compound. Non-limiting examples of lipid compounds include fatty
acids and their derivatives, including straight chain, branched
chain, saturated and unsaturated fatty acids, carotenoids,
terpenes, bile acids, and steroids, including cholesterol and
derivatives or analogs thereof.
[0470] The term "folate" as used herein, refers to analogs and
derivatives of folic acid, for example antifolates, dihydrofloates,
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.
[0471] The term "compounds with neutral charge" as used herein,
refers to compositions which are neutral or uncharged at neutral or
physiological pH. Examples of such compounds are cholesterol and
other steroids, cholesteryl hemisuccinate (CHEMS), dioleoyl
phosphatidyl choline, distearoylphosphotidyl choline (DSPC), fatty
acids such as oleic acid, phosphatidic acid and its derivatives,
phosphatidyl serine, polyethylene glycol-conjugated
phosphatidylamine, phosphatidylcholine, phosphatidylethanolamine
and related variants, prenylated compounds including farnesol,
polyprenols, tocopherol, and their modified forms, diacylsuccinyl
glycerols, fusogenic or pore forming peptides,
dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.
[0472] The term "lipid aggregate" as used herein refers to a
lipid-containing composition wherein the lipid is in the form of a
liposome, micelle (non-lamellar phase) or other aggregates with one
or more lipids.
[0473] The term "biological system" as used herein, refers to a
eukaryotic system or a prokaryotic system, can be a bacterial cell,
plant cell or a mammalian cell, or can be of plant origin,
mammalian origin, yeast origin, Drosophila origin, or
archebacterial origin.
[0474] The term "systemic administration" as used herein refers to
the in vivo systemic absorption or accumulation of drugs in the
blood stream followed by distribution throughout the entire body.
Administration routes which lead to systemic absorption include,
without limitations: intravenous, subcutaneous, intraperitoneal,
inhalation, oral, intrapulmonary and intramuscular. Each of these
administration routes expose the desired negatively charged
polymers, e.g., nucleic acids, to an accessible diseased tissue.
The rate of entry of a drug into the circulation has been shown to
be a function of molecular weight or size. The use of a liposome or
other drug carrier comprising the compounds of the instant
invention can potentially localize the drug, for example, in
certain tissue types, such as the tissues of the reticular
endothelial system (RES). A liposome formulation which can
facilitate the association of drug with the surface of cells, such
as, lymphocytes and macrophages is also useful. This approach can
provide enhanced delivery of the drug to target cells by taking
advantage of the specificity of macrophage and lymphocyte immune
recognition of abnormal cells, such as the cancer cells.
[0475] The term "pharmacological composition" or "pharmaceutical
formulation" refers to a composition or formulation in a form
suitable for administration, for example, systemic administration,
into a cell or patient, preferably a human. Suitable forms, in
part, depend upon the use or the route of entry, for example oral,
transdermal, or by injection. Such forms should not prevent the
composition or formulation to reach a target cell (i.e., a cell to
which the negatively charged polymer is targeted).
[0476] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0477] The drawings will be first described briefly.
DRAWINGS
[0478] FIG. 1 shows examples of chemically stabilized ribozyme
motifs. HH Rz, represents hammerhead ribozyme motif (Usman et al.,
1996, Curr. Op. Struct. Bio., 1, 527); NCH Rz represents the NCH
ribozyme motif (Ludwig & Sproat, International PCT Publication
No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif
(Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, Eckstein
et al., International PCT publication No. WO 99/16871). N or n,
represent independently a nucleotide which can be same or different
and have complementarity to each other; rI, represents ribo-Inosine
nucleotide; arrow indicates the site of cleavage within the target.
Position 4 of the HH Rz and the NCH Rz is shown as having
2'-C-allyl modification, but those skilled in the art will
recognize that this position can be modified with other
modifications well known in the art, so long as such modifications
do not significantly inhibit the activity of the ribozyme.
[0479] FIG. 2 shows an example of the Amberzyme ribozyme motif that
is chemically stabilized (see for example Beigelman et al.,
International PCT publication No. WO 99/55857).
[0480] FIG. 3 shows an example of the Zinzyme A ribozyme motif that
is chemically stabilized (see for example Beigelman et al.,
Beigelman et al., International PCT publication No. WO
99/55857).
[0481] FIG. 4 shows an example of a DNAzyme motif described by
Santoro et al., 1997, PNAS, 94, 4262.
[0482] FIG. 5 shows a synthetic scheme for the synthesis of a
folate conjugate of the instant invention.
[0483] FIG. 6 shows representative examples of fludarabine-folate
conjugate molecules of the invention.
[0484] FIG. 7 shows a synthetic scheme for post-synthetic
modification of a nucleic acid molecule to produce a folate
conjugate.
[0485] FIG. 8 shows a synthetic scheme for generating a protected
pteroic acid synthon of the invention.
[0486] FIG. 9 shows a synthetic scheme for generating a
2-dithiopyridyl activated folic acid synthon of the invention.
[0487] FIG. 10 shows a synthetic scheme for generating an
oligonucleotide or nucleic acid-folate conjugate.
[0488] FIG. 11 shows an alternative synthetic scheme for generating
an oligonucleotide or nucleic acid-folate conjugate.
[0489] FIG. 12 shows an alternative synthetic scheme for
post-synthetic modification of a nucleic acid molecule to produce a
folate conjugate.
[0490] FIG. 13 shows a non-limiting example of a synthetic scheme
for the synthesis of a N-acetyl-D-galactosamine-2'-aminouridine
phosphoramidite conjugate of the invention.
[0491] FIG. 14 shows a non-limiting example of a synthetic scheme
for the synthesis of a N-acetyl-D-galactosamine-D-threoninol
phosphoramidite conjugate of the invention.
[0492] FIG. 15 shows a non-limiting example of a
N-acetyl-D-galactosamine siNA nucleic acid conjugate and a
N-acetyl-D-galactosamine enzymatic nucleic acid conjugate of the
invention. W shown in the example refers to a biodegradable linker,
for example a nucleic acid dimer, trimer, or tetramer comprising
ribonucleotides and/or deoxyribonucleotides. The siNA can be
conjugated at the 3', 5' or both 3' and 5' ends of the sense strand
of a double stranded siNA and/or the 3'-end of the antisense strand
of the siNA. A single stranded siNA molecule can be conjugated at
the 3'-end of the siNA.
[0493] FIG. 16 shows a non-limiting example of a synthetic scheme
for the synthesis of a dodecanoic acid derived conjugate linker of
the invention.
[0494] FIG. 17 shows a non-limiting example of a synthetic scheme
for the synthesis of an oxime linked nucleic acid/peptide conjugate
of the invention.
[0495] FIG. 18 shows non-limiting examples of phospholipid derived
nucleic acid conjugates of the invention. W shown in the examples
refers to a biodegradable linker, for example a nucleic acid dimer,
trimer, or tetramer comprising ribonucleotides and/or
deoxyribonucleotides. The siNA can be conjugated at the 3', 5' or
both 3' and 5' ends of the sense strand of a double stranded siNA
and/or the 3'-end of the antisense strand of the siNA. A single
stranded siNA molecule can be conjugated at the 3'-end of the
siNA.
[0496] FIG. 19 shows a non-limiting example of a synthetic scheme
for preparing a phospholipid derived siNA conjugates of the
invention.
[0497] FIG. 20 shows a non-limiting example of a synthetic scheme
for preparing a polyethylene glycol (PEG) derived enzymatic nucleic
acid conjugates of the invention.
[0498] FIG. 21 shows PK data of a 40K PEG conjugated enzymatic
nucleic acid molecule compared to the corresponding non-conjugated
enzymatic nucleic acid molecule. The graph is a time course of
serum concentration in mice dosed with 30 mg/kg of Angiozyme.TM. or
40-kDa-PEG-Angiozyme.TM.. The hybridization method was used to
quantitate Angiozyme.TM. levels.
[0499] FIG. 22 shows PK data of a phospholipid conjugated enzymatic
nucleic acid molecule compared to the corresponding non-conjugated
enzymatic nucleic acid molecule.
[0500] FIG. 23 shows a non-limiting example of a synthetic scheme
for preparing a poly-N-acetyl-D-galactosamine nucleic acid
conjugate of the invention.
[0501] FIG. 24a-b shows a non-limiting example of a synthetic
approach for synthesizing peptide or protein conjugates to PEG
utilizing a biodegradable linker using oxime and morpholino
linkages.
[0502] FIG. 25 shows a non-limiting example of a synthetic approach
for synthesizing peptide or protein conjugates to PEG utilizing a
biodegradable linker using oxime and phosphoramidate linkages
[0503] FIG. 26a-b shows a non-limiting example of a synthetic
approach for synthesizing peptide or protein conjugates to PEG
utilizing a biodegradable linker using phosphoramidate
linkages.
[0504] FIG. 27 shows non-limiting examples of phospholipid derived
protein/peptide conjugates of the invention. W shown in the
examples refers to a biodegradable linker, for example a nucleic
acid dimer, trimer, or tetramer comprising ribonucleotides and/or
deoxyribonucleotides.
[0505] FIG. 28 shows a non-limiting example of an
N-acetyl-D-galactosamine peptide/protein conjugate of the
invention, the example shown is with a peptide. W shown in the
example refers to a biodegradable linker, for example a nucleic
acid dimer, trimer, or tetramer comprising ribonucleotides and/or
deoxyribonucleotides.
[0506] FIG. 29 shows a non-limiting example of a synthetic approach
for synthesizing peptide or protein conjugates to PEG utilizing a
biodegradable linker using phosphoramidate linkages via coupling a
protein phosphoramidite to a PEG conjugated nucleic acid
linker.
[0507] FIG. 30 shows a non-limiting example of the synthesis of
siNA cholesterol conjugates of the invention using a
phosphoramidite approach.
[0508] FIG. 31 shows a non-limiting example of the synthesis of
siNA PEG conjugates of the invention using NHS ester coupling.
[0509] FIG. 32 shows a non-limiting example of the synthesis of
siNA cholesterol conjugates of the invention using NHS ester
coupling.
[0510] FIG. 33 shows a non-limiting example of various siNA
cholesterol conjugates of the invention.
[0511] FIG. 34 shows a non-limiting example of various siNA
cholesterol conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a double stranded siNA molecule.
[0512] FIG. 35 shows a non-limiting example of various siNA
cholesterol conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a double stranded siNA molecule.
[0513] FIG. 36 shows a non-limiting example of various siNA
cholesterol conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a single stranded siNA molecule.
[0514] FIG. 37 shows a non-limiting example of various siNA
phospholipid conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a double stranded siNA molecule.
[0515] FIG. 38 shows a non-limiting example of various siNA
phospholipid conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a single stranded siNA molecule.
[0516] FIG. 39 shows a non-limiting example of various siNA
galactosamine conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a double stranded siNA molecule.
[0517] FIG. 40 shows a non-limiting example of various siNA
galactosamine conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a single stranded siNA molecule.
[0518] FIG. 41 shows a non-limiting example of various generalized
siNA conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a double stranded siNA molecule. CONJ in the figure
refers to any biologically active compound or any other conjugate
compound as described herein and in the Formulae herein.
[0519] FIG. 42 shows a non-limiting example of various generalized
siNA conjugates of the invention in which various linker
chemistries and/or cleavable linkers can be utilized at different
positions of a single stranded siNA molecule. CONJ in the figure
refers to any biologically active compound or any other conjugate
compound as described herein and in the Formulae herein.
[0520] FIG. 43 shows a non-limiting example of the pharmacokinetic
distribution of intact siNA in liver after administration of
conjugated or unconjugated siNA molecules in mice.
[0521] FIG. 44 shows a non-limiting example of the activity of
conjugated siNA constructs compared to matched chemistry
unconjugated siNA constructs in an HBV cell culture system without
the use of transfection lipid. As shown in the Figure, siNA
conjugates provide efficacy in cell culture without the need for
transfection reagent.
[0522] FIG. 45 shows a non-limiting example of a scheme for the
synthesis of a mono galactosamine phosphoramidite of the invention
that can be used to generate galactosamine conjugated nucleic acid
molecules.
[0523] FIG. 46 shows a non-limiting example of a scheme for the
synthesis of a tri-galactosamine phosphoramidite of the invention
that can be used to generate tri-galactosamine conjugated nucleic
acid molecules.
[0524] FIG. 47 shows a non-limiting example of a scheme for the
synthesis of another tri-galactosamine phosphoramidite of the
invention that can be used to generate tri-galactosamine conjugated
nucleic acid molecules.
[0525] FIG. 48 shows a non-limiting example of an alternate scheme
for the synthesis of a tri-galactosamine phosphoramidite of the
invention that can be used to generate tri-galactosamine conjugated
nucleic acid molecules.
[0526] FIG. 49 shows a non-limiting example of a scheme for the
synthesis of a cholesterol NHS ester of the invention that can be
used to generate cholesterol conjugated nucleic acid molecules.
METHOD OF USE
[0527] The compositions and conjugates of the instant invention can
be used to administer pharmaceutical agents. Pharmaceutical agents
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state in
a patient.
[0528] Generally, the compounds of the instant invention are
introduced by any standard means, with or without stabilizers,
buffers, and the like, to form a pharmaceutical composition. For
use of a liposome delivery mechanism, standard protocols for
formation of liposomes can be followed. The compositions of the
present invention can also be formulated and used as tablets,
capsules or elixirs for oral administration; suppositories for
rectal administration; sterile solutions; suspensions for
injectable administration; and the like.
[0529] The present invention also includes pharmaceutically
acceptable formulations of the compounds described above,
preferably in combination with the molecule(s) to be delivered.
These formulations include salts of the above compounds, e.g., acid
addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and benzene sulfonic acid.
[0530] In one embodiment, the invention features the use of the
compounds of the invention in a composition comprising
surface-modified liposomes containing poly (ethylene glycol) lipids
(PEG-modified, or long-circulating liposomes or stealth liposomes).
In another embodiment, the invention features the use of compounds
of the invention covalently attached to polyethylene glycol. These
formulations offer a method for increasing the accumulation of
drugs in target tissues. This class of drug carriers resists
opsonization and elimination by the mononuclear phagocytic system
(MPS or RES), thereby enabling longer blood circulation times and
enhanced tissue exposure for the encapsulated drug (Lasic et al.
Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull.
1995, 43, 1005-1011). Such compositions have been shown to
accumulate selectively in tumors, presumably by extravasation and
capture in the neovascularized target tissues (Lasic et al.,
Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys.
Acta, 1238, 86-90). The long-circulating compositions enhance the
pharmacokinetics and pharmacodynamics of therapeutic compounds,
such as DNA and RNA, particularly compared to conventional cationic
liposomes which are known to accumulate in tissues of the MPS (Liu
et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al.,
International PCT Publication No. WO 96/10391; Ansell et al.,
International PCT Publication No. WO 96/10390; Holland et al.,
International PCT Publication No. WO 96/10392). Long-circulating
compositions are also likely to protect drugs from nuclease
degradation to a greater extent compared to cationic liposomes,
based on their ability to avoid accumulation in metabolically
aggressive MPS tissues such as the liver and spleen.
[0531] The present invention also includes a composition(s)
prepared for storage or administration that includes a
pharmaceutically effective amount of the desired compound(s) in a
pharmaceutically acceptable carrier or diluent. Acceptable carriers
or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985) hereby incorporated by reference herein. For example,
preservatives, stabilizers, dyes and flavoring agents can be
included in the composition. Examples of such agents include but
are not limited to sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. In addition, antioxidants and suspending
agents can be included in the composition.
[0532] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors which those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the negatively charged polymer. Furthermore, the
compounds of the invention and formulations thereof can be
administered to a fetus via administration to the mother of a
fetus.
[0533] The compounds of the invention and formulations thereof can
be administered orally, topically, parenterally, by inhalation or
spray or rectally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and vehicles. The term parenteral as used herein includes
percutaneous, subcutaneous, intravascular (e.g., intravenous),
intramuscular, or intrathecal injection or infusion techniques and
the like. In addition, there is provided a pharmaceutical
formulation comprising a nucleic acid molecule of the invention and
a pharmaceutically acceptable carrier. One or more nucleic acid
molecules of the invention can be present in association with one
or more non-toxic pharmaceutically acceptable carriers and/or
diluents and/or adjuvants, and if desired other active ingredients.
The pharmaceutical compositions containing nucleic acid molecules
of the invention can be in a form suitable for oral use, for
example, as tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsion, hard or
soft capsules, or syrups or elixirs.
[0534] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be, for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate can be
employed.
[0535] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0536] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0537] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0538] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0539] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example, sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0540] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0541] The compounds of the invention can also be administered in
the form of suppositories, e.g., for rectal administration of the
drug. These compositions can be prepared by mixing the drug with a
suitable non-irritating excipient that is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols.
[0542] Compounds of the invention can be administered parenterally
in a sterile medium. The drug, depending on the vehicle and
concentration used, can either be suspended or dissolved in the
vehicle. Advantageously, adjuvants such as local anesthetics,
preservatives and buffering agents can be dissolved in the
vehicle.
[0543] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
patient per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. Dosage unit forms will generally contain between
from about 1 mg to about 500 mg of an active ingredient.
[0544] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the particular disease undergoing therapy.
[0545] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0546] The compounds of the present invention can also be
administered to a patient in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
Synthesis of Nucleic acid Molecules
[0547] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
nucleic acid motifs ("small refers to nucleic acid motifs less than
about 100 nucleotides in length, preferably less than about 80
nucleotides in length, and more preferably less than about 50
nucleotides in length; e.g., antisense oligonucleotides, hammerhead
or the NCH ribozymes) are preferably used for exogenous delivery.
The simple structure of these molecules increases the ability of
the nucleic acid to invade targeted regions of RNA structure.
Exemplary molecules of the instant invention are chemically
synthesized, and others can similarly be synthesized.
[0548] Oligonucleotides (eg; antisense GeneBlocs) are synthesized
using protocols known in the art as described in Caruthers et al.,
1992, Methods in Enzymology 211, 3-19, Thompson et al.,
International PCT Publication No. WO 99/54459, Wincott et al.,
1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997,
Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol
Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of
these references are incorporated herein by reference. The
synthesis of oligonucleotides makes use of common nucleic acid
protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end. In a non-limiting
example, small scale syntheses are conducted on a 394 Applied
Biosystems, Inc. synthesizer using a 0.2 mmol scale protocol with a
2.5 min coupling step for 2'-O-methylated nucleotides and a 45 sec
coupling step for 2'-deoxy nucleotides. Table II outlines the
amounts and the contact times of the reagents used in the synthesis
cycle. Alternatively, syntheses at the 0.2 .mu.mol scale can be
performed on a 96-well plate synthesizer, such as the instrument
produced by Protogene (Palo Alto, Calif.) with minimal modification
to the cycle. In a non-limiting example, a 33-fold excess (60 .mu.L
of 0.11 M=6.6 .mu.mol) of 2'-O-methyl phosphoramidite and a
105-fold excess of S-ethyl tetrazole (60 .mu.L of 0.25 M=15 mmol)
can be used in each coupling cycle of 2'-O-methyl residues relative
to polymer-bound 5'-hydroxyl. In a non-limiting example, a 22-fold
excess (40 .mu.L of 0.11 M=4.4 .mu.mol) of deoxy phosphoramidite
and a 70-fold excess of S-ethyl tetrazole (40 .mu.L of 0.25 M=10
.mu.mol) can be used in each coupling cycle of deoxy residues
relative to polymer-bound 5'-hydroxyl. Average coupling yields on
the 394 Applied Biosystems, Inc. synthesizer, determined by
colorimetric quantitation of the trityl fractions, are typically
97.5-99%. Other oligonucleotide synthesis reagents for the 394
Applied Biosystems, Inc. synthesizer include but are not limited
to; detritylation solution is 3% TCA in methylene chloride (ABI);
capping is performed with 16% N-methyl imidazole in THF (ABI) and
10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation
solution is 16.9 mM I.sub.2, 49 mM pyridine, 9% water in THF
(PERSEPTIVE.TM.). Burdick & Jackson Synthesis Grade
acetonitrile is used directly from the reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from
the solid obtained from American International Chemical, Inc.
Alternately, for the introduction of phosphorothioate linkages,
Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in
acetonitrile) is used.
[0549] Deprotection of the antisense oligonucleotides is performed
as follows: the polymer-bound trityl-on oligoribonucleotide is
transferred to a 4 mL glass screw top vial and suspended in a
solution of 40% aq. methylamine (1 mL) at 65.degree. C. for 10 min.
After cooling to -20.degree. C., the supernatant is removed from
the polymer support. The support is washed three times with 1.0 mL
of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added
to the first supernatant. The combined supernatants, containing the
oligoribonucleotide, are dried to a white powder. Standard drying
or lyophilization methods known to those skilled in the art can be
used.
[0550] The method of synthesis used for normal RNA including
certain enzymatic nucleic acid molecules follows the procedure as
described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845;
Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et
al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997,
Methods Mol. Bio., 74, 59, and makes use of common nucleic acid
protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end. In a non-limiting
example, small scale syntheses are conducted on a 394 Applied
Biosystems, Inc. synthesizer using a 0.2 .mu.mol scale protocol
with a 7.5 min coupling step for alkylsilyl protected nucleotides
and a 2.5 min coupling step for 2'-O-methylated nucleotides. Table
II outlines the amounts and the contact times of the reagents used
in the synthesis cycle. Alternatively, syntheses at the 0.2 .mu.mol
scale can be done on a 96-well plate synthesizer, such as the
instrument produced by Protogene (Palo Alto, Calif.) with minimal
modification to the cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6
.mu.mol) of 2'-O-methyl phosphoramidite and a 75-fold excess of
S-ethyl tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in
each coupling cycle of 2'-O-methyl residues relative to
polymer-bound 5'-hydroxyl. A 66-fold excess (120 .mu.L of 0.11
M=13.2 .mu.mol) of alkylsilyl (ribo) protected phosphoramidite and
a 150-fold excess of S-ethyl tetrazole (120 .mu.L of 0.25 M=30
.mu.mol) can be used in each coupling cycle of ribo residues
relative to polymer-bound 5'-hydroxyl. Average coupling yields on
the 394 Applied Biosystems, Inc. synthesizer, determined by
colorimetric quantitation of the trityl fractions, are typically
97.5-99%. Other oligonucleotide synthesis reagents for the 394
Applied Biosystems, Inc. synthesizer include; detritylation
solution is 3% TCA in methylene chloride (ABI); capping is
performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic
anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9
mM I.sub.2, 49 mM pyridine, 9% water in THF (PERSEPTIVE.TM.).
Burdick & Jackson Synthesis Grade acetonitrile is used directly
from the reagent bottle. S-Ethyltetrazole solution (0.25 M in
acetonitrile) is made up from the solid obtained from American
International Chemical, Inc. Alternately, for the introduction of
phosphorothioate linkages, Beaucage reagent
(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is
used.
[0551] Deprotection of the RNA is performed using either a two-pot
or one-pot protocol. For the two-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw
top vial and suspended in a solution of 40% aq. methylamine (1 mL)
at 65.degree. C. for 10 min. After cooling to -20.degree. C., the
supernatant is removed from the polymer support. The support is
washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and
the supernatant is then added to the first supernatant. The
combined supernatants, containing the oligoribonucleotide, are
dried to a white powder. The base deprotected oligoribonucleotide
is resuspended in anhydrous TEA/HF/NMP solution (300 .mu.L of a
solution of 1.5 mL N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL
TEA.3HF to provide a 1.4 M HF concentration) and heated to
65.degree. C. After 1.5 h, the oligomer is quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0552] Alternatively, for the one-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw
top vial and suspended in a solution of 33% ethanolic
methylamine/DMSO: 1/1 (0.8 mL) at 65.degree. C. for 15 min. The
vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is
heated at 65.degree. C. for 15 min. The sample is cooled at
-20.degree. C. and then quenched with 1.5 M NH.sub.4HCO.sub.3.
[0553] For purification of the trityl-on oligomers, the quenched
NH.sub.4HCO.sub.3 solution is loaded onto a C-18 containing
cartridge that had been prewashed with acetonitrile followed by 50
mM TEAA. After washing the loaded cartridge with water, the RNA is
detritylated with 0.5% TFA for 13 min. The cartridge is then washed
again with water, salt exchanged with 1 M NaCl and washed with
water again. The oligonucleotide is then eluted with 30%
acetonitrile.
[0554] Inactive hammerhead ribozymes or binding attenuated control
((BAC) oligonucleotides) are synthesized by substituting a U for
G.sub.5 and a U for A.sub.14 (numbering from Hertel, K. J., et al.,
1992, Nucleic Acids Res., 20, 3252). Similarly, one or more
nucleotide substitutions can be introduced in other enzymatic
nucleic acid molecules to inactivate the molecule and such
molecules can serve as a negative control.
[0555] The average stepwise coupling yields are typically >98%
(Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of
ordinary skill in the art will recognize that the scale of
synthesis can be adapted to be larger or smaller than the example
described above including, but not limited to, 96 well format, with
the ratio of chemicals used in the reaction being adjusted
accordingly.
[0556] Alternatively, the nucleic acid molecules of the present
invention can be synthesized separately and joined together
post-synthetically, for example by ligation (Moore et al., 1992,
Science 256, 9923; Draper et al., International PCT publication No.
WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19,
4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951;
Bellon et al., 1997, Bioconjugate Chem. 8, 204).
[0557] The nucleic acid molecules of the present invention are
modified extensively to enhance stability by modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl,
T-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,
163). Ribozymes are purified by gel electrophoresis using general
methods or are purified by high pressure liquid chromatogaphy
(HPLC; See Wincott et al., Supra, the totality of which is hereby
incorporated herein by reference) and are re-suspended in
water.
Optimizing Activity of the Nucleic Acid Molecule of the
Invention.
[0558] Chemically synthesizing nucleic acid molecules with
modifications (base, sugar and/or phosphate) that prevent their
degradation by serum ribonucleases can increase their potency (see
e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science
253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17,
334; Usman et al., International Publication No. WO 93/15187; and
Rossi et al., International Publication No. WO 91/03162; Sproat,
U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
herein). Modifications which enhance their efficacy in cells, and
removal of bases from nucleic acid molecules to shorten
oligonucleotide synthesis times and reduce chemical requirements
are desired. (All these publications are hereby incorporated by
reference herein).
[0559] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H, nucleotide base
modifications (for a review see Usman and Cedergren, 1992, TIBS.
17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163;
Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification
of nucleic acid molecules have been extensively described in the
art (see Eckstein et al., International Publication PCT No. WO
92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.
Science, 1991, 253, 314-317; Usman and Cedergren, Trends in
Biochem. Sci., 1992, 17, 334-339; Usman et al. International
Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711
and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman
et al., International PCT publication No. WO 97/26270; Beigelman et
al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.
5,627,053; Woolf et al., International PCT Publication No. WO
98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed
on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39,
1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences),
48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,
99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010;
all of the references are hereby incorporated in their totality by
reference herein). Such publications describe general methods and
strategies to determine the location of incorporation of sugar,
base and/or phosphate modifications and the like into ribozymes
without inhibiting catalysis, and are incorporated by reference
herein. In view of such teachings, similar modifications can be
used as described herein to modify the nucleic acid molecules of
the instant invention.
[0560] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorothioate,
and/or 5'-methylphosphonate linkages improves stability, too many
of these modifications may cause some toxicity. Therefore, when
designing nucleic acid molecules the amount of these
internucleotide linkages should be minimized. Without being bound
by any particular theory, the reduction in the concentration of
these linkages should lower toxicity resulting in increased
efficacy and higher specificity of these molecules.
[0561] Nucleic acid molecules having chemical modifications that
maintain or enhance activity are provided. Such nucleic acid is
also generally more resistant to nucleases than unmodified nucleic
acid. Thus, in a cell and/or in vivo the activity can not be
significantly lowered. Therapeutic nucleic acid molecules (e.g.,
enzymatic nucleic acid molecules and antisense nucleic acid
molecules) delivered exogenously are optimally stable within cells
until translation of the target RNA has been inhibited long enough
to reduce the levels of the undesirable protein. This period of
time varies between hours to days depending upon the disease state.
The nucleic acid molecules should be resistant to nucleases in
order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of RNA and DNA (Wincott et
al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,
Methods in Enzymology 211, 3-19 (incorporated by reference herein)
have expanded the ability to modify nucleic acid molecules by
introducing nucleotide modifications to enhance their nuclease
stability as described above.
[0562] Use of the nucleic acid-based molecules of the invention can
lead to better treatment of the disease progression by affording
the possibility of combination therapies (e.g., multiple antisense
or enzymatic nucleic acid molecules targeted to different genes,
nucleic acid molecules coupled with known small molecule
inhibitors, or intermittent treatment with combinations of
molecules (including different motifs) and/or other chemical or
biological molecules). The treatment of patients with nucleic acid
molecules can also include combinations of different types of
nucleic acid molecules.
[0563] In another embodiment, nucleic acid catalysts having
chemical modifications that maintain or enhance enzymatic activity
are provided. Such nucleic acids are also generally more resistant
to nucleases than unmodified nucleic acid. Thus, in a cell and/or
in vivo the activity of the nucleic acid can not be significantly
lowered. As exemplified herein such enzymatic nucleic acids are
useful in a cell and/or in vivo even if activity over all is
reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090).
Such enzymatic nucleic acids herein are said to "maintain" the
enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.
[0564] In another aspect the nucleic acid molecules comprise a 5'
and/or a 3'-cap structure.
[0565] In another embodiment the 3'-cap includes, for example
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;
4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0566] In one embodiment, the invention features modified enzymatic
nucleic acid molecules with phosphate backbone modifications
comprising one or more phosphorothioate, phosphorodithioate,
methylphosphonate, morpholino, amidate carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a
review of oligonucleotide backbone modifications see Hunziker and
Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in
Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994,
Novel Backbone Replacements for Oligonucleotides, in Carbohydrate
Modifications in Antisense Research, ACS, 24-39. These references
are hereby incorporated by reference herein.
[0567] In connection with 2'-modified nucleotides as described for
the invention, by "amino" is meant 2'-NH.sub.2 or 2'-O--NH.sub.2,
which can be modified or unmodified. Such modified groups are
described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695
and Matulic-Adamic et al., WO 98/28317, respectively, which are
both incorporated by reference in their entireties.
[0568] Various modifications to nucleic acid (e.g., antisense and
ribozyme) structure can be made to enhance the utility of these
molecules. For example, such modifications can enhance shelf-life,
half-life in vitro, stability, and ease of introduction of such
oligonucleotides to the target site, including e.g., enhancing
penetration of cellular membranes and conferring the ability to
recognize and bind to targeted cells.
[0569] Use of these molecules can lead to better treatment of
disease progression by affording the possibility of combination
therapies (e.g., multiple enzymatic nucleic acid molecules targeted
to different genes, enzymatic nucleic acid molecules coupled with
known small molecule inhibitors, or intermittent treatment with
combinations of enzymatic nucleic acid molecules (including
different enzymatic nucleic acid molecule motifs) and/or other
chemical or biological molecules). The treatment of patients with
nucleic acid molecules can also include combinations of different
types of nucleic acid molecules. Therapies can be devised which
include a mixture of enzymatic nucleic acid molecules (including
different enzymatic nucleic acid molecule motifs), antisense and/or
2-5A chimera molecules to one or more targets to alleviate symptoms
of a disease.
Indications
[0570] Particular disease states that can be treated using
compounds and compositions of the invention include, but are not
limited to, cancers and cancerous conditions such as breast, lung,
prostate, colorectal, brain, esophageal, stomach, bladder,
pancreatic, cervical, hepatocellular, head and neck, and ovarian
cancer, melanoma, lymphoma, glioma, multidrug resistant cancers;
ocular conditions such as macular degeneration and diabetic
retinopathy, and/or viral infections including HIV, HBV, HCV, CMV,
RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus,
severe acute respiratory syndrome (SARS) virus, Ebola virus, foot
and mouth virus, and papilloma virus infection.
[0571] The molecules of the invention can be used in conjunction
with other known methods, therapies, or drugs. For example, the use
of monoclonal antibodies (eg; mAb IMC C225, mAB ABX-EGF) treatment,
tyrosine kinase inhibitors (TKIs), for example OSI-774 and ZD1839,
chemotherapy, and/or radiation therapy, are all non-limiting
examples of a methods that can be combined with or used in
conjunction with the compounds of the instant invention. Common
chemotherapies that can be combined with nucleic acid molecules of
the instant invention include various combinations of cytotoxic
drugs to kill the cancer cells. These drugs include, but are not
limited to, paclitaxel (Taxol), docetaxel, cisplatin, methotrexate,
cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate,
gemcitabine, vinorelbine etc. Those skilled in the art will
recognize that other drug compounds and therapies can be similarly
be readily combined with the compounds of the instant invention are
hence within the scope of the instant invention.
Diagnostic Uses
[0572] The compounds of this invention, for example, nucleic acid
conjugate molecules, can be used as diagnostic tools to examine
genetic drift and mutations within diseased cells or to detect the
presence of a disease related RNA in a cell. The close relationship
between, for example, enzymatic nucleic acid molecule activity and
the structure of the target RNA allows the detection of mutations
in any region of the molecule which alters the base-pairing and
three-dimensional structure of the target RNA. By using multiple
enzymatic nucleic acid molecules conjugates of the invention, one
can map nucleotide changes which are important to RNA structure and
function in vitro, as well as in cells and tissues. Cleavage of
target RNAs with enzymatic nucleic acid molecules can be used to
inhibit gene expression and define the role (essentially) of
specified gene products in the progression of disease. In this
manner, other genetic targets can be defined as important mediators
of the disease. These experiments can lead to better treatment of
the disease progression by affording the possibility of
combinational therapies (e.g., multiple enzymatic nucleic acid
molecules targeted to different genes, enzymatic nucleic acid
molecules coupled with known small molecule inhibitors, or
intermittent treatment with combinations of enzymatic nucleic acid
molecules and/or other chemical or biological molecules). Other in
vitro uses of enzymatic nucleic acid molecules of this invention
are well known in the art, and include detection of the presence of
mRNAs associated with a disease-related condition. Such RNA is
detected by determining the presence of a cleavage product after
treatment with an enzymatic nucleic acid molecule using standard
methodology.
[0573] In a specific example, enzymatic nucleic acid molecules that
are delivered to cells as conjugates and which cleave only
wild-type or mutant forms of the target RNA are used for the assay.
The first enzymatic nucleic acid molecule is used to identify
wild-type RNA present in the sample and the second enzymatic
nucleic acid molecule is used to identify mutant RNA in the sample.
As reaction controls, synthetic substrates of both wild-type and
mutant RNA are cleaved by both enzymatic nucleic acid molecules to
demonstrate the relative enzymatic nucleic acid molecule
efficiencies in the reactions and the absence of cleavage of the
"non-targeted" RNA species. The cleavage products from the
synthetic substrates also serve to generate size markers for the
analysis of wild-type and mutant RNAs in the sample population.
Thus each analysis requires two enzymatic nucleic acid molecules,
two substrates and one unknown sample which is combined into six
reactions. The presence of cleavage products is determined using an
RNAse protection assay so that full-length and cleavage fragments
of each RNA can be analyzed in one lane of a polyacrylamide gel. It
is not absolutely required to quantify the results to gain insight
into the expression of mutant RNAs and putative risk of the desired
phenotypic changes in target cells. The expression of mRNA whose
protein product is implicated in the development of the phenotype
is adequate to establish risk. If probes of comparable specific
activity are used for both transcripts, then a qualitative
comparison of RNA levels will be adequate and will decrease the
cost of the initial diagnosis. Higher mutant form to wild-type
ratios are correlated with higher risk whether RNA levels are
compared qualitatively or quantitatively. The use of enzymatic
nucleic acid molecules in diagnostic applications contemplated by
the instant invention is more fully described in George et al.,
U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No.
5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and
Ellington, International PCT publication No. WO 00/24931, Breaker
et al., International PCT Publication Nos. WO 00/26226 and
98/27104, and Sullenger et al., International PCT publication No.
WO 99/29842.
Additional Uses
[0574] Potential uses of sequence-specific enzymatic nucleic acid
molecules of the instant invention that are delivered to cells as
conjugates can have many of the same applications for the study of
RNA that DNA restriction endonucleases have for the study of DNA
(Nathans et al., 1975 Ann Rev. Biochem. 44:273). For example, the
pattern of restriction fragments can be used to establish sequence
relationships between two related RNAs, and large RNAs can be
specifically cleaved to fragments of a size more useful for study.
The ability to engineer sequence specificity of the enzymatic
nucleic acid molecule is ideal for cleavage of RNAs of unknown
sequence. Applicant has described the use of nucleic acid molecules
to down-regulate gene expression of target genes in bacterial,
microbial, fungal, viral, and eukaryotic systems including plant,
or mammalian cells.
Example 1
Synthesis of
O.sup.1-(4-monomethoxytrityl)-N-(6-(N-(.alpha.-OFm-L-glutamyl)aminocaproy-
l))-D-threoninol-N.sup.2-iBu-N.sup.10-TFA-pteroic acid conjugate
3'-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20) (FIG.
5)
[0575] General. All reactions were carried out under a positive
pressure of argon in anhydrous solvents. Commercially available
reagents and anhydrous solvents were used without further
purification. .sup.1H (400.035 MHz) and .sup.31P (161.947 MHz) NMR
spectra were recorded in CDCl.sub.3, unless stated otherwise, and
chemical shifts in ppm refer to TMS and H.sub.3PO.sub.4,
respectively. Analytical thin-layer chromatography (TLC) was
performed with Merck Art.5554 Kieselgel 60 F.sub.254 plates and
flash column chromatography using Merck 0.040-0.063 mm silica gel
60.
[0576] N--(N-Fmoc-6-aminocaproyl)-D-threoninol (13).
N-Fmoc-6-aminocaproic acid (10 g, 28.30 mmol) was dissolved in DMF
(50 ml) and N-hydroxysuccinimide (3.26 g, 28.30 mmol) and
1,3-dicyclohexylcarbodiimide (5.84 g, 28.3 mmol) were added to the
solution. The reaction mixture was stirred at RT (about 23.degree.
C.) overnight and the precipitated 1,3-dicyclohexylurea filtered
off. To the filtrate D-threoninol (2.98 g, 28.30 mmol) was added
and the reaction mixture stirred at RT overnight. The solution was
reduced to ca half the volume in vacuo, the residue diluted with
about m ml of ethyl acetate and extracted with about x ml of 5%
NaHCO.sub.3, followed by washing with brine. The organic layer was
dried (Na.sub.2SO.sub.4), evaporated to a syrup and chromatographed
by silica gel column chromatography using 1-10% gradient of
methanol in ethyl acetate. Fractions containing the product were
pooled and evaporated to a white solid (9.94 g, 80%). .sup.1H-NMR
(DMSO-d.sub.6-D.sub.2O) 7.97-7.30 (m, 8H, aromatic), 4.34 (d,
J=6.80, 2H, Fm), 4.26 (t, J=6.80, 1H, Fm), 3.9 (m, 1H, H3 Thr),
3.69 (m, 1H, H2 Thr), 3.49 (dd, J=10.6, J=7.0, 1H, H1 Thr), 3.35
(dd, J=10.6, J=6.2, 1H, H1' Thr), 3.01 (m, 2H, CH.sub.2CO Acp),
2.17 (m, 2H, CH.sub.2NH Acp), 1.54 (m, 2H, CH.sub.2 Acp), 1.45 (m,
2H, CH.sub.2 Acp), 1.27 (m, 2H, CH.sub.2 Acp), 1.04 (d, J=6.4, 3H,
CH.sub.3). MS/ESI.sup.+ m/z 441.0 (M+H).sup.+.
[0577]
O.sup.1-(4-Monomethoxytrityl)-N--(N-Fmoc-6-aminocaproyl)-D-threonin-
ol (14). To the solution of 13 (6 g, 13.62 mmol) in dry pyridine
(80 ml) p-anisylchlorodiphenyl-methane (6 g, 19.43 mmol) was added
and the reaction mixture stirred at RT overnight. Methanol was
added (20 ml) and the solution concentrated in vacuo. The residual
syrup was partitioned between about x ml of dichloromethane and
about x ml of 5% NaHCO.sub.3, the organic layer was washed with
brine, dried (Na.sub.2SO.sub.4) and evaporated to dryness. Flash
column chromatography using 1-3% gradient of methanol in
dichloromethane afforded 14 as a white foam (6 g, 62%). .sup.1H-NMR
(DMSO) 7.97-6.94 (m, 22H, aromatic), 4.58 (d, 1H, J=5.2, OH), 4.35
(d, J=6.8, 2H, Fm), 4.27 (t, J=6.8, 1H, Fm), 3.97 (m, 2H, H2, H3
Thr), 3.80 (s, 3H, OCH.sub.3), 3.13 (dd, J=8.4, J=5.6, 1H, H1 Thr),
3.01 (m, 2H, CH.sub.2C0 Acp), 2.92 (m, dd, J=8.4, J=6.4, 1H, H1'
Thr), 2.21 (m, 2H, CH.sub.2NH Acp), 1.57 (m, 2H, CH.sub.2 Acp),
1.46 (m, 2H, CH.sub.2 Acp), 1.30 (m, 2H, CH.sub.2 Acp), 1.02 (d,
J=5.6, 3H, CH.sub.3). MS/ESI.sup.+ m/z 735.5 (M+Na).sup.+.
[0578]
O.sup.1-(4-Monomethoxytrityl)-N-(6-aminocaproyl)-D-threoninol (15).
14 (9.1 g, 12.77 mmol) was dissolved in DMF (100 ml) containing
piperidine (10 ml) and the reaction mixture was kept at RT for
about 1 hour. The solvents were removed in vacuo and the residue
purified by silica gel column chromatography using 1-10% gradient
of methanol in dichloromethane to afford 15 as a syrup (4.46 g,
71%). .sup.1H-NMR 457.48-6.92 (m, 14H, aromatic), 6.16 (d, J=8.8,
1H, NH), 4.17 (m, 1H, H3 Thr), 4.02 (m, 1H, H2 Thr), 3.86 (s, 3H,
OCH.sub.3), 3.50 (dd, J=9.7, J=4.4, 1H, H1 Thr), 3.37 (dd, J=9.7,
J=3.4, 1H, Thr), 2.78 (t, J=6.8, 2H, CH.sub.2CO Acp), 2.33 (t,
J=7.6, 2H, CH.sub.2NH Acp), 1.76 (m, 2H, CH.sub.2 Acp), 1.56 (m,
2H, CH.sub.2 Acp), 1.50 (m, 2H, CH.sub.2 Acp), 1.21 (d, J=6.4, 3H,
CH.sub.3). MS/ESI.sup.+ m/z 491.5 (M+H).sup.+.
[0579]
O.sup.1-(4-Monomethoxytrityl)-N-(6-(N--(N-Boc-.alpha.-OFm-L-glutamy-
l)aminocaproyl))-D-threoninol (16). To the solution of
N-Boc-.alpha.-OFm-glutamic acid (Bachem) (1.91 g, 4.48 mmol) in DMF
(10 ml) N-hydroxysuccinimide (518 mg, 4.50 mmol) and
1,3-dicyclohexylcarbodiimide (928 mg, 4.50 mmol) was added and the
reaction mixture was stirred at RT overnight. 1,3-Dicyclohexylurea
was filtered off and to the filtrate 15 (2 g, 4.08 mmol) and
pyridine (2 ml) were added. The reaction mixture was stirred at RT
for 3 hours and than concentrated in vacuo. The residue was
partitioned between ethyl acetate and 5% Na.sub.2HCO.sub.3, the
organic layer extracted with brine as previously described, dried
(Na.sub.2SO.sub.4) and evaporated to a syrup. Column chromatography
using 2-10% gradient of methanol in dichlotomethane afforded 16 as
a white foam (3.4 g, 93%). .sup.1II-NMR 7.86-6.91 (m, 22H,
aromatic), 6.13 (d, J=8.8, 1H, NH), 5.93 (br s, 1H, NH), 5.43 (d,
J=8.4, 1H, NH), 4.63 (dd, J=10.6, J=6.4, 1H, Fm), 4.54 (dd, J=10.6,
J=6.4, 1H, Fm), 4.38 (m, 1H, Glu), 4.3 (t, J=6.4, 1H, Fm), 4.18 (m,
1H, H3 Thr), 4.01 (m, 1H, H2 Thr), 3.88 (s, 3H, OCH.sub.3), 3.49
(dd, J=9.5, J=4.4, 1H, H1 Thr), 3.37 (dd, J=9.5, J=3.8, 1H, H1'
Thr), 3.32 (m, 2H, CH.sub.2CO Acp), 3.09 (br s, 1H, OH), 2.32 (m,
2H, CH.sub.2NH Acp), 2.17 (m, 3H, Glu), 1.97 (m, 1H, Glu), 1.77 (m,
2H, CH.sub.2 Acp), 1.61 (m, 2H, CH.sub.2 Acp), 1.52 (s, 9H, t-Bu),
1.21 (d, J=6.4, 3H, CH.sub.3). MS/ESI.sup.+ m/z 920.5
(M+Na).sup.+.
[0580] N-(6-(N-.alpha.-OFm-L-glutamyl)aminocaproyl))-D-threoninol
hydrochloride (17). 16 (2 g, 2.23 mmol) was dissolved in methanol
(30 ml) containing anisole (10 ml) and to this solution x ml of 4M
HCl in dioxane was added. The reaction mixture was stirred for 3
hours at RT and then concentrated in vacuo. The residue was
dissolved in ethanol and the product precipitated by addition of x
ml of ether. The precipitate was washed with ether and dried to
give 17 as a colorless foam (1 g, 80%). .sup.1H-NMR
(DMSO-d.sub.6-D.sub.2O) 0.97-7.40 (m, 8H, aromatic), 4.70 (m, 1H,
Fm), 4.55 (m, 1H, Fm), 4.40 (t, J=6.4, 1H, Fm), 4.14 (t, J=6.6, 1H,
Glu), 3.90 (dd, J=2.8, J=6.4, 1H, H3 Thr), 3.68 (m, 1H, H2 Thr),
3.49 (dd, J=10.6, J=7.0, 1H, H1 Thr), 3.36 (dd, J=10.6, J=6.2, 1H,
H1' Thr), 3.07 (m, 2H, CH.sub.2C0 Acp), 2.17 m, 3H), 1.93 (m, 2H),
1.45 (m, 2H), 1.27 (m, 2H), 1.04 (d, J=6.4, 3H Thr). MS/ESI.sup.+
m/z 526.5 (M+H).sup.+.
[0581]
N-(6-(N-.alpha.-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N.sup.2--
iBu-N.sup.10-TFA-pteroic acid conjugate (18). To the solution of
N.sup.2-iBu-N.sup.10-TFA-pteroic acid.sup.1 (480 mg, 1 mmol) in DMF
(5 ml) 1-hydroxybenzotriazole (203 mg, 1.50 mmol), EDCI (288 mg,
1.50 mmol) and 17 (free base, 631 mg, 1.2 mmol) are added. The
reaction mixture is stirred at RT for 2 hours, then concentrated to
ca 3 ml and loaded on the column of silica gel. Elution with
dichloromethane, followed by 1-20% gradient of methanol in
dichloromethane afforded 18 (0.5 g, 51%). .sup.1H-NMR
(DMSO-d.sub.6-D.sub.2O) .delta. 9.09 (d, J=6.8, 1H, NH) 8.96 (s,
1H, H7 pteroic acid), 8.02-7.19 (m, 13H, aromatic, NH), 5.30 (s,
2H, pteroic acid), 4.50 (m, 1H, Glu), 4.41 (d, J=6.8, 2H, Fm), 4.29
(t, J=6.8, 1H, Fm), 3.89 (dd, J=6.2, J=2.8, 1H, H3 Thr), 3.68 (m,
1H, H2 Thr), 3.48 (dd, J=10.4, J=7.0, 1H, H1 Thr), 3.36 (dd,
J=10.4, J=6.2, 1H Thr), 106 (m, 2H, CH.sub.2CO Acp), 2.84 (m, 1H,
iBu), 2.25 (m, 2H, CH.sub.2NH Acp), 2.16 (m, 3H, Glu), 1.99 (m, 1H,
Glu), 1.52 (m, 2H Acp), 1.42 (m, 2H Acp), 1.27 (m, 2H Acp), 1.20
(s, 3H iBu), 1.19 (s, 3H, iBu), 1.03 (d, J=6.2, 3H Thr).
MS/ESI.sup.- m/z 984.5 (M-H).sup.-.
[0582]
O.sup.1-(4-monomethoxytrityl)-N-(6-(N-.alpha.-OFm-L-glutamyl)aminoc-
aproyl))-D-threoninol-N.sup.2-iBu-N.sup.10-TFA-pteroic acid
conjugate (19). To the solution of conjugate 18 (1 g, 1.01 mmol) in
dry pyridine (15 ml) p-anisylchlorodiphenylmethane (405 mg) was
added and the reaction mixture was stirred, protected from
moisture, at RT overnight. Methanol (3 ml) was added and the
reaction mixture concentrated to a syrup in vacuo. The residue was
partitioned between dichloromethane and 5% NaHCO.sub.3, the organic
layer washed with brine, dried (Na.sub.2SO.sub.4) and evaporated to
dryness. Column chromatography using 0.5-10% gradient of methanol
in dichloromethane afforded 19 as a colorless foam (0.5 g, 39%.
.sup.1H-NMR (DMSO-d.sub.6-D.sub.2O .delta.9.09 (d, J=6.8, 1H, NH)
8.94 (s, 1H, H7 pteroic acid), 8.00-6.93 (m, 27H, aromatic, NH),
5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.40 (d, J=6.8, 2H,
Fm), 4.29 (t, J=6.8, 1H, Fm), 3.94 (m, 2H, H3, H2 Thr), 3.79 (s,
3H, OCH.sub.3) 3.11 (dd, J=8.6, J=5.8, 1H, H1 Thr), 3.04 (m, 2H,
CH.sub.2CO Acp), 2.91 (dd, J=8.6, J=6.4, 1H, H1' Thr), 2.85 (m, 1H,
iBu), 2.25 (m, 2H, CH.sub.2NH Acp), 2.19 (m, 2H, Glu), 2.13 (m, 1H,
Glu), 1.98 (m, 1H, Glu), 1.55 (m, 2H Acp), 1.42 (m, 2H Acp), 1.29
(m, 2H Acp), 1.20 (s, 3H iBu), 1.18 (s, 3H, iBu), 1.00 (d, J=6.4,
3H Thr). MS/ESI.sup.- m/z 1257.0 (M-H).sup.-.
[0583]
O.sup.1-(4-monomethoxytrityl)-N-(6-(N-.alpha.-OFm-L-glutamyl)aminoc-
aproyl))-D-threoninol-N.sup.2-iBu-N.sup.10-TFA-pteroic acid
conjugate 3'-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20).
To the solution of 19 (500 mg, 0.40 mmol) in dichloromethane (2 ml)
2-cyanoethyl tetraisopropylphosphordiamidite (152 .mu.L, 0.48 mmol)
was added followed by pyridinium trifluoroacetate (93 mg, 0.48
mmol). The reaction mixture was stirred at RT for 1 hour and than
loaded on the column of silica gel in hexanes. Elution using ethyl
acetate-hexanes 1:1, followed by ethyl acetate and ethyl
acetate-acetone 1:1 in the presence of 1% pyridine afforded 20 as a
colorless foam (480 mg, 83%). .sup.31P NMR .delta. 149.4 (s), 149.0
(s).
Example 2
Synthesis of 2-dithiopyridyl activated folic acid (30) (FIG. 9)
[0584] Synthesis of the cysteamine modified folate 30 is presented
in FIG. 9. Monomethoxytrityl cysteamine 21 was prepared by
selective tritylation of the thiol group of cysteamine with
4-methoxytrityl alcohol in trifluoroacetic acid. Peptide coupling
of 21 with Fmoc-Glu-OtBu (Bachem Bioscience Inc., King of Prussia,
Pa.) in the presence of PyBOP yielded 22 in a high yield. N-Fmoc
group was removed smoothly with piperidine to give 23. Condensation
of 23 with p-(4-methoxytrityl)aminobenzoic acid, prepared by
reaction of p-aminobenzoic acid with 4-methoxytrityl chloride in
pyridine, afforded the fully protected conjugate 24. Selective
cleavage of N-MMTr group with acetic acid afforded 25 in
quantitative yield. Shiff base formation between 25 and
N.sup.2-iBu-6-formylpterin 26,.sup.9 followed by reduction with
borane-pyridine complex proceeded with a good yield to give fully
protected cysteamine-folate adduct 27..sup.12 The consecutive
cleavage of protecting groups of 27 with base and acid yielded
thiol derivative 29. The thiol exchange reaction of 29 with
2,2-dipyridyl disulfide afforded the desired S-pyridyl activated
synthon 30 as a yellow powder; Isolated as a TEA.sup.+ salt:
.sup.1H NMR spectrum for 10 in D.sub.2O: .delta. 8.68 (s, 1H, H-7),
8.10 (d, J=3.6, 1H, pyr), 7.61 (d, J=8.8, 2H, PABA), 7.43 (m, 1H,
pyr), 7.04 (d, J=7.6, 1H, pyr), 6.93 (m, 1H, pyr), 6.82 (d, J=8.8,
1H, PABA), 4.60 (s, 2H, 6-CH.sub.2), 4.28 (m, 1H, Glu), 3.30-3.08
(m, 2H, cysteamine), 3.05 (m, 6H, TEA), 2.37 (m, 2H, cysteamine),
2.10 (m, 4H, Glu), 1.20 (m, 9H, TEA). MS/ESI.sup.- m/z 608.02
[M-H].sup.-. It is worth noting that the isolation of 30 as its
TEA.sup.+ or Na.sup.+ salt made it soluble in DMSO and/or water,
which is an important requirement for its use in conjugation
reactions.
Example 3
Post Synthetic Conjugation of Enzymatic Nucleic Acid to Form
Nucleic Acid-Folate Conjugate (33) (FIG. 10)
[0585] Oligonucleotide synthesis, deprotection and purification was
performed as described herein. 5'-Thiol-Modifier C6 (Glen Research,
Sterling, Va.) was coupled as the last phosphoramidite to the
5'-end of a growing oligonucleotide chain. After cleavage from the
solid support and base deprotection, the disulfide modified
enzymatic nucleic acid molecule 31 (FIG. 10) was purified using ion
exchange chromatography. The thiol group was unmasked by reduction
with dithiothreitol (DTT) to afford 32 which was purified by gel
filtration and immediately conjugated with 30. The resulting
conjugate 33 was separated from the excess folate by gel filtration
and then purified by RP HPLC using gradient of acetonitrile in 50
mM triethylammonium acetate (TEAA). Desalting was performed by RP
HPLC. Reactions were conducted on 400 mg of disulfide modified
enzymatic nucleic acid molecule 31 to afford 200-250 mg (50-60%
yield) of conjugate 33. MALDI TOF MS confirmed the structure: 13
[M-H].sup.- 12084.74 (calc. 12083.82). An alternative approach to
this synthesis is shown in FIG. 11.
[0586] As shown in Examples 2 and 3, a folate-cysteamine adduct can
be prepared by a scaleable solution phase synthesis in a good
overall yield. Disulfide conjugation of this novel targeting ligand
to the thiol-modified oligonucleotide is suitable for the
multi-gram scale synthesis. The 9-atom spacer provides a useful
spatial separation between folate and attached oligonucleotide
cargo. Importantly, conjugation of folate to the oligonucleotide
through a disulfide bond should permit intermolecular separation
which was suggested to be required for the functional cytosolic
entry of a protein drug.
Example 4
Synthesis of Galactose and N-acetyl-Galactosamine Conjugates (FIGS.
13, 14, and 15)
[0587] Applicant has designed both nucleoside and
non-nucleoside-N-acetyl-D-galactosamine conjugates suitable for
incorporation at any desired position of an oligonucleotide.
Multiple incorporations of these monomers could result in a
"glycoside cluster effect".
[0588] All reactions were carried out under a positive pressure of
argon in anhydrous solvents. Commercially available reagents and
anhydrous solvents were used without further purification.
N-acetyl-D-galactosamine was purchased from Pfanstiel (Waukegan,
Ill.), folic acid from Sigma (St. Louis, Mo.), D-threoninol from
Aldrich (Milwaukee, Wis.) and N-Boc-.alpha.-OFm glutamic acid from
Bachem. .sup.1H (400.035 MHz) and .sup.31P (161.947 MHz) NMR
spectra were recorded in CDCl.sub.3, unless stated otherwise, and
chemical shifts in ppm refer to TMS and H3PO4, respectively.
Analytical thin-layer chromatography (TLC) was performed with Merck
Art.5554 Kieselgel 60 F.sub.254 plates and flash column
chromatography using Merck 0.040-0.063 mm silica gel 60. The
general procedures for RNA synthesis, deprotection and purification
are described herein. MALDI-TOF mass spectra were determined on
PerSeptive Biosystems Voyager spectrometer. Electrospray mass
spectrometry was run on the PE/Sciex API365 instrument.
[0589]
2'-(N-L-lysyl)amino-5'-O-4,4'-dimethoxytrityl-2'-deoxyuridine (2).
2'-(N-.alpha.,.epsilon.-bis-Fmoc-L-lysyl)amino-5'-O-4,4'-dimethoxytrityl--
2'-deoxyuridine (1) (4 g, 3.58 mmol) was dissolved in anhydrous DMF
(30 ml) and diethylamine (4 ml) was added. The reaction mixture was
stirred at rt for 5 hours and than concentrated (oil pump) to a
syrup. The residue was dissolved in ethanol and ether was added to
precipitate the product (1.8 g, 75%). .sup.1H-NMR
(DMSO-d.sub.6-D.sub.2O) .delta. 7.70 (d, J.sub.6,5=8.4, 1H, 116),
7.48-6.95 (m, 13H, aromatic), 5.93 (d, J1',2'=8.4, 1H, H1'), 5.41
(d, J.sub.5,6=8.4, 1H, H5), 4.62 (m, 1H, H2'), 4.19 (d, 1H, H3'),
3.81 (s, 6H, 2.times.OMe), 3.30 (m, 4H, 2H5', CH.sub.2), 1.60-1.20
(m, 6H, 3.times.CH.sub.2). MS/ESI.sup.+ m/z 674.0 (M+H).sup.+.
[0590]
N-Acetyl-1,4,6-tri-O-acetyl-2-amino-2-deoxy-.beta.-D-galactospyrano-
se (3). N-Acetyl-D-galac-tosamine (6.77 g, 30.60 mmol) was
suspended in acetonitrile (200 ml) and triethylamine (50 ml, 359
mmol) was added. The mixture was cooled in an ice-bath and acetic
anhydride (50 ml, 530 mmol)) was added dropwise under cooling. The
suspension slowly cleared and was then stirred at rt for 2 hours.
It was than cooled in an ice-bath and methanol (60 ml) was added
and the stirring continued for 15 min. The mixture was concentrated
under reduced pressure and the residue partitioned between
dichloromethane and 1 N HCl. Organic layer was washed twice with 5%
NaHCO.sub.3, followed by brine, dried (Na2SO4) and evaporated to
dryness to afford 10 g (84%) of 3 as a colorless foam. .sup.1H NMR
was in agreement with published data (Findeis, 1994, Int. J.
Peptide Protein Res., 43, 477-485.
[0591] 2-Acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactospyranose
(4). This compound was prepared from 3 as described by Findeis
supra.
[0592] Benzyl 12-Hydroxydodecanoate (5). To a cooled (0.degree. C.)
and stirred solution of 12-hydroxydodecanoic acid (10.65 g, 49.2
mmol) in DMF (70 ml) DBU (8.2 ml, 54.1 mmol) was added, followed by
benzyl bromide (6.44 ml, 54.1 mmol). The mixture was left overnight
at rt, than concentrated under reduced pressure and partitioned
between 1 N HCl and ether. Organic phase was washed with saturated
NaHCO.sub.3, dried over Na.sub.2SO.sub.4 and evaporated. Flash
chromatography using 20-30% gradient of ethyl acetate in hexanes
afforded benzyl ester as a white powder (14.1 g, 93.4%).
.sup.1H-NMR spectral data were in accordance with the published
values..sup.33
[0593] 12'-Benzyl
hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.beta.-D-galacto-
pyrano-se (6). 1-Chloro sugar 4 (4.26 g, 11.67 mmol) and benzyl
12-hydroxydodecanoate (5) (4.3 g, 13.03 mmol) were dissolved in
nitromethane-toluene 1:1 (122 ml) under argon and Hg(CN).sub.2
(3.51 g, 13.89 mmol) and powdered molecular sieves 4A (1.26 g) were
added. The mixture was stirred at rt for 24 h, filtered and the
filtrate concentrated under reduced pressure. The residue was
partitioned between dichloromethane and brine, organic layer was
washed with brine, followed by 0.5 M KBr, dried (Na.sub.2SO.sub.4)
and evaporated to a syrup. Flash silica gel column chromatography
using 15-30% gradient of acetone in hexanes yielded product 6 as a
colorless foam (6 g, 81%). .sup.1H-NMR .delta. 7.43 (m, 5H,
phenyl), 5.60 (d, 1H, J.sub.NH,2=8.8, NH), 5.44 (d, J.sub.4,3=3.2,
1H, H4), 5.40 (dd, J.sub.3,4=3.2, J.sub.3,2=10.8, 1H, H3), 5.19 (s,
2H, CH.sub.2Ph), 4.80 (d, J.sub.1,2=8.0, 1H, H1), 4.23 (m, 2H,
CH.sub.2), 3.99 (m, 3H, H2, H6), 3.56 (m, 1H, H5), 2.43 (t, J=7.2,
2H, CH.sub.2), 2.22 (s, 3H, Ac), 2.12 (s, 3H, Ac), 2.08 (s, 3H,
Ac), 2.03 (s, 3H, Ac), 1.64 (m, 4H, 2.times.CH.sub.2), 1.33 (br m,
14H, 7.times.CH.sub.2). MS/ESI.sup.- m/z 634.5 (M-H).sup.-.
12'-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.beta.-D-gala-
ctopyranose (7)
[0594] Conjugate 6 (2 g, 3.14 mmol)) was dissolved in ethanol (50
ml) and 5% Pd--C (0.3 g) was added. The reaction mixture was
hydrogenated overnight at 45 psi H.sub.2, the catalyst was filtered
off and the filtrate evaporated to dryness to afford pure 7 (1.7 g,
quantitative) as a white foam. .sup.1H-NMR 5.73 (d, 1H,
J.sub.NH,2=8.4, NH), 5.44 (d, J.sub.4,3=3.0, 1H, H4), 5.40 (dd,
J.sub.3,4=3.0, J.sub.3,2=11.2, 1H, H3), 4.78 (d, J.sub.1,2=8.8, 1H,
H1), 4.21 (m, 2H, CH.sub.2), 4.02 (m, 3H, H2, H6), 3.55 (m, 1H,
H5), 2.42 (m, 2H, CH.sub.2), 2.23 (s, 3H, Ac), 2.13 (s, 3H, Ac),
2.09 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.69 (m, 4H, 2.times.CH.sub.2),
1.36 (br m, 14H, 7.times.CH.sub.2). MS/ESI.sup.- m/z 544.0
(M-H).sup.-.
[0595]
2'-(N-.alpha.,.epsilon.-bis-(12'-Hydroxydodecanoyl-2-acetamido-3,4,-
6-tri-O-acetyl-2-deoxy-.beta.-D-galac-topyranose)-L-lysyl)amino-2'-deoxy-5-
'-dimethoxytrityl uridine (9). 7 (1.05 g, 1.92 mmol) was dissolved
in anhydrous THF and N-hydroxysuccinimide (0.27 g, 2.35 mmol) and
1,3-dicyclohexylcarbodiimide (0.55 g, 2.67 mmol) were added. The
reaction mixture was stirred at rt overnight, then filtered through
Celite pad and the filtrate concentrated under reduced pressure.
The crude NHSu ester 8 was dissolved in dry DMF (13 ml) containing
diisopropylethylamine (0.67 ml, 3.85 mmol) and to this solution
nucleoside 2 (0.64 g, 0.95 mmol was added). The reaction mixture
was stirred at rt overnight and than concentrated under reduced
pressure. The residue was partitioned between water and
dichloromethane, the aqueous layer extracted with dichloromethane,
the organic layers combined, dried (Na.sub.2SO.sub.4) and
evaporated to a syrup. Flash silica gel column chromatography using
2-3% gradient of methanol in ethyl acetate yielded 9 as a colorless
foam (1.04 g, 63%). .sup.1H-NMR .delta. 7.42 (d, J.sub.65=8.4, 1H,
H6 Urd), 7.53-6.97 (m, 13H, aromatic), 6.12 (d, 1H, H-1'), 5.41 (m,
3H, 115 Urd, H.sub.4NAcGal), 5.15 (dd, J.sub.3,4=3.6,
J.sub.3,2=11.2, 2H, H.sub.3NAcGal), 4.87 (dd, 1H, H2'), 4.63 (d,
J.sub.1,2=8.0, 2H, H.sub.1NAcGal), 4.42 (d, J.sub.3,2=5.6, 1H,
H3'), 4.29-4.04 (m, 9H, H4', H.sub.2NAcGal, H5NacGal, CH.sub.2),
3.95-3.82 (m, 8H, H6 NAcGal, 2.times.OMe), 3.62-3.42 (m, 4H, H5',
H6 NAcGal), 3.26 (m, 2H, CH.sub.2), 2.40-1.97 (m, 28H, CH.sub.2,
Ac), 1.95-1.30 (m, 50H, CH.sub.2). MS/ESI.sup.- m/z 1727.0
(M-H).sup.-.
[0596]
2'-(N-.alpha.,.epsilon.-bis-(12'-Hydroxydodecanoyl-2-acetamido-3,4,-
6-tri-O-acetyl-2-deoxy-.beta.-D-galac-topyranose)-L-lysyl)amino-2'-deoxy-5-
'-O-4,4'-dimethoxytrityl uridine 3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) (10). Conjugate 9 (0.87 g, 0.50
mmol) was dissolved in dry dichloromethane (10 ml) under argon and
diisopropylethylamine (0.36 ml, 2.07 mmol) and 1-methylimidazole
(21 .mu.L, 0.26 mmol) were added. The solution was cooled to
0.degree. C. and 2-cyanoethyl diisopropylchlorophosphoramidite
(0.19 ml, 0.85 mmol) was added. The reaction mixture was stirred at
rt for 1 hour, than cooled to 0.degree. C. and quenched with
anhydrous ethanol (0.5 ml). After stirring for 10 min the solution
was concentrated under reduced pressure (40.degree. C.) and the
residue dissolved in dichloromethane and chromatographed on the
column of silica gel using hexanes-ethyl acetate 1:1, followed by
ethyl acetate and finally ethyl acetate-acetone 1:1 (1%
triethylamine was added to solvents) to afford the phosphoramidite
10 (680 mg, 69%). .sup.31P-NMR .delta. 152.0 (s), 149.3 (s).
MS/ESI.sup.- m/z 1928.0 (M-H).sup.-.
[0597]
N-(12'-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.be-
ta.-D-galactopyranose)-D-threoninol (11).
12'-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-.beta.-D-gal-
ac-topyranose 7 (850 mg, 1.56 mmol) was dissolved in DMF (5 ml) and
to the solution N-hydroxysuccinimide (215 mg, 1.87 mmol) and
1,3-dicyclohexylcarbodimide (386 mg, 1.87 mmol) were added. The
reaction mixture was stirred at rt overnight, the precipitate was
filtered off and to the filtrate D-threoninol (197 mg, 1.87 mmol)
was added. The mixture was stirred at rt overnight and concentrated
in vacuo. The residue was partitioned between dichloromethane and
5% NaHCO.sub.3, the organic layer was washed with brine, dried
(Na.sub.2SO.sub.4) and evaporated to a syrup. Silica gel column
chromatography using 1-10% gradient of methanol in dichloromethane
afforded 11 as a colorless oil (0.7 g, 71%). .sup.1H-NMR .delta.
6.35 (d, J=7.6, 1H, NH), 5.77 (d, J=8.0, 1H, NH), 5.44 (d,
J.sub.4,3=3.6, 1H, H4), 5.37 (dd, J.sub.3,2=11.2, 1H, H3), 4.77 (d,
J.sub.1,2=8.0, 1H, H1), 4.28-4.18 (m, 3H, CH.sub.2, CH), 4.07-3.87
(m, 6H), 3.55 (m, 1H, H5), 3.09 (d, J=3.2, 1H, OH), 3.02 (t, J=4.6,
1H, OH), 2.34 (t, J=7.4 2H, CH.sub.2), 2.23 (s, 3H, Ac), 2.10 (s,
3H, Ac), 2.04 (s, 3H, Ac), 1.76-1.61 (m, 2.times.CH.sub.2), 1.35
(m, 14H, 7.times.CH.sub.2), 1.29 (d, J=6.4, 3H, CH.sub.3).
MS/ESI.sup.- m/z (M-H).sup.-.
[0598]
1-O-(4-Monomethoxytrityl)-N-(12'-hydroxydodecanoyl-2-acetamido-3,4,-
6-tri-O-acetyl-2-deoxy-.beta.-D-galactopyranose)-D-threoninol (12).
To the solution of 11 (680 mg, 1.1 mmol) in dry pyridine (10 ml)
p-anisylchlorotriphenylmethane (430 mg, 1.39 mmol) was added and
the reaction mixture was stirred, protected from moisture,
overnight. Methanol (3 ml) was added and the solution stirred for
15 min and evaporated in vacuo. The residue was partitioned between
dichloromethane and 5% NaHCO.sub.3, the organic layer was washed
with brine, dried (Na.sub.2SO.sub.4) and evaporated to a syrup.
Silica gel column chromatography using 1-3% gradient of methanol in
dichloromethane afforded 12 as a white foam (0.75 g, 77%).
.sup.1H-NMR .delta. 7.48-6.92 (m, 14H, aromatic), 6.15 (d, J=8.8,
1H, NH), 5.56 (d, J=8.0, 1H, NH), 5.45 (d, J.sub.4,3=3.2, 1H, H4),
5.40 (dd, J.sub.3,4=3.2, J.sub.3,2=11.2, 1H, H3), 4.80 (d,
J.sub.1,2=8.0, 1H, H1), 4.3-4.13 (m, 3H, CH.sub.2, CH), 4.25-3.92
(m, 4H, H6, H2, CH), 3.89 (s, 3H, OMe), 3.54 (m, 2H, H5, CH), 3.36
(dd, J=3.4, J=9.8, 1H, CH), 3.12 (d, J=2.8, 1H, OH), 2.31 (t,
J=7.6, 2H, CH.sub.2), 2.22 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.03 (s,
3H, Ac), 1.80-1.55 (m, 2.times.CH.sub.2), 1.37 (m, 14H,
7.times.CH.sub.2), 1.21 (d, J=6.4, 3H, CH.sub.3). MS/ESI.sup.- m/z
903.5 (M-H).sup.-.
[0599]
1-O-(4-Monomethoxytrityl)-N-(12'-hydroxydodecanoyl-2-acetamido-3,4,-
6-tri-.beta.-acetyl-2-deoxy-.beta.-D-galactopyranose)-D-threoninol
3-O-(2-cyanoethyl N,N-diisopropylphosphorami-dite) (13). Conjugate
12 (1.2 g, 1.33 mmol) was dissolved in dry dichloromethane (15 ml)
under argon and diisopropylethylamine (0.94 ml, 5.40 mmol) and
1-methylimidazole (55 pt, 0.69 mmol) were added. The solution was
cooled to 0.degree. C. and 2-cyanoethyl
N,N-diisopropyl-chlorophosphoramidite (0.51 ml, 2.29 mmol) was
added. The reaction mixture was stirred at rt for 2 hours, than
cooled to 0.degree. C. and quenched with anhydrous ethanol (0.5
ml). After stirring for 10 min. the solution was concentrated under
reduced pressure (40.degree. C.) and the residue dissolved in
dichloromethane and chromatographed on the column of silica gel
using 50-80% gradient of ethyl acetate in hexanes (1%
triethylamine) to afford the phosphoramidite 13 (1.2 g, 82%).
.sup.31P-NMR 149.41 (s), 149.23 (s).
Oligonucleotide Synthesis
[0600] Phosphoramidites 10, and 13, were used along with standard
2'-O-TBDMS and 2'-O-methyl nucleoside phosphoramidites. Synthesis
were conducted on a 394 (ABI) synthesizer using modified 2.5
.mu.mol scale protocol with a 5 min coupling step for 2'-O-TBDMS
protected nucleotides and 2.5 min coupling step for 2'-O-methyl
nucleosides. Coupling efficiency for the phosphoramidite 10 was
lower than 50% while coupling efficiencies for phosphoramidite 13
was typically greater than 95% based on the measurement of released
trityl cations. Once the synthesis was completed, the
oligonucleotides were deprotected. The 5'-trityl groups were left
attached to the oligomers to assist purification. Cleavage from the
solid support and the removal of the protecting groups was
performed as described herein with the exception of using 20%
piperidine in DMF for 15 min for the removal of Fm protection prior
methylamine treatment. The 5'-tritylated oligomers were separated
from shorter (trityl-off) failure sequences using a short column of
SEP-PAK C-18 adsorbent. The bound, tritylated oligomers were
detritylated on the column by treatment with 1% trifluoroacetic
acid, neutralized with triethylammonium acetate buffer, and than
eluted. Further purification was achieved by reverse-phase HPLC. An
example of a N-acetyl-D-galactosamine conjugate that can be
synthesized using phosphoramidite 13 is shown in FIG. 15.
[0601] Structures of the ribozyme conjugates were confirmed by
MALDI-TOF MS.
Monomer Synthesis
[0602] 2'-Amino-2'-deoxyuridine-N-acetyl-D-galactosamine conjugate.
The bis-Fmoc protected lysine linker was attached to the 2'-amino
group of 2'-amino-2'-deoxyuridine using the EEDQ catalyzed peptide
coupling. The 5'-OH was protected with 4,4'-dimethoxytrityl group
to give 1, followed by the cleavage of N-Fmoc groups with
diethylamine to afford synthon 2 in the high overall yield.
[0603] 2-acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactopyranose
4 was synthesized with minor modifications according to the
reported procedure (Findeis supra). Mercury salt catalyzed
glycosylation of 4 with the benzyl ester of 12-hydroxydodecanoic
acid 5 afforded glycoside 6 in 81% yield. Hydrogenolysis of benzyl
protecting group yielded 7 in a quantitative yield. The coupling of
the sugar derivative with the nucleoside synthon was achieved
through preactivation of the carboxylic function of 7 as
N-hydroxysuccinimide ester 8, followed by coupling to
lysyl-2'-aminouridine conjugate 2. The final conjugate 9 was than
phosphitylated under standard conditions to afford the
phosphoramidite 10 in 69% yield.
[0604] D-Threoninol-N-acetyl-D-galactosamine conjugate Using the
similar strategy as described above, D-threoninol was coupled to 7
to afford conjugate 11 in a good yield. Monomethoxytritylation,
followed by phosphitylation yielded the desired phosphoramidite
13.
Example 2
Synthesis of Oxime Linked Nucleic Acid/Peptide Conjugates (FIGS. 16
and 17)
[0605] 12-Hydroxydodecanoic acid benzyl ester Benzyl bromide (10.28
ml, 86.45 mmol) was added dropwise to a solution of
12-hydroxydodecanoic acid (17 g, 78.59 mmol) and DBU (12.93 ml,
86.45 mmol) in absolute DMF (120 ml) under vigorous stirring at
0.degree. C. After completeion of the addition reaction mixture was
warmed to a room temperature and left overnight under stirring. TLC
(hexane-ethylacetate 3:1) indicated complete transformation of the
starting material. DMF was removed under reduced pressure and the
residue was partitioned between ethyl ether and 1N HCl. Organic
phase was separated, washed with saturated aq sodium bicarbonate
and dried over sodium sulfate. Sodium sulfate was filtered off,
filtrate was evaporated to dryness. The residue was crystallized
from hexane to give 21.15 g (92%) of the title compound as a white
powder.
[0606] 12-O--N-Phthaloyl-dodecanoic acid benzyl ester (15).
Diethylazodicarboxylate (DEAD, 16.96 ml, 107.7 mmol) was added
dropwise to the mixture of 12-Hydroxydodecanoic acid benzyl ester
(21 g, 71.8 mmol), triphenylphosphine (28.29 g, 107.7 mmol) and
N-hydroxyphthalimide (12.88 g, 78.98 mmol) in absolute THF (250 ml)
at -20.degree.--30.degree. C. under stirring. The reaction mixture
was stirred at this temperature for additional 2-3 h, after which
time TLC (hexane-ethylacetate 3:1) indicated reaction completion.
The solvent was removed in vacuo and the residue was treated ether
(250 ml). Formed precipitate of triphenylphosphine oxide was
filtered off, mother liquor was evaporated to dryness- and the
residue was dissolved in methylene chloride and purified by flash
chromatography on silica gel in hexane-ethyl acetate (7:3).
Appropriate fractions were pooled and evaporated to dryness to
afford 26.5 g(84.4%) of compound 15.
[0607] 12-O--N-Phthaloyl-dodecanoic acid (16). Compound 15 (26.2 g,
59.9 mmol) was dissolved in 225 ml of ethanol-ethylacetate (3.5:1)
mixture and 10% Pd/C (2.6 g) was added. The reaction mixture was
hydrogenated in Parr apparatus for 3 hours. Reaction mixture was
filtered through celite and evaporated to dryness. The residue was
crystallized from methanol to provide 15.64 g (75%) of compound
16.
[0608] 12-O--N-Phthaloyl-dodecanoic acid 2,3-di-hydroxy-propylamide
(18) The mixture of compound 16 (15.03 g, 44.04 mmol),
dicyclohexylcarbodiimide (10.9 g, 52.85 mmol) and
N-hydroxysuccinimide (6.08 g, 52.85 mmol) in absolute DMF (150 ml)
was stirred at room temperature overnight. TLC (methylene
chloride-methanol 9:1) indicated complete conversion of the
starting material and formation of NHS ester 17. Then
aminopropanediol (4.01 g, 44 mmol) was added and the reaction
mixture was stirred at room temperature for another 2 h. The formed
precipitate of dicyclohexylurea was removed by filtration, filtrate
was evaporated under reduced pressure. The residue was partitioned
between ethyl acetate and saturated aq sodium bicarbonate. The
whole mixture was filtered to remove any insoluble material and
clear layers were separated. Organic phase was concentrated in
vacuo until formation of crystalline material. The precipitate was
filtered off and washed with cold ethylacetate to produce 10.86 g
of compound 17. Combined mother liquor and washings were evaporated
to dryness and crystallized from ethylacetate to afford 3.21 g of
compound 18. Combined yield--14.07 g (73.5%).
[0609] 12-O--N-Phthaloyl-dodecanoic acid 2-hydroxy,
3-dimethoxytrityloxy-propylamide (19) Dimethoxytrityl chloride
(12.07 g, 35.62 mmol) was added to a stirred solution of compound
18 (14.07 g, 32.38 mmol) in absolute pyridine (130 ml) at 0.degree.
C. The reaction solution was kept at 0.degree. C. overnight. Then
it was quenched with MeOH (10 ml) and evaporated to dryness. The
residue was dissolved in methylene chloride and washed with
saturated aq sodium bicarbonate. Organic phase was separated, dried
over sodium sulfate and evaporated to dryness. The residue was
purified by flash chromatography on silica gel using step gradient
of acetone in hexanes (3:7 to 1:1) as an eluent. Appropriate
fractions were pooled and evaporated to provide 14.73 g (62%) of
compound 19, as a colorless oil.
[0610] 12-O--N-Phthaloyl-dodecanoic acid
2-O-(cyanoethyl-N,N-diisopropylamino-phosphoramidite),
3-dimethoxytrityloxy-propylamide (20). Phosphitylated according to
Sanghvi, et al., 2000, Organic Process Research and Development, 4,
175-81.
[0611] Purified by flash chromatography on silica gel using step
gradient of acetone in hexanes (1:4 to 3:7) containing 0.5% of
triethylamine. Yield--82%, colourless oil.
Oxidation of Peptides
[0612] Peptide (3.3 mg, 3.3 .mu.mol) was dissolved in 10 mM AcONa
and 2 eq of sodium periodate (100 mM soln in water) was added.
Final reaction volume--0.5 ml. After 10 minutes reaction mixture
was purified using analytical HPLC on Phenomenex Jupiter 5u C18
300A (150.times.4.6 mm) column; solvent A: 50 mM KH.sub.2PO.sub.4
(pH 3); solvent B: 30% of solvent A in MeCN; gradient B over 30
min. Appropriate fractions were pooled and concentrated on a
SpeedVac to dryness. Yield: quantitative.
Conjugation Reaction of Herzyme-ONH2-Linker with N-glyoxyl Peptide
(FIG. 17)
[0613] Herzyme (SEQ ID NO: 13) with a 5'-terminal linker (1000D)
was mixed with oxidized peptide (3-5 eq) in 50 mM KH2PO4 (pH3,
reaction volume 1 ml) and kept at room temperature for 24-48 h. The
reaction mixture was purified using analytical HPLC on a Phenomenex
Jupiter 5u C18 300A (150.times.4.6 mm) column; solvent A: 10 mM
TEAA; solvent B: 10 mM TEAA/MeCN. Appropriate fractions were pooled
and concentrated on a SpeedVac to dryness to provide desired
conjugate. ESMS: calculated: 12699, determined: 12698.
Example 5
Synthesis of Phospholipid Enzymatic Nucleic Acid Conjugates (FIG.
19)
[0614] A phospholipid enzymatic nucleic acid conjugate (see FIG.
19) was prepared by coupling a C18H37 phosphoramidite to the 5'-end
of an enzymatic nucleic acid molecule (Angiozyme.TM., SEQ ID NO:
24) during solid phase oligonucleotide synthesis on an ABI 394
synthesizer using standard synthesis chemistry. A 5'-terminal
linker comprising 3'-AdT-di-Glycerol-5', where A is Adenosine, dT
is 2'-deoxy Thymidine, and di-Glycerol is a di-DMT-Glycerol linker
(Chemgenes CAT number CLP-5215), is used to attach two C18H37
phosphoramidites to the enzymatic nucleic acid molecule using
standard synthesis chemistry. Additional equivalents of the C18H37
phosphoramidite were used for the bis-coupling. Similarly, other
nucleic acid conjugates as shown in FIG. 18 can be prepared
according to similar methodology.
Example 6
Synthesis of PEG Enzymatic Nucleic Acid Conjugates (FIG. 20)
[0615] A 40K-PEG enzymatic nucleic acid conjugate (see FIG. 20) was
prepared by post synthetic N-hydroxysuccinimide ester coupling of a
PEG derivative (Shearwater Polymers Inc, CAT number PEG2-NHS) to
the 5'-end of an enzymatic nucleic acid molecule (Angiozyme.TM.,
SEQ ID NO: 24). A 5'-terminal linker comprising 3'-AdT-C6-amine-5',
where A is Adenosine, dT-C6-amine is 2'-deoxy Thymidine with a C5
linked six carbon amine linker (Glen Research CAT number
10-1039-05), is used to attach the PEG derivative to the enzymatic
nucleic acid molecule using NHS coupling chemistry. Angiozyme.TM.
with the C6dT-NH2 at the 5' end was synthesized and deprotected
using standard oligonucleotide synthesis procedures as described
herein. The crude sample was subsequently loaded onto a reverse
phase column and rinsed with sodium chloride solution (0.5 M). The
sample was then desalted with water on the column until the
concentration of sodium chloride was close to zero. Acetonitrile
was used to elute the sample from the column. The crude product was
then concentrated and lyophilized to dryness.
[0616] The crude material (Angiozyme.TM.) with 5'-amino linker (50
mg) was dissolved in sodium borate buffer (1.0 mL, pH 9.0). The PEG
NHS ester (200 mg) was dissolved in anhydrous DMF (1.0 mL). The
Angiozyme.TM. buffer solution was then added to the PEG NHS ester
solution. The mixture was immediately vortexed for 5 minutes.
Sodium acetate buffer solution (5 mL, pH 5.2) was used to quench
the reaction. Conjugated material was then purified by ion-exchange
and reverse phase chromatography.
Example 7
Phamacokinetics of PEG Ribozyme Acid Conjugate (FIG. 21)
[0617] Forty-eight female C57Bl/6 mice were given a single
subcutaneous (SC) bolus of 30 mg/kg Angiozyme.TM. and 30 mg/kg
Angiozyme.TM./40K PEG conjugate. Plasma was collected out to 24
hours post ribozyme injection. Plasma samples were analyzed for
full length ribozyme by a hybridization assay.
[0618] Oligonucleotides complimentary to the 5' and 3' ends of
Angiozyme.TM. were synthesized with biotin at one oligo, and FITC
on the other oligo. A biotin oligo and FITC labeled oligo pair are
incubated at 1 ug/ml with known concentrations of Angiozyme.TM. at
75 degrees C. for 5 min. After 10 minutes at RT, the mixture is
allowed to bind to streptavidin coated wells of a 96-wll plate for
two hours. The plate is washed with Tris-saline and detergent, and
peroxidase labeled anti-FITC antibody is added. After one hour, the
wells are washed, and the enzymatic reaction is developed, then
read on an ELISA plate reader. Results are shown in FIG. 21.
Example 8
Phamacokinetics of Phospholipid Ribozyme Conjugate (FIG. 22)
[0619] Seventy-two female C57Bl/6 mice were given a single
intravenous (4) bolus of 30 mg/kg Angiozyme.TM. and 30 mg/kg
Angiozyme.TM. conjugated with phospholipid (FIG. 19). Plasma was
collected out to 3 hours post ribozyme injection. Plasma samples
were analyzed for full length ribozyme by a hybridization
assay.
[0620] Oligonucleotides complimentary to the 5' and 3' ends of
Angiozyme.TM. were synthesized with biotin at one oligo, and FITC
on the other oligo. A biotin oligo and FITC labeled oligo pair are
incubated at 1 ug/ml with known concentrations of Angiozyme.TM. at
75 degrees C. for 5 min. After 10 minutes at RT, the mixture is
allowed to bind to streptavidin coated wells of a 96-wll plate for
two hours. The plate is washed with Tris-saline and detergent, and
peroxidase labeled anti-FITC antibody is added. After one hr, the
wells are washed, and the enzymatic reaction is developed, then
read on an ELISA plate reader. Results are shown in FIG. 22.
Example 9
Synthesis of Protein or Peptide Conjugates with Biodegradable
Linkers (FIGS. 24-26, and 29)
[0621] Proteins and Peptides can be Conjugated with Various
Molecules, Including Peg, via biodegradable nucleic acid linker
molecules of the invention, using oxime and morpholino linkages.
For example, a therapeutic antibody can be conjugated with PEG to
improve the FIG. 24 shows a non-limiting example of a synthetic
approach for synthesizing peptide or protein conjugates to PEG
utilizing a biodegradable linker, the example shown is for a
protein conjugate. Other conjugates can be synthesized in a similar
manner where the protein or peptide is conjugated to molecules
other than PEG, such as small molecules, toxins, radioisotopes,
peptides or other proteins. (a) The protein of interest, such as an
antibody or interferon, is synthesized with a terminal Serine or
Threonine moiety that is oxidized, for example with sodium
periodate. The oxidized protein is then coupled to a nucleic acid
linker molecule that is designed to be biodegradable, for example a
cytidine-deoxythymidine, cytidine-deoxyuridine,
adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that
contains an oxyamino (O--NH.sub.2) function. Other biodegradable
nucleic acid linkers can be similarly used, for example other
dimers, trimers, tetramers etc. that are designed to be
biodegradable. The example shown makes use of a 5'-oxyamino moiety,
however, other examples can utilize an oxyamino at other positions
within the nucleic acid molecule, for example at the 2'-position,
3'-position, or at a nucleic acid base position. (b) The
protein/nucleic acid conjugate is then oxidized to generate a
dialdehyde function that is coupled to PEG molecule comprising an
amino group (H.sub.2N-PEG), for example a PEG molecule with an
amino linker. Other amino containing molecules can be conjugated as
shown in the figure, for example small molecules, toxins, or
radioisotope labeled molecules.
[0622] Proteins and peptides can be conjugated with various
molecules, including PEG, via biodegradable nucleic acid linker
molecules of the invention, using oxime and phosphoramidate
linkages. FIG. 25 shows a non-limiting example of a synthetic
approach for synthesizing peptide or protein conjugates to PEG
utilizing a biodegradable linker, the example shown is for a
protein conjugate. Other conjugates can be synthesized in a similar
manner where the protein or peptide is conjugated to molecules
other than PEG, such as small molecules, toxins, radioisotopes,
peptides or other proteins. The protein of interest, such as an
antibody or interferon, is synthesized with a terminal Serine or
Threonine moiety that is oxidized, for example with sodium
periodate. The oxidized protein is then coupled to a nucleic acid
linker molecule that is designed to be biodegradable, for example a
cytidine-deoxythymidine, cytidine-deoxyuridine,
adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that
contains an oxyamino (O--NH.sub.2) function and a terminal
phosphate group. Terminal phosphate groups can be introduced during
synthesis of the nucleic acid molecule using chemical
phosphorylation reagents, such as Glen Research Cat Nos.
10-1909-02, 10-1913-02, 10-1914-02, and 10-1918-02. Other
biodegradable nucleic acid linkers can be similarly used, for
example other dimers, trimers, tetramers etc. that are designed to
be biodegradable. The example shown makes use of a 5'-oxyamino
moiety, however, other examples can utilize an oxyamino at other
positions within the nucleic acid molecule, for example at the
2'-position, 3'-position, or at a nucleic acid base position. The
protein/nucleic acid conjugate terminal phosphate group is then
activated with an activator reagent, such as NMI and/or tetrazole,
and coupled a PEG molecule comprising an amino group
(H.sub.2N-PEG), for example a PEG molecule with an amino linker.
Other amino containing molecules can be conjugated as shown in the
figure, for example small molecules, toxins, or radioisotope
labeled molecules.
[0623] Proteins and peptides can be conjugated with various
molecules, including PEG, via biodegradable nucleic acid linker
molecules of the invention, using phosphoramidate linkages. FIG. 26
shows a non-limiting example of a synthetic approach for
synthesizing peptide or protein conjugates to PEG utilizing a
biodegradable linker, the example shown is for a protein conjugate.
Other conjugates can be synthesized in a similar manner where the
protein or peptide is conjugated to molecules other than PEG, such
as small molecules, toxins, radioisotopes, peptides or other
proteins. (a) A nucleic acid linker molecule that is designed to be
biodegradable, for example a cytidine-deoxythymidine,
cytidine-deoxyuridine, adenosine-deoxythymidine, or
adenosine-deoxyuridine dimer, is synthesized with a terminal
phosphate group. Other biodegradable nucleic acid linkers can be
similarly used, for example other dimers, trimers, tetramers etc.
that are designed to be biodegradable. The protein/nucleic acid
conjugate terminal phosphate group is then activated with an
activator reagent, such as NMI and/or tetrazole, and coupled a PEG
molecule comprising an amino group (H.sub.2N-PEG), for example a
PEG molecule with an amino linker. Other amino containing molecules
can be conjugated as shown in the figure, for example small
molecules, toxins, or radioisotope labeled molecules. The terminal
protecting group, for example a dimethoxytrityl group, is removed
from the conjugate and a terminal phosphite group is introduced
with a phosphitylating reagent, such as
N,N-diisopropyl-2-cyanoethyl chlorophosphoramidite. (b) The
PEG/nucleic acid conjugate is then coupled to a peptide or protein
comprising an amino group, such as the amino terminus or amino side
chain of a suitably protected peptide or protein or via an amino
linker. The conjugate is then oxidized and any protecting groups
are removed to yield the protein/PEG conjugate comprising a
biodegradable linker.
[0624] Proteins and peptides can be conjugated with various
molecules, including PEG, via biodegradable nucleic acid linker
molecules of the invention, using phosphoramidate linkages from
coupling protein-based phosphoramidites. FIG. 29 shows a
non-limiting example of a synthetic approach for synthesizing
peptide or protein conjugates to PEG utilizing a biodegradable
linker, the example shown is for a protein conjugate. Other
conjugates can be synthesized in a similar manner where the protein
or peptide is conjugated to molecules other than PEG, such as small
molecules, toxins, radioisotopes, peptides or other proteins. The
protein of interest, such as an antibody or interferon, is
synthesized with a terminal Serine, Threonin, or Tyrosine moiety
that is phosphitylated, for example with
N,N-diisopropyl-2-cyanoethyl chlorophosphoramidite. The
phosphitylated protein is then coupled to a nucleic acid linker
molecule that is designed to be biodegradable, for example a
cytidine-deoxythymidine, cytidine-deoxyuridine,
adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that
contains conjugated PEG molecule as described in FIG. 18. Other
biodegradable nucleic acid linkers can be similarly used, for
example other dimers, trimers, tetramers etc. that are designed to
be biodegradable.
Example 10
Galactosamine Ribozyme Conjugate Targeting HBV
[0625] A nuclease-resistance ribozyme directed against the
Heptatitis B viral RNA (HBV) (HepBzyme.TM.) is in early stages of
preclinical development. HepBzyme, which targets site 273 of the
Hepatitis B viral RNA, has produced statistically significant
decreases in serum HBV levels in a HBV transgenic mouse model in a
dose-dependent manner (30 and 100 mg/kg/day). In an effort to
improve hepatic uptake by targeting the asialoglycoprotein
receptor, a series of 5 branched galactosamine residues were
attached via phosphate linkages to the 5'-terminus of HepBzyme
(Gal-HepBzyme). The affect of the galactosamine conjugation on
HepBzyme was assessed by quantitation of .sup.32P-labeled HepBzyme
and Gal-HepBzyme in plasma, liver and kidney of mice following a
single SC bolus administration of 30 mg/kg. The plasma disposition
of the intact ribozyme was similar between Gal-HepBzyme and
HepBzyme. An approximate three-fold increase in the maximum
observed concentration of intact ribozyme in liver (C.sub.max) was
observed in liver for Gal-HepBzyme (6.1.+-.1.8 ng/mg) vs. HepBzyme
(2.2.+-.0.8 ng/mg) (p<0.05). The area under the curve (AUCall)
for Gal-HepBzyme was also increased by approximately two-fold. This
was accompanied by a substantial decrease (approximately 40%) in
the AUC.sub.all for intact ribozyme in kidney. In addition to the
significant increase in C.sub.max observed for intact Gal-HepBzyme
in the liver, there was an increase in the total number of ribozyme
equivalents, which may be suggestive of increased affinity of both
the intact ribozyme and metabolites for asialoglycoprotein receptor
and galactose-specific receptors in the liver. These data
demonstrate that conjugation of a ribozyme with galactosamine
produces a compound with a more favorable disposition profile, and
illustrates the utility of conjugated ribozymes with improved in
vivo pharmacokinetics and biodistribution.
Example 11
Synthesis of siNA Conjugates
[0626] siNA molecules can be designed to interact with various
sites in a target RNA message, for example, target sequences within
the RNA sequence. The sequence of one strand of the siNA
molecule(s) is complementary to the target site sequences. The siNA
molecules can be chemically synthesized using methods described
herein. Inactive siNA molecules that are used as control sequences
can be synthesized by scrambling the sequence of the siNA molecules
such that it is not complementary to the target sequence.
Generally, siNA constructs can by synthesized using solid phase
oligonucleotide synthesis methods as described herein (see for
example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071;
5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158;
Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all
incorporated by reference herein in their entirety).
[0627] In a non-limiting example, RNA oligonucleotides are
synthesized in a stepwise fashion using the phosphoramidite
chemistry as is known in the art. Standard phosphoramidite
chemistry involves the use of nucleosides comprising any of
5'-O-dimethoxytrityl, 2'-O-tert-butyldimethylsilyl,
3'-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and
exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4
acetyl cytidine, and N2-isobutyryl guanosine). Alternately,
2'-O--Silyl Ethers can be used in conjunction with acid-labile
2'-O-orthoester protecting groups in the synthesis of RNA as
described by Scaringe supra. Differing 2' chemistries can require
different protecting groups, for example 2'-deoxy-2'-amino
nucleosides can utilize N-phthaloyl protection as described by
Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference
herein in its entirety).
[0628] During solid phase synthesis, each nucleotide is added
sequentially (3'- to 5'-direction) to the solid support-bound
oligonucleotide. The first nucleoside at the 3'-end of the chain is
covalently attached to a solid support (e.g., controlled pore glass
or polystyrene) using various linkers. The nucleotide precursor, a
ribonucleoside phosphoramidite, and activator are combined
resulting in the coupling of the second nucleoside phosphoramidite
onto the 5'-end of the first nucleoside. The support is then washed
and any unreacted 5'-hydroxyl groups are capped with a capping
reagent such as acetic anhydride to yield inactive 5'-acetyl
moieties. The trivalent phosphorus linkage is then oxidized to a
more stable phosphate linkage. At the end of the nucleotide
addition cycle, the 5'-O-protecting group is cleaved under suitable
conditions (e.g., acidic conditions for trityl-based groups and
Fluoride for silyl-based groups). The cycle is repeated for each
subsequent nucleotide.
[0629] Modification of synthesis conditions can be used to optimize
coupling efficiency, for example by using differing coupling times,
differing reagent/phosphoramidite concentrations, differing contact
times, differing solid supports and solid support linker
chemistries depending on the particular chemical composition of the
siNA to be synthesized. Deprotection and purification of the siNA
can be performed as is generally described in Deprotection and
purification of the siNA can be performed as is generally described
in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098,
U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.
6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or
Scaringe supra, incorporated by reference herein in their
entireties. Additionally, deprotection conditions can be modified
to provide the best possible yield and purity of siNA constructs.
For example, applicant has observed that oligonucleotides
comprising 2'-deoxy-2'-fluoro nucleotides can degrade under
inappropriate deprotection conditions. Such oligonucleotides are
deprotected using aqueous methylamine at about 35.degree. C. for 30
minutes. If the 2'-deoxy-2'-fluoro containing oligonucleotide also
comprises ribonucleotides, after deprotection with aqueous
methylamine at about 35.degree. C. for 30 minutes, TEA-HF is added
and the reaction maintained at about 65.degree. C. for an
additional 15 minutes.
[0630] The introduction of conjugate moieties is accomplished
either during solid phase synthesis using phosphoramidite chemistry
described above, or post-synthetically using, for example,
N-hydroxysuccinimide (NHS) ester coupling to an amino linker
present in the siNA. Typically, a conjugate introduced during solid
phase synthesis will be added to the 5'-end of a nucleic acid
sequence as the final coupling reaction in the synthesis cycle
using the phosphoramidite approach. Coupling conditions can be
optimized for high yield coupling, for example by modification of
coupling times and reagent concentrations to effectuate efficient
coupling. As such, the 5'-end of the sense strand of a siNA
molecule is readily conjugated with a conjugate moiety having a
reactive phosphorus group available for coupling (e.g., a compound
having Formulae 1, 5, 8, 55, 56, 57, 60, 86, 92, 104, 110, 113,
115, 116, 117, 118, 120, or 122) using the phosphoramidite
approach, providing a 5'-terminal conjugate (see for example FIG.
41).
[0631] Conjugate precursors having a reactive phosphorus group and
a protected hydroxyl group can be used to incorporate a conjugate
moiety anywhere in the siNA sequence, such as in the loop portion
of a single stranded hairpin siNA construct (see for example FIG.
42). For example, using the phosphoramidite approach, a conjugate
moiety comprising a phosphoramidite and protected hydroxyl (e.g., a
compound having Formulae 86, 92, 104, 113, 115, 116, 117, 118, 120,
or 122 herein) is first coupled at the desired position within the
siNA sequence using solid phase synthesis phosphoramidite coupling.
Second, removal of the protecting group (e.g., dimethoxytrityl)
allows coupling of additional nucleotides to the siNA sequence.
This approach allows the conjugate moiety to be positioned anywhere
within the siNA molecule.
[0632] Conjugate derivatives can also be introduced to a siNA
molecule post synthetically. Post synthetic conjugation allows a
conjugate moiety to be introduced at any position within the siNA
molecule where an appropriate functional group is present (e.g., a
C5 alkylamine linker present on a nucleotide base or a
2'-alkylamine linker present on a nucleotide sugar can provide a
point of attachment for an NHS-conjugate moiety). Generally, a
reactive chemical group present in the siNA molecule is unmasked
following synthesis, thus allowing post-synthetic coupling of the
conjugate to occur. In a non-limiting example, an protected amino
linker containing nucleotide (e.g., TFA protected C5 propylamino
thymidine) is introduced at a desired position of the siNA during
solid phase synthesis. Following cleavage and deprotection of the
siNA, the free amine is made available for NHS ester coupling of
the conjugate at the desired position within the siNA sequence,
such as at the 3'-end of the sense and/or antisense strands, the 3'
and/or 5'-end of the sense strand, or within the siNA sequence,
such as in the loop portion of a single stranded hairpin siNA
sequence.
[0633] A conjugate moiety can be introduced at different locations
within a siNA molecule using both solid phase synthesis and
post-synthetic coupling approaches. For example, solid phase
synthesis can be used to introduce a conjugate moiety at the 5'-end
of the siNA (e.g. sense strand) and post-synthetic coupling can be
used to introduce a conjugate moiety at the 3'-end of the siNA
(e.g. sense strand and/or antisense strand). As such, a siNA sense
strand having 3' and 5' end conjugates can be synthesized (see for
example FIG. 41). Conjugate moieties can also be introduced in
other combinations, such as at the 5'-end, 3'-end and/or loop
portions of a siNA molecule (see for example FIG. 42).
Example 12
Phamacokinetics of siNA Conjugates (FIG. 43)
[0634] Three nuclease resistant siNA molecule targeting site 1580
of hepatitis B virus (HBV) RNA were designed using Stab 7/8
chemistry (see Table 4) and a 5'-terminal conjugate moiety.
[0635] One siNA conjugate comprises a branched cholesterol
conjugate linked to the sense strand of the siNA. The "cholesterol"
siNA conjugate molecule has the structure shown below:
##STR00199##
[0636] where T stands for thymidine, B stands for inverted
deoxyabasic, G stands for 2'-deoxy guanosine, A stands for 2'-deoxy
adenosine, G stands for 2'-O-methyl guanosine, A stands for
2'-O-methyl adenosine, u stands for 2'-fluoro uridine, c stands for
2'-fluoro cytidine, a stands for adenosine, and s stands for
phosphorothioate linkage.
[0637] Another siNA conjugate comprises a branched phospholipid
conjugate linked to the sense strand of the siNA. The
"phospholipid" siNA conjugate molecule has the structure shown
below:
##STR00200##
[0638] where T stands for thymidine, B stands for inverted
deoxyabasic, G stands for 2'-deoxy guanosine, A stands for 2'-deoxy
adenosine, G stands for 2'-O-methyl guanosine, A stands for
2'-O-methyl adenosine, u stands for 2'-fluoro uridine, c stands for
2'-fluoro cytidine, a stands for adenosine, and s stands for
phosphorothioate linkage.
[0639] Another siNA conjugate comprises a polyethylene glycol (PEG)
conjugate linked to the sense strand of the siNA. The "PEG" siNA
conjugate molecule has the structure shown below:
##STR00201##
[0640] where T stands for thymidine, B stands for inverted
deoxyabasic, G stands for 2'-deoxy guanosine, A stands for 2'-deoxy
adenosine, G stands for 2'-O-methyl guanosine, A stands for
2'-O-methyl adenosine, u stands for 2'-fluoro uridine, c stands for
2'-fluoro cytidine, a stands for adenosine, and s stands for
phosphorothioate linkage.
[0641] The Cholesterol, Phospholipid, and PEG conjugates were
evaluated for pharmakokinetic properties in mice compared to a
non-conjugated siNA construct having matched chemistry and
sequence. This study was conducted in female CD-1 mice
approximately 26 g (6-7 weeks of age). Animals were housed in
groups of 3. Food and water were provided ad libitum. Temperature
and humidity were according to Pharmacology Testing Facility
performance standards (SOP's) which are in accordance with the 1996
Guide for the Care and Use of Laboratory Animals (NRC). Animals
were acclimated to the facility for at least 3 days prior to
experimentation.
[0642] Absorbance at 260 nm was used to determine the actual
concentration of the stock solution of pre-annealed HBV siNA. An
appropriate amount of HBV siNA was diluted in sterile veterinary
grade normal saline (0.9%) based on the average body weight of the
mice. A small amount of the antisense (Stab 7) strand was
internally labeled with gamma 32P-ATP. The 32P-labeled stock was
combined with excess sense strand (Stab 8) and annealed. Annealing
was confirmed prior to combination with unlabled drug. Each mouse
received a subcutaneous bolus of 30 mg/kg (based on duplex) and
approximately 10 million cpm (specific activity of approximately 15
cpm/ng).
[0643] Three animals per timepoint (1, 4, 8, 24, 72, 96 h) were
euthanized by CO2 inhalation followed immediately by
exsanguination. Blood was sampled from the heart and collected in
heparinized tubes. After exsanguination, animals were perfused with
10-15 mL of sterile veterinary grade saline via the heart. Samples
of liver were then collected and frozen.
[0644] Tissue samples were homogenized in a digestion buffer prior
to compound quantitation. Quantitation of intact compound was
determined by scintillation counting followed by PAGE and
phosphorimage analysis. Results are shown in FIG. 43. As shown in
the figure, the conjugated siNA constructs shown vastly improved
liver PK compared to the unconjugated siNA construct.
Example 13
Cell Culture of siNA Conjugates (FIG. 44)
[0645] The Cholesterol conjugates and Phospholipid conjugated siNA
constructs described in Example 12 above were evaluated for cell
culture efficacy in a HBV cell culture system.
Transfection of HepG2 Cells with psHBV-1 and siNA
[0646] The human hepatocellular carcinoma cell line Hep G2 was
grown in Dulbecco's modified Eagle media supplemented with 10%
fetal calf serum, 2 mM glutamine, 0.1 mM nonessential amino acids,
1 mM sodium pyruvate, 25 mM Hepes, 100 units penicillin, and 100
.mu.g/ml streptomycin. To generate a replication competent cDNA,
prior to transfection the HBV genomic sequences are excised from
the bacterial plasmid sequence contained in the psHBV-1 vector.
Other methods known in the art can be used to generate a
replication competent cDNA. This was done with an EcoRI and Hind
III restriction digest. Following completion of the digest, a
ligation was performed under dilute conditions (20 .mu.g/ml) to
favor intermolecular ligation. The total ligation mixture was then
concentrated using Qiagen spin columns.
siNA Activity Screen and Dose Response Assay
[0647] Transfection of the human hepatocellular carcinoma cell
line, Hep G2, with replication-competent HBV DNA results in the
expression of HBV proteins and the production of virions. To test
the efficacy of siNA conjugates targeted against HBV RNA, the
Cholesterol siNA conjugate and Phospholipid siNA conjugate
described in Example 12 were compared to a non-conjugated control
siNA (see FIG. 44). An inverted sequence duplex was used as a
negative control for the unconjugated siNA. Dose response studies
were performed in which HBV genomic DNA was transfected with HBV
genomic DNA with lipid at 12.5 ug/ml into Hep G2 cells. 24 hours
after transfection with HBV DNA, cell culture media was removed and
siNA duplexes were added to cells without lipid at 10 uM, 5, uM,
2.5 uM, 1 uM, and 100 nm and the subsequent levels of secreted HBV
surface antigen (HBsAg) were analyzed by ELISA 72 hours post
treatment (see FIG. 44). To determine siNA activity, HbsAg levels
were measured following transfection with siNA. Immulon 4 (Dynax)
microtiter wells were coated overnight at 4.degree. C. with
anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1 .mu.g/ml in
Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5). The wells
were then washed 4.times. with PBST (PBS, 0.05% Tween.RTM. 20) and
blocked for 1 hr at 37.degree. C. with PBST, 1% BSA. Following
washing as above, the wells were dried at 37.degree. C. for 30 min.
Biotinylated goat ant-HBsAg (Accurate YVS 1807) was diluted 1:1000
in PBST and incubated in the wells for 1 hr. at 37.degree. C. The
wells were washed 4.times. with PBST. Streptavidin/Alkaline
Phosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in
PBST, and incubated in the wells for 1 hr. at 37.degree. C. After
washing as above, p-nitrophenyl phosphate substrate (Pierce 37620)
was added to the wells, which were then incubated for 1 hour at
37.degree. C. The optical density at 405 nm was then determined. As
shown in FIG. 44, the phospholipid and cholesterol conjugates
demonstrate marked dose dependent inhibition of HBsAg expression
compared to the unconjugated siNA construct when delivered to cells
without any transfection agent (lipid).
[0648] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and compositions described herein are
exemplary and are not intended as limitations on the scope of the
invention. Changes therein and other uses will occur to those
skilled in the art, which are encompassed within the spirit of the
invention, are defined by the scope of the claims.
[0649] It will be readily apparent to one skilled in the art that
varying substitutions and modifications can be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. Thus, such additional embodiments are
within the scope of the present invention and the following
claims.
[0650] The invention illustratively described herein suitably can
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by various
embodiments, optional features, modification and variation of the
concepts herein disclosed may be resorted to by those skilled in
the art, and that such modifications and variations are considered
to be within the scope of this invention as defined by the
description and the appended claims.
[0651] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0652] Other embodiments are within the following claims.
TABLE-US-00001 TABLE I Characteristics of naturally occurring
ribozymes Group I Introns Size: ~150 to >1000 nucleotides.
Requires a U in the target sequence immediately 5' of the cleavage
site. Binds 4-6 nucleotides at the 5'-side of the cleavage site.
Reaction mechanism: attack by the 3'-OH of guanosine to generate
cleavage products with 3'-OH and 5'-guanosine. Additional protein
cofactors required in some cases to help folding and maintenance of
the active structure. Over 300 known members of this class. Found
as an intervening sequence in Tetrahymena thermophila rRNA, fungal
mitochondria, chloroplasts, phage T4, blue-green algae, and others.
Major structural features largely established through phylogenetic
comparisons, mutagenesis, and biochemical studies [.sup.i,
.sup.ii]. Complete kinetic framework established for one ribozyme
[.sup.iii, .sup.iv, .sup.v, .sup.vi]. Studies of ribozyme folding
and substrate docking underway [.sup.vii, .sup.viii, .sup.ix].
Chemical modification investigation of important residues well
established [.sup.x, .sup.xi]. The small (4-6 nt) binding site may
make this ribozyme too non-specific for targeted RNA cleavage,
however, the Tetrahymena group I intron has been used to repair a
"defective" .beta.-galactosidase message by the ligation of new
.beta.- galactosidase sequences onto the defective message
[.sup.xii]. RNAse P RNA (M1 RNA) Size: ~290 to 400 nucleotides. RNA
portion of a ubiquitous ribonucleoprotein enzyme. Cleaves tRNA
precursors to form mature tRNA [.sup.xiii]. Reaction mechanism:
possible attack by M.sup.2+-OH to generate cleavage products with
3'-OH and 5'-phosphate. RNAse P is found throughout the prokaryotes
and eukaryotes. The RNA subunit has been sequenced from bacteria,
yeast, rodents, and primates. Recruitment of endogenous RNAse P for
therapeutic applications is possible through hybridization of an
External Guide Sequence (EGS) to the target RNA [.sup.xiv, .sup.xv]
Important phosphate and 2' OH contacts recently identified
[.sup.xvi, .sup.xvii] Group II Introns Size: >1000 nucleotides.
Trans cleavage of target RNAs recently demonstrated [.sup.xviii,
.sup.xix]. Sequence requirements not fully determined. Reaction
mechanism: 2'-OH of an internal adenosine generates cleavage
products with 3'-OH and a "lariat" RNA containing a 3'-5' and a
2'-5' branch point. Only natural ribozyme with demonstrated
participation in DNA cleavage [.sup.xx, .sup.xxi] in addition to
RNA cleavage and ligation. Major structural features largely
established through phylogenetic comparisons [.sup.xxii]. Important
2' OH contacts beginning to be identified [.sup.xxiii] Kinetic
framework under development [.sup.xxiv] Neurospora VS RNA Size:
~144 nucleotides. Trans cleavage of hairpin target RNAs recently
demonstrated [.sup.xxv]. Sequence requirements not fully
determined. Reaction mechanism: attack by 2'-OH 5' to the scissile
bond to generate cleavage products with 2',3'-cyclic phosphate and
5'-OH ends. Binding sites and structural requirements not fully
determined. Only 1 known member of this class. Found in Neurospora
VS RNA. Hammerhead Ribozyme (see text for references) Size: ~13 to
40 nucleotides. Requires the target sequence UH immediately 5' of
the cleavage site. Binds a variable number nucleotides on both
sides of the cleavage site. Reaction mechanism: attack by 2'-OH 5'
to the scissile bond to generate cleavage products with
2',3'-cyclic phosphate and 5'-OH ends. 14 known members of this
class. Found in a number of plant pathogens (virusoids) that use
RNA as the infectious agent. Essential structural features largely
defined, including 2 crystal structures [.sup.xxvi, .sup.xxvii]
Minimal ligation activity demonstrated (for engineering through in
vitro selection) [.sup.xxviii] Complete kinetic framework
established for two or more ribozymes [.sup.xxix]. Chemical
modification investigation of important residues well established
[.sup.xxx]. Hairpin Ribozyme Size: ~50 nucleotides. Requires the
target sequence GUC immediately 3' of the cleavage site. Binds 4-6
nucleotides at the 5'-side of the cleavage site and a variable
number to the 3'-side of the cleavage site. Reaction mechanism:
attack by 2'-OH 5' to the scissile bond to generate cleavage
products with 2',3'-cyclic phosphate and 5'-OH ends. 3 known
members of this class. Found in three plant pathogen (satellite
RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory
yellow mottle virus) which uses RNA as the infectious agent.
Essential structural features largely defined [.sup.xxxi,
.sup.xxxii, .sup.xxxiii, .sup.xxxiv] Ligation activity (in addition
to cleavage activity) makes ribozyme amenable to engineering
through in vitro selection [.sup.xxxv] Complete kinetic framework
established for one ribozyme [.sup.xxxvi]. Chemical modification
investigation of important residues begun [.sup.xxxvii,
.sup.xxxviii]. Hepatitis Delta Virus (HDV) Ribozyme Size: ~60
nucleotides. Trans cleavage of target RNAs demonstrated
[.sup.xxxix]. Binding sites and structural requirements not fully
determined, although no sequences 5' of cleavage site are required.
Folded ribozyme contains a pseudoknot structure [.sup.xl]. Reaction
mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage products with 2',3'-cyclic phosphate and 5'-OH ends. Only
2 known members of this class. Found in human HDV. Circular form of
HDV is active and shows increased nuclease stability [.sup.xli]
.sup.i Michel, Francois; Westhof, Eric. Slippery substrates. Nat.
Struct. Biol. (1994), 1(1), 5-7. .sup.ii Lisacek, Frederique; Diaz,
Yolande; Michel, Francois. Automatic identification of group I
intron cores in genomic DNA sequences. J. Mol. Biol. (1994),
235(4), 1206-17. .sup.iii Herschlag, Daniel; Cech, Thomas R. .
Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme.
1. Kinetic description of the reaction of an RNA substrate
complementary to the active site. Biochemistry (1990), 29(44),
10159-71. .sup.iv Herschlag, Daniel; Cech, Thomas R. . Catalysis of
RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic
description of the reaction of an RNA substrate that forms a
mismatch at the active site. Biochemistry (1990), 29(44), 10172-80.
.sup.v Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the
Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent
pKa. Biochemistry (1996), 35(5), 1560-70. .sup.vi Bevilacqua,
Philip C.; Sugimoto, Naoki; Turner, Douglas H. . A mechanistic
framework for the second step of splicing catalyzed by the
Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58. .sup.vii
Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H. .
Thermodynamic and activation parameters for binding of a
pyrene-labeled substrate by the Tetrahymena ribozyme: docking is
not diffusion-controlled and is driven by a favorable entropy
change. Biochemistry (1995), 34(44), 14394-9. .sup.viii Banerjee,
Aloke Raj; Turner, Douglas H. . The time dependence of chemical
modification reveals slow steps in the folding of a group I
ribozyme. Biochemistry (1995), 34(19), 6504-12. .sup.ix Zarrinkar,
Patrick P.; Williamson, James R. . The P9.1-P9.2 peripheral
extension helps guide folding of the Tetrahymena ribozyme. Nucleic
Acids Res. (1996), 24(5), 854-8. .sup.x Strobel, Scott A.; Cech,
Thomas R. . Minor groove recognition of the conserved G.cntdot.U
pair at the Tetrahymena ribozyme reaction site. Science
(Washington, D.C.) (1995), 267(5198), 675-9. .sup.xi Strobel, Scott
A.; Cech, Thomas R. . Exocyclic Amine of the Conserved G.cntdot.U
Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes
to 5'-Splice Site Selection and Transition State Stabilization.
Biochemistry (1996), 35(4), 1201-11. .sup.xii Sullenger, Bruce A.;
Cech, Thomas R. . Ribozyme-mediated repair of defective mRNA by
targeted trans-splicing. Nature (London) (1994), 371(6498), 619-22.
.sup.xiii Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol.
Chem., 247, 5243-5251 (1972). .sup.xiv Forster, Anthony C.; Altman,
Sidney. External guide sequences for an RNA enzyme. Science
(Washington, D.C., 1883-) (1990), 249(4970), 783-6. .sup.xv Yuan,
Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human
RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10. .sup.xvi
Harris, Michael E.; Pace, Norman R. . Identification of phosphates
involved in catalysis by the ribozyme RNase P RNA. RNA (1995),
1(2), 210-18. .sup.xvii Pan, Tao; Loria, Andrew; Zhong, Kun.
Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts
between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A.
(1995), 92(26), 12510-14. .sup.xviii Pyle, Anna Marie; Green,
Justin B. . Building a Kinetic Framework for Group II Intron
Ribozyme Activity: Quantitation of Interdomain Binding and Reaction
Rate. Biochemistry (1994), 33(9), 2716-25. .sup.xix Michels,
William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron
into a New Multiple-Turnover Ribozyme that Selectively Cleaves
Oligonucleotides: Elucidation of Reaction Mechanism and
Structure/Function Relationships. Biochemistry (1995), 34(9),
2965-77. .sup.xx Zimmerly, Steven; Guo, Huatao; Eskes, Robert;
Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M. . A group II
intron RNA is a catalytic component of a DNA endonuclease involved
in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
.sup.xxi Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams
J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave
DNA and RNA linkages with similar efficiency, and lack contacts
with substrate 2'-hydroxyl groups. Chem. Biol. (1995), 2(11),
761-70. .sup.xxii Michel, Francois; Ferat, Jean Luc. Structure and
activities of group II introns. Annu. Rev. Biochem. (1995), 64,
435-61. .sup.xxiii Abramovitz, Dana L.; Friedman, Richard A.; Pyle,
Anna Marie. Catalytic role of 2'-hydroxyl groups within a group II
intron active site. Science (Washington, D.C.) (1996), 271(5254),
1410-13. .sup.xxiv Daniels, Danette L.; Michels, William J., Jr.;
Pyle, Anna Marie. Two competing pathways for self-splicing by group
II introns: a quantitative analysis of in vitro reaction rates and
products. J. Mol. Biol. (1996), 256(1), 31-49. .sup.xxv Guo, Hans
C. T.; Collins, Richard A. . Efficient trans-cleavage of a
stem-loop RNA substrate by a ribozyme derived from Neurospora VS
RNA. EMBO J. (1995), 14(2), 368-76. .sup.xxvi Scott, W. G., Finch,
J. T., Aaron, K. The crystal structure of an all RNA hammerhead
ribozyme: A proposed mechanism for RNA catalytic cleavage. Cell,
(1995), 81, 991-1002. .sup.xxvii McKay, Structure and function of
the hammerhead ribozyme: an unfinished story. RNA, (1996), 2,
395-403. .sup.xxviii Long, D., Uhlenbeck, O., Hertel, K. Ligation
with hammerhead ribozymes. U.S. Pat. No. 5,633,133. .sup.xxix
Hertel, K. J., Herschlag, D., Uhlenbeck, O. A kinetic and
thermodynamic framework for the hammerhead ribozyme reaction.
Biochemistry, (1994) 33, 3374-3385. Beigelman, L., et al., Chemical
modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270,
25702-25708. .sup.xxx Beigelman, L., et al., Chemical modifications
of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.
.sup.xxxi Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz,
Phillip. `Hairpin` catalytic RNA model: evidence for helixes and
sequence requirement for substrate RNA. Nucleic Acids Res. (1990),
18(2), 299-304. .sup.xxxii Chowrira, Bharat M.; Berzal-Herranz,
Alfredo; Burke, John M. . Novel guanosine requirement for catalysis
by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
.sup.xxxiii Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira,
Bharat M.; Butcher, Samuel E.; Burke, John M. . Essential
nucleotide sequences and secondary structure elements of the
hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73. .sup.xxxiv
Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.;
Butcher, Samuel E. . Substrate selection rules for the hairpin
ribozyme determined by in vitro selection, mutation, and analysis
of mismatched substrates. Genes Dev. (1993), 7(1), 130-8. .sup.xxxv
Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M. . In vitro
selection of active hairpin ribozymes by sequential RNA-catalyzed
cleavage and ligation reactions. Genes Dev. (1992), 6(1), 129-34.
.sup.xxxvi Hegg, Lisa A.; Fedor, Martha J. . Kinetics and
Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes.
Biochemistry (1995), 34(48), 15813-28. .sup.xxxvii Grasby, Jane A.;
Mersmann, Karin; Singh, Mohinder; Gait, Michael J. . Purine
Functional Groups in Essential Residues of the Hairpin Ribozyme
Required for Catalytic Cleavage of RNA. Biochemistry (1995),
34(12), 4068-76. .sup.xxxviii Schmidt, Sabine; Beigelman, Leonid;
Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik S.; Gait,
Michael J. . Base and sugar requirements for RNA cleavage of
essential nucleoside residues in internal loop B of the hairpin
ribozyme: implications for secondary
structure. Nucleic Acids Res. (1996), 24(4), 573-81. .sup.xxxix
Perrotta, Anne T.; Been, Michael D. . Cleavage of
oligoribonucleotides by a ribozyme derived from the hepatitis
.delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.
.sup.xl Perrotta, Anne T.; Been, Michael D. . A pseudoknot-like
structure required for efficient self-cleavage of hepatitis delta
virus RNA. Nature (London) (1991), 350(6317), 434-6. .sup.xli
Puttaraju, M.; Perrotta, Anne T.; Been, Michael D. . A circular
trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res.
(1993), 21(18), 4253-8.
TABLE-US-00002 TABLE II A. 2.5 .mu.mol Synthesis Cycle ABI 394
Instrument Reagent Equivalents Amount Wait Time* DNA Wait Time*
2'-O-methyl Wait Time*RNA Phosphoramidites 6.5 163 .mu.L 45 sec 2.5
min 7.5 min S-Ethyl Tetrazole 23.8 238 .mu.L 45 sec 2.5 min 7.5 min
Acetic Anhydride 100 233 .mu.L 5 sec 5 sec 5 sec N-Methyl Imidazole
186 233 .mu.L 5 sec 5 sec 5 sec TCA 176 2.3 mL 21 sec 21 sec 21 sec
Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 .mu.L 100
sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2 .mu.mol
Synthesis Cycle ABI 394 Instrument Reagent Equivalents Amount Wait
Time* DNA Wait Time* 2'-O-methyl Wait Time*RNA Phosphoramidites 15
31 .mu.L 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 .mu.L 45
sec 233 min 465 sec Acetic Anhydride 655 124 .mu.L 5 sec 5 sec 5
sec N-Methyl Imidazole 1245 124 .mu.L 5 sec 5 sec 5 sec TCA 700 732
.mu.L 10 sec 10 sec 10 sec Iodine 20.6 244 .mu.L 15 sec 15 sec 15
sec Beaucage 7.7 232 .mu.L 100 sec 300 sec 300 sec Acetonitrile NA
2.64 mL NA NA NA C. 0.2 .mu.mol Synthesis Cycle 96 well Instrument
Equivalents: DNA/ Amount: DNA/ Reagent 2'-O-methyl/Ribo
2'-O-methyl/Ribo Wait Time* DNA Wait Time* 2'-O-methyl Wait Time*
Ribo Phosphoramidites 22/33/66 40/60/120 .mu.L 60 sec 180 sec 360
sec S-Ethyl Tetrazole 70/105/210 40/60/120 .mu.L 60 sec 180 min 360
sec Acetic Anhydride 265/265/265 50/50/50 .mu.L 10 sec 10 sec 10
sec N-Methyl Imidazole 502/502/502 50/50/50 .mu.L 10 sec 10 sec 10
sec TCA 238/475/475 250/500/500 .mu.L 15 sec 15 sec 15 sec Iodine
6.8/6.8/6.8 80/80/80 .mu.L 30 sec 30 sec 30 sec Beaucage 34/51/51
80/120/120 100 sec 200 sec 200 sec Acetonitrile NA 1150/1150/1150
.mu.L NA NA NA *Wait time does not include contact time during
delivery.
TABLE-US-00003 TABLE 3 Peptides for Conjugation SEQ ID Peptide
Sequence NO ANTENNAP RQI KIW FQN RRM KWK K amide 14 EDIA Kaposi AAV
ALL PAV LLA LLA P + VQR 15 fibroblast KRQ KLMP growth factor caiman
MGL GLH LLV LAA ALQ GA 16 crocodylus Ig(5) light chain HIVenvelope
GAL FLG FLG AAG STM GA + PKS 17 glycoprotein KRK 5 (NLS of the
SV40) gp41 HIV-1 Tat RKK RRQ RRR 18 Influenza
GLFEAIAGFIENGWEGMIDGGGYC 19 hemagglutinin envelop glycoprotein RGD
peptide X-RGD-X 20 where X is any amino acid or peptide transportan
A GWT LNS AGY LLG KIN LKA LAA 21 LAK KIL Somatostatin (S)FC YWK TCT
22 (tyr-3- octreotate) Pre-S-peptide (S)DH QLN PAF 23 (S)optional
Serine for coupling Italic = optional D isomer for stability
TABLE-US-00004 TABLE 4 Non-limiting examples of Stabilization
Chemistries for chemically modified siNA constructs Chemistry
pyrimidine Purine cap p = S Strand "Stab 1" Ribo Ribo -- 5 at
5'-end S/AS 1 at 3'-end "Stab 2" Ribo Ribo -- All linkages Usually
AS "Stab 3" 2'-fluoro Ribo -- 4 at 5'-end Usually S 4 at 3'-end
"Stab 4" 2'-fluoro Ribo 5' and 3'-ends -- Usually S "Stab 5"
2'-fluoro Ribo -- 1 at 3'-end Usually AS "Stab 6" 2'-O-Methyl Ribo
5' and 3'-ends -- Usually S "Stab 7" 2'-fluoro 2'-deoxy 5' and
3'-ends -- Usually S "Stab 8" 2'-fluoro 2'-O-Methyl -- 1 at 3'-end
S or AS "Stab 9" Ribo Ribo 5' and 3'-ends -- Usually S "Stab 10"
Ribo Ribo -- 1 at 3'-end Usually AS "Stab 11" 2'-fluoro 2'-deoxy --
1 at 3'-end Usually AS Stab 12 2'-fluoro LNA 5' and 3'-ends Usually
S "Stab 13" 2'-fluoro LNA 1 at 3'-end Usually AS "Stab 14"
2'-fluoro 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end "Stab 15"
2'-deoxy 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end "Stab 16 Ribo
2'-O-Methyl 5' and 3'-ends Usually S "Stab 17" 2'-O-Methyl
2'-O-Methyl 5' and 3'-ends Usually S "Stab 18" 2'-fluoro
2'-O-Methyl 1 at 3'-end Usually AS CAP = any terminal cap, such as
inverted deoxy abasic, glyceryl, or a conjugate moiety. All Stab
1-18 chemistries can comprise 3'-terminal thymidine (TT) residues
All Stab 1-18 chemistries typically comprise 21 nucleotides, but
can vary as described herein. S = sense strand AS = antisense
strand
Sequence CWU 1
1
24110RNAArtificial SequenceDescription of Artificial Sequence
Example of a Stem II region 1gccguuaggc 10215RNAArtificial
SequenceDescription of Artificial Sequence Generic Target Nucleic
Acid 2nnnnnnunnn nnnnn 15336RNAArtificial SequenceDescription of
Artificial Sequence Enzymatic Nucleic Acid 3nnnnnnncug augagnnnga
aannncgaaa nnnnnn 36414RNAArtificial SequenceDescription of
Artificial Sequence Generic Target Nucleic Acid 4nnnnncnnnn nnnn
14535RNAArtificial SequenceDescription of Artificial Sequence
Enzymatic Nucleic Acid 5nnnnnnncug augagnnnga aannncgaan nnnnn
35615RNAArtificial SequenceDescription of Artificial Sequence
Generic Target Nucleic Acid 6nnnnnnngnn nnnnn 15735RNAArtificial
SequenceDescription of Artificial Sequence Enzymatic Nucleic Acid
7nnnnnnnuga uggcaugcac uaugcgcgnn nnnnn 35848RNAArtificial
SequenceDescription of Artificial Sequence Enzymatic Nucleic Acid
8gugugcaacc ggaggaaacu cccuucaagg acgaaagucc gggacggg
48916RNAArtificial SequenceDescription of Artificial Sequence
Target Nucleic Acid 9gccguggguu gcacac 161036RNAArtificial
SequenceDescription of Artificial Sequence Enzymatic Nucleic Acid
10gugccuggcc gaaaggcgag ugaggucugc cgcgcn 361115RNAArtificial
SequenceDescription of Artificial Sequence Target Nucleic Acid
11gcgcggcgca ggcac 151216DNAArtificial SequenceDescription of
Artificial Sequence Enzymatic Nucleic Acid Motif 12rggctagcta
caacga 161333RNAArtificial SequenceDescription of Artificial
Sequence Enzymatic Nucleic Acid 13gcaguggccg aaaggcgagu gaggucuagc
uca 331416PRTArtificial Sequencemisc_featureSynthetic peptide 14Arg
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
151526PRTArtificial Sequencemisc_featureSynthetic peptide 15Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Val
Gln Arg Lys Arg Gln Lys Leu Met Pro 20 251617PRTArtificial
Sequencemisc_featureSynthetic peptide 16Met Gly Leu Gly Leu His Leu
Leu Val Leu Ala Ala Ala Leu Gln Gly1 5 10 15Ala1724PRTArtificial
Sequencemisc_featureSynthetic peptide 17Gly Ala Leu Phe Leu Gly Phe
Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Pro Lys Ser Lys Arg
Lys Val 20189PRTArtificial Sequencemisc_featureSynthetic peptide
18Arg Lys Lys Arg Arg Gln Arg Arg Arg1 51924PRTArtificial
Sequencemisc_featureSynthetic peptide 19Gly Leu Phe Glu Ala Ile Ala
Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Gly Gly
Tyr Cys 20205PRTArtificial Sequencemisc_feature(1)..(1)Xaa stands
for any amino acid 20Xaa Arg Gly Asp Xaa1 52127PRTArtificial
Sequencemisc_featureSynthetic peptide 21Gly Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Lys Ile Asn Leu1 5 10 15Lys Ala Leu Ala Ala Leu
Ala Lys Lys Ile Leu 20 25229PRTArtificial
Sequencemisc_feature(1)..(1)Ser stands for optional Serine for
coupling 22Ser Phe Cys Tyr Trp Lys Thr Cys Thr1 5239PRTArtificial
Sequencemisc_feature(1)..(1)Ser stands for optional Serine for
coupling 23Ser Asp His Gln Leu Asn Pro Ala Phe1 52434RNAArtificial
SequenceDescription of Artificial Sequence Nucleic Acid
24gaguugcuga ugaggccgaa aggccgaaag ucug 34
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