U.S. patent application number 12/969393 was filed with the patent office on 2011-06-16 for sirna and methods of manufacture.
This patent application is currently assigned to OPKO OPHTHALMICS, LLC. Invention is credited to N. Nicole Endejann, Samuel Jotham Reich.
Application Number | 20110143400 12/969393 |
Document ID | / |
Family ID | 38983407 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143400 |
Kind Code |
A1 |
Reich; Samuel Jotham ; et
al. |
June 16, 2011 |
SIRNA AND METHODS OF MANUFACTURE
Abstract
Double-stranded RNA of about 19 to about 25 nucleotides in
length capable of regulating gene expression by RNA interference is
provided. Such double-stranded RNA are particularly useful for
treating disease or conditions associated with a target mRNA or
gene. Methods of manufacture and methods of use of the
double-stranded RNA are also provided.
Inventors: |
Reich; Samuel Jotham; (Miami
Beach, FL) ; Endejann; N. Nicole; (Streetsboro,
OH) |
Assignee: |
OPKO OPHTHALMICS, LLC
Miami
FL
|
Family ID: |
38983407 |
Appl. No.: |
12/969393 |
Filed: |
December 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11851145 |
Sep 6, 2007 |
7872118 |
|
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12969393 |
|
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60824953 |
Sep 8, 2006 |
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Current U.S.
Class: |
435/91.3 ;
536/24.5 |
Current CPC
Class: |
C12N 2310/14 20130101;
C12N 15/1136 20130101; C12N 2330/30 20130101; C12N 15/111
20130101 |
Class at
Publication: |
435/91.3 ;
536/24.5 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C07H 21/02 20060101 C07H021/02 |
Claims
1. A method of preparing a double-stranded RNA molecule comprising
(a) synthesizing a first set of two or more RNA sub-strands,
wherein the combined length of the first set of sub-strands is from
about 19 to about 25 nucleotides, (b) synthesizing a second set of
two or more RNA sub-strands, wherein the combined length of the
second set of sub-strands is from about 19 to about 25 nucleotides;
(c) combining the synthesized first and second set of RNA
sub-strands under conditions, wherein a double-stranded RNA
molecule is formed
2. The method according to claim 1, wherein the RNA strands are
chemically synthesized.
3. The method according to claim 1, wherein the RNA strands are
enzymatically synthesized.
4. The method of claim 1, wherein the double-stranded RNA molecule
comprises a single double stranded region and single stranded
regions of about 1 to about 5 nucleotides at the 3' ends of at
least one of the strands of the double-stranded RNA molecule.
5. The method of claim 1, wherein both strands of the
double-stranded RNA molecule each have a 3'-overhang from about 1
to about 5 nucleotides.
6. The method of claim 1, wherein both strands of the
double-stranded RNA molecule each have a 3'-overhang of about 2
nucleotides.
7. The method of claim 1, wherein each strand has a length from
20-22 nucleotides.
8. The method of claim 1, wherein the double-stranded RNA molecule
comprises at least one sugar-modified nucleotide, wherein the 2'-OH
group of said sugar-modified nucleotide is replaced by a group
selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, N(R).sub.2 or
CN, wherein R is C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo
is F, C, Br or I.
9. The method of claim 1, wherein the double stranded RNA molecule
comprises at least one backbone-modified nucleotide containing a
phosphorothioate group.
10.-18. (canceled)
19. A method of preparing a double-stranded RNA molecule comprising
(a) synthesizing a first set of two or more RNA sub-strands,
wherein the combined length of the first set of sub-strands is 25
or fewer nucleotides, (b) synthesizing a second set of two or more
RNA sub-strands, wherein the combined length of the second set of
sub-strands is 25 or fewer nucleotides; (c) combining the
synthesized first and second set of RNA sub-strands under
conditions, wherein a double-stranded RNA molecule is formed.
20. The method of claim 19, wherein the double-stranded RNA
molecule comprises a single double stranded region and single
stranded regions of about 1 to about 5 nucleotides at the 3' ends
of at least one of the strands of the double-stranded RNA
molecule.
21. The method of claim 20, wherein each strand has a length from
20-22 nucleotides.
22.-24. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Application No.
60/824,953 filed Sep. 8, 2006 entitled "siRNA and Methods of
Manufacture", herein incorporated by reference in its entirety.
GOVERNMENT INTERESTS
[0002] Not applicable
PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not applicable
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
BACKGROUND OF THE INVENTION
[0004] Not Applicable
Field of Invention
[0005] Not applicable
Description of Related Art
[0006] Not applicable
SUMMARY OF THE INVENTION
[0007] One embodiment of the present invention provides methods of
manufacturing isolated siRNA that may be useful as therapeutic
agents. In certain embodiments, such methods may include the
synthesis of two or more nucleotide strands. In further
embodiments, the method may include the synthesis of three, four or
more nucleotide sub-strands, wherein the total length of the sets
of sub-strands is from about 19 to about 25 nucleotides in
length.
[0008] In one embodiment, the method of preparing a double-stranded
RNA molecule comprises synthesizing a first set of two or more RNA
sub-strands, wherein the combined length of the first set of
sub-strands is from about 19 to about 25 nucleotides, synthesizing
a second set of two or more RNA sub-strands, wherein the combined
length of the second set of sub-strands is from about 19 to about
25 nucleotides; and combining the synthesized first and second set
of RNA sub-strands under conditions, wherein a double-stranded RNA
molecule is formed which is at 19 to at 25 nucleotides in length,
and further may have single stranded regions of about 1 to about 5
nucleotides at the 3' ends of at least one of the strands of the
double-stranded RNA molecule.
[0009] In another embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing one RNA strand
having a length from about 19 to about 25 nucleotides, (b)
synthesizing a second RNA strand and a third RNA strand, wherein
the combined length of the second RNA strand and the third RNA
strand is from about 19 to about 25 nucleotides; and combining the
synthesized RNA strands under conditions, wherein a double-stranded
RNA molecule is formed, wherein the double-stranded RNA molecule
consists of a single double stranded region and single stranded
regions of about 1 to about 5 nucleotides at the 3' ends of at
least one of the strands of the double-stranded RNA molecule.
[0010] In a further embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing a first set of
two or more RNA sub-strands, wherein the combined length of the
first set of sub-strands is 25 or fewer nucleotides, synthesizing a
second set of two or more RNA sub-strands, wherein the combined
length of the second set of sub-strands is 25 or fewer nucleotides;
and combining the synthesized first and second set of RNA
sub-strands under conditions, wherein a double-stranded RNA
molecule is formed.
[0011] In another embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing one RNA strand
having a length from 25 or fewer nucleotides, synthesizing a second
RNA strand and a third RNA strand, wherein the combined length of
the second RNA strand and the third RNA strand is 25 or fewer
nucleotides; and combining the synthesized RNA strands under
conditions, wherein a double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule consists of a single
double stranded region and single stranded regions of about 1 to
about 5 nucleotides at the 3' ends of at least one of the strands
of the double-stranded RNA molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] For a fuller understanding of the nature and advantages of
the present invention, reference should be had to the following
detailed description taken in connection with the accompanying
drawings, in which:
[0013] FIG. 1 is a bar graph comparing the efficacy of a specific
siRNA targeting VEGF prepared by synthesizing and combining two
strands of RNA (Cand5) and a specific siRNA targeting VEGF prepared
by synthesizing and combining three strands of RNA without ligase
(Cand5 mixture) or with ligase (Cand 5 mixture-ligated) 293 cells
at various concentrations.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Before the present compositions and methods are described,
it is to be understood that this invention is not limited to the
particular processes, compositions, or methodologies described, as
these may vary. It is also to be understood that the terminology
used in the description is for the purpose of describing the
particular versions or embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims. Unless defined otherwise, all technical and
scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of embodiments of the
present invention, the preferred methods, devices, and materials
are now described. All publications mentioned herein are
incorporated by reference in their entirety. Nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0015] It must also be noted that as used herein and in the
appended claims, the singular forms "a", "an", and "the" include
plural reference unless the context clearly dictates otherwise.
Thus, for example, reference to a "molecule" is a reference to one
or more molecules and equivalents thereof known to those skilled in
the art, and so forth. As used herein, the term "about" means plus
or minus 10% of the numerical value of the number with which it is
being used. Therefore, about 50% means in the range of 45%-55%.
[0016] Unless otherwise indicated, all nucleic acid sequences
herein are given in the 5' to 3' direction. Also, all
deoxyribonucleotides in a nucleic acid sequence are represented by
capital letters (e.g., deoxythymidine is "T"), and ribonucleotides
in a nucleic acid sequence are represented by lower case letters
(e.g., uridine is "u").
[0017] RNA interference (hereinafter "RNAi") is a method of
post-transcriptional gene regulation that is conserved throughout
many eukaryotic organisms. RNAi is induced by short (i.e., <30
nucleotide) double stranded RNA ("dsRNA") molecules which are
present in the cell. These short dsRNA molecules, called "short
interfering RNA" or "siRNA," cause the destruction of messenger
RNAs ("mRNAs") which share sequence homology with the siRNA to
within one nucleotide resolution. It is believed that the siRNA and
the targeted mRNA bind to an "RNA-induced silencing complex" or
"RISC", which cleaves the targeted mRNA. The siRNA is apparently
recycled much like a multiple-turnover enzyme, with 1 siRNA
molecule capable of inducing cleavage of approximately 1000 mRNA
molecules. siRNA-mediated RNAi degradation of an mRNA is therefore
more effective than currently available technologies for inhibiting
expression of a target gene.
[0018] Synthetic siRNA of 21 and 22 nucleotides in length, and
which have short 3' overhangs, are able to induce RNAi of target
mRNA in a Drosophila cell lysate. Cultured mammalian cells also
exhibit RNAi degradation with synthetic siRNA, and RNAi degradation
induced by synthetic siRNA has recently been shown in living mice.
The therapeutic potential of siRNA-induced RNAi degradation has
been demonstrated in several recent in vitro studies, including the
siRNA-directed inhibition of HIV-1 infection and reduction of
neurotoxic polyglutamine disease protein expression. siRNA-directed
inhibition of VEGF has also been demonstrated as set forth in U.S.
Pat. No. 7,148,342, filed Nov. 4, 2002, herein incorporated by
reference in its entirety.
[0019] One aspect of the present invention provides isolated siRNA
comprising short double-stranded RNA from about 17 nucleotides to
about 29 nucleotides in length, preferably from about 19 to about
25 nucleotides in length, that are targeted to a given mRNA. The
siRNA comprise a sense RNA strand and a complementary antisense RNA
strand annealed together by standard Watson-Crick base-pairing
interactions (hereinafter "base-paired"). In one embodiment, as is
described in more detail below, the sense strand comprises a
nucleic acid sequence which is identical to a target sequence
contained within the target mRNA. In a further embodiment, the
sequence of the double-stranded RNA molecule of the present
invention has to have a sufficient identity to a nucleic acid
target molecule in order to mediate target-specific RNAi and/or DNA
methylation. Preferably, the sequence has an identity of at least
50%, particularly of at least 70% to the desired target molecule in
the double-stranded portion of the RNA molecule. More preferably,
the identity is a least 85% and most preferably 100% in the
double-stranded portion of the RNA molecule. The identity of a
double-stranded RNA molecule to a predetermined nucleic acid target
molecule, e.g. an mRNA target molecule may be determined as
follows:
I=n.times.100
wherein I is the identity in percent, n is the number of identical
nucleotides in the double-stranded portion of the dsRNA and the
target and L is the length of the sequence overlap of the
double-stranded portion of the dsRNA and the target. Alternatively,
the identity of the double-stranded RNA molecule to the target
sequence may also be defined including the 3' overhand,
particularly an overhang having a length from 1-3 nucleotides. In
this case the sequence identity is preferably at least 50%, more
preferably at least 70% and most preferably at least 85% to the
target sequence. For example, the nucleotides from the 3' overhang
and up to 2 nucleotides from the 5' and/or 3' terminus of the
double strand may be modified without significant loss of
activity.
[0020] The sense and antisense strands of the present siRNA can
comprise two complementary, single-stranded RNA molecules or can
comprise a single molecule in which two complementary portions are
base-paired and are covalently linked by a single-stranded
"hairpin" area. Without wishing to be bound by any theory, it is
believed that the hairpin area of the latter type of siRNA molecule
is cleaved intracellularly by the "Dicer" protein (or its
equivalent) to form an siRNA of two individual base-paired RNA
molecules.
[0021] As used herein, "isolated" means altered or removed from the
natural state through human intervention. For example, an siRNA
naturally present in a living animal is not "isolated," but a
synthetic siRNA, or an siRNA partially or completely separated from
the coexisting materials of its natural state is "isolated." An
isolated siRNA can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a cell into
which the siRNA has been delivered.
[0022] The target gene to which the RNA molecule of the invention
is directed may be associated with a pathological condition. For
example, the gene may be a pathogen-associated gene, e.g. a viral
gene, a tumor-associated gene or an autoimmune disease-associated
gene. The target gene may also be a heterologous gene expressed in
a recombinant cell or a genetically altered organism. By
determining or modulating, particularly, inhibiting the function of
such a gene valuable information and therapeutic benefits in the
agricultural field or in the medicine or veterinary medicine field
may be obtained. In a preferred embodiment, the target gene
selected from VEGF (see U.S. Pat. No. 7,148,342, herein
incorporated by reference in its entirety), HIF-1 .alpha. (see U.S.
Publication No. 20040180357 (10/699,557) herein incorporated by
reference in its entirety), ICAM-1 (see U.S. Publication No.
20040220129 (10/759,878), herein incorporated by reference in its
entirety), Angiopoietin 1, Angiopoietin 2 and Tie 2 (see U.S.
Publication No. 20040248174 (10/827,759), herein incorporated by
reference in its entirety) and complement, including C3 (see U.S.
application Ser. No. 11/615,554, herein incorporated by reference
in its entirety).
[0023] Genes can be analyzed for further alternative splice forms
using techniques well-known in the art. Such techniques include
reverse transcription-polymerase chain reaction (RT-PCR), northern
blotting and in-situ hybridization. Techniques for analyzing mRNA
sequences are described, for example, in Busting S A (2000), J.
Mol. Endocrinol, 25: 169-193, the entire disclosure of which is
herein incorporated by reference. Representative techniques for
identifying alternatively spliced mRNAs are also described
below.
[0024] For example, databases that contain nucleotide sequences
related to a given disease gene can be used to identify
alternatively spliced mRNA. Such databases include GenBank, Embase,
and the Cancer Genome Anatomy Project (CGAP) database. The CGAP
database, for example, contains expressed sequence tags (ESTs) from
various types of human cancers. An mRNA or gene sequence from the
target genes can be used to query such a database to determine
whether ESTs representing alternatively spliced mRNAs have been
found for a these genes.
[0025] A technique called "RNAse protection" can also be used to
identify alternatively spliced mRNAs. RNAse protection involves
translation of a gene sequence into synthetic RNA, which is
hybridized to RNA derived from other cells; for example, cells from
tissue at or near the site of neovascularization. The hybridized
RNA is then incubated with enzymes that recognize RNA:RNA hybrid
mismatches. Smaller than expected fragments indicate the presence
of alternatively spliced mRNAs. The putative alternatively spliced
mRNAs can be cloned and sequenced by methods well known to those
skilled in the art.
[0026] RT-PCR can also be used to identify alternatively spliced in
RNAs. In RT-PCR, mRNA from the diseased tissue is converted into
cDNA by the enzyme reverse transcriptase, using methods well-known
to those of ordinary skill in the art. The entire coding sequence
of the cDNA is then amplified via PCR using a forward primer
located in the 3' untranslated region, and a reverse primer located
in the 5' untranslated region. The amplified products can be
analyzed for alternative splice forms, for example by comparing the
size of the amplified products with the size of the expected
product from normally spliced mRNA, e.g., by agarose gel
electrophoresis. Any change in the size of the amplified product
can indicate alternative splicing.
[0027] mRNA produced from mutant genes can also be readily
identified through the techniques described above for identifying
alternative splice forms. As used herein, "mutant" genes or mRNA
include genes or mRNA which differ in sequence from the known
sequences. Thus, allelic forms of these genes, and the mRNA
produced from them, are considered "mutants" for purposes of this
invention.
[0028] It is understood that a given mRNA may contain target
sequences in common with their respective alternative splice forms,
cognates or mutants. A single siRNA comprising such a common
targeting sequence can therefore induce RNAi-mediated degradation
of different RNA types which contain the common targeting
sequence.
[0029] The siRNA can comprise partially purified RNA, substantially
pure RNA, synthetic RNA, or recombinantly produced RNA, as well as
altered RNA that differs from naturally-occurring RNA by the
addition, deletion, substitution and/or alteration of one or more
nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the siRNA or to
one or more internal nucleotides of the siRNA, including
modifications that make the siRNA resistant to nuclease
digestion.
[0030] One or both strands of the siRNA can also comprise a 3'
overhang. As used herein, a "3' overhang" refers to at least one
unpaired nucleotide extending from the 3'-end of a duplexed RNA
strand.
[0031] Thus in one embodiment, the siRNA of the invention comprises
at least one 3' overhang of from 1 to about 6 nucleotides (which
includes ribonucleotides or deoxynucleotides) in length, preferably
from 1 to about 5 nucleotides in length, more preferably from 1 to
about 4 nucleotides in length, or from about 1 to about 3
nucleotides in length and particularly preferably from about 2 to
about 4 nucleotides in length, and more preferably about 2
nucleotides in length.
[0032] In the embodiment in which both strands of the siRNA
molecule comprise a 3' overhang, the length of the overhangs can be
the same or different for each strand. In a most preferred
embodiment, the 3' overhang is present on both strands of the
siRNA, and is 2 nucleotides in length. For example, each strand of
the siRNA of the invention can comprise 3' overhangs of
dithymidylic acid (TT) or diuridylic acid ("uu").
[0033] In order to enhance the stability of the present siRNA, the
3' overhangs can be also stabilized against degradation. In one
embodiment, the overhangs are stabilized by including purine
nucleotides, such as adenosine or guanosine nucleotides.
Alternatively, for example 2'-deoxythymidine is tolerated and does
not affect the efficiency of RNAi degradation. In particular, the
absence of a 2' hydroxyl in the 2'-deoxythymidine significantly
enhances the nuclease resistance of the 3' overhang in tissue
culture medium.
[0034] In certain embodiments, the siRNA comprises the sequence
AA(N19)TT or NA(N21), where N is any nucleotide. These siRNA
comprise approximately 30-70% GC, and preferably comprise
approximately 50% G/C. The sequence of the sense siRNA strand
corresponds to (N19)TT or N21 (i.e., positions 3 to 23),
respectively. In the latter case, the 3' end of the sense siRNA is
converted to TT. The rationale for this sequence conversion is to
generate a symmetric duplex with respect to the sequence
composition of the sense and antisense strand 3' overhangs. The
antisense RNA strand is then synthesized as the complement to
positions 1 to 21 of the sense strand.
[0035] Because position 1 of the 23-nt sense strand in these
embodiments is not recognized in a sequence-specific manner by the
antisense strand, the 3'-most nucleotide residue of the antisense
strand can be chosen deliberately. However, the penultimate
nucleotide of the antisense strand (complementary to position 2 of
the 23-nt sense strand in either embodiment) is generally
complementary to the targeted sequence.
[0036] In another embodiment, the siRNA comprises the sequence
NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a
pyrimidine C or U/T). The respective 21-nt sense and antisense RNA
strands of this embodiment therefore generally begin with a purine
nucleotide. Such siRNA can be expressed from pol III expression
vectors without a change in targeting site, as expression of RNAs
from pol III promoters is only believed to be efficient when the
first transcribed nucleotide is a purine.
[0037] The siRNA can be targeted to any stretch of approximately
19-25 contiguous nucleotides in any of the target mRNA sequences
(the "target sequence"). Techniques for selecting target sequences
for siRNA are given, for example, in Tuschl T et al., "The siRNA
User Guide," revised the entire disclosure of which is herein
incorporated by reference. "The siRNA User Guide" is available on
the world wide web at a website maintained by Dr. Thomas Tuschl,
Department of Cellular Biochemistry, AG 105, Max-Planck-Institute
for Biophysical Chemistry, 37077 Gottingen, Germany, and can be
found by accessing the website of the Max Planck Institute and
searching with the keyword "siRNA." Thus, the sense strand of the
present siRNA comprises a nucleotide sequence identical to any
contiguous stretch of about 19 to about 25 nucleotides in the
target mRNA.
[0038] Generally, a target sequence on the target mRNA can be
selected from a given cDNA sequence corresponding to the target
mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'
direction) from the start codon. The target sequence can, however,
be located in the 5' or 3' untranslated regions, or in the region
nearby the start codon (see, e.g., the target sequences of SEQ ID
NOS: 73 and 74 in Table 1 below, which are within 100 nt of the
5'-end of the VEGF.sub.121 cDNA).
[0039] For example, a suitable target sequence in the VEGF.sub.121
cDNA sequence is:
TABLE-US-00001 TCATCACGAAGTGGTGAAG (SEQ ID NO: 1)
[0040] Thus, an siRNA of the invention targeting this sequence, and
which has 3' uu overhangs on each strand (overhangs shown in bold),
is:
TABLE-US-00002 5'-ucaucacgaaguggugaaguu-3' (SEQ ID NO: 2)
3'-uuaguagugcuucaccacuuc-5' (SEQ ID NO: 3)
[0041] An siRNA of the invention targeting this same sequence, but
having 3' TT overhangs on each strand (overhangs shown in bold)
is:
TABLE-US-00003 5'-ucaucacgaaguggugaagTT-3' (SEQ ID NO: 4)
3'-TTaguagugcuucaccacuuc-5' (SEQ ID NO: 5)
[0042] Preferably, the siRNA are chemically synthesized using
appropriately protected ribonucleoside phosphoramidites and a
conventional DNA/RNA synthesizer. The siRNA can be synthesized as
two separate, complementary RNA molecules, or as a single RNA
molecule with two complementary regions. Commercial suppliers of
synthetic RNA molecules or synthesis reagents include Proligo
(Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA),
Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen
Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and
Cruachem (Glasgow, UK).
[0043] In one embodiment, the double-stranded RNA molecule may be
prepared by synthesizing two RNA strands each having a length from
about 19 to about 25, e.g. from about 19 to about 23, nucleotides,
wherein said RNA strands are capable of forming a double-stranded
RNA molecule, wherein preferably at least one strand has a
3'-overhang from 1-5 nucleotides, and combining the synthesized RNA
strands under conditions, where a double-stranded RNA molecule is
formed, which is capable of mediating target-specific nucleic acid
modifications, particularly RNA interference and/or DNA
methylation.
[0044] In another embodiment, the double-stranded RNA molecule may
be prepared by synthesizing multiple RNA strands and combining the
multiple strands under conditions where a double-stranded RNA
molecule is formed that is capable of mediating target-specific
nucleic acid modifications, particularly RNA interference and/or
DNA methylation. In one embodiment, three RNA strands may be
synthesized, wherein one RNA strand has a length from about 19 to
about 25 nucleotides and the second RNA strand, a first sub-strand,
has a length from about 1 to about 24 nucleotides and the third RNA
strand, a second sub-strand, has a length from about 1 to about 24
nucleotides, such that when the RNA sub-strands are combined they
have a total length of about 19 to about 25 nucleotides in length.
For example, one RNA strand may have a length from about 19
nucleotides, a first RNA sub-strand may have a length of two
nucleotides and a second RNA sub-strand may have a length of about
17 nucleotides. In a further example, one RNA strand may have a
length from about 19 nucleotides, a second RNA strand may have a
length of 3 nucleotides and a third RNA strand may have a length of
about 16 nucleotides.
[0045] In one preferred embodiment, the double-stranded RNA
molecule may be prepared by synthesizing three RNA strands, each of
a different length, for example, one RNA strand has a length of
about 21 nucleotides, a second RNA strand has a length of about 10
nucleotides and a third RNA strand has a length of about 11
nucleotides. The three synthesized RNA strands are combined under
conditions where a double-stranded RNA molecule is formed that has
a 2 nucleotide overhang on the 3' ends of each RNA strand. In an
additional embodiment, a ligase may be added to ligate the two RNA
strands of the shortest length.
[0046] In another embodiment, the double-stranded RNA molecule may
be prepared by synthesizing multiple RNA sub-strands and combining
the multiple strands under conditions where a double-stranded RNA
molecule is formed that is capable of mediating target-specific
nucleic acid modifications, particularly RNA interference and/or
DNA methylation. For example, four or more RNA sub-strands may be
synthesized and combined to form a double-stranded molecule that is
capable of RNA interference, wherein the total length of each of
the two strands, after combining the sub-strands, that make up the
double-stranded molecule has a length of about 19 to about 25
nucleotides.
[0047] In one embodiment, the method of preparing a double-stranded
RNA molecule comprises synthesizing a first set of two or more RNA
sub-strands, wherein the combined length of the first set of
sub-strands is from about 19 to about 25 nucleotides, synthesizing
a second set of two or more RNA sub-strands, wherein the combined
length of the second set of sub-strands is from about 19 to about
25 nucleotides; and combining the synthesized first and second set
of RNA sub-strands under conditions, wherein a double-stranded RNA
molecule is formed.
[0048] In a further embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing one RNA strand
having a length from about 19 to about 25 nucleotides, synthesizing
a second RNA strand and a third RNA strand, wherein the combined
length of the second RNA strand and the third RNA strand is from
about 19 to about 25 nucleotides; combining the synthesized RNA
strands under conditions, wherein a double-stranded RNA molecule is
formed, wherein the double-stranded RNA molecule consists of a
single double stranded region and single stranded regions of about
1 to about 5 nucleotides at the 3' ends of at least one of the
strands of the double-stranded RNA molecule.
[0049] One embodiment comprises synthesizing a first set of two or
more RNA sub-strands, wherein the combined length of the first set
of sub-strands is from about 19 to about 25 nucleotides,
synthesizing a second set of two or more RNA sub-strands, wherein
the combined length of the second set of sub-strands is from about
19 to about 25 nucleotides; and combining the synthesized first and
second set of RNA sub-strands under conditions, wherein a
double-stranded RNA molecule is formed.
[0050] In one embodiment, the method of preparing a double-stranded
RNA molecule comprises synthesizing a first set of two or more RNA
sub-strands, wherein the combined length of the first set of
sub-strands is from about 19 to about 25 nucleotides, synthesizing
a second set of two or more RNA sub-strands, wherein the combined
length of the second set of sub-strands is from about 19 to about
25 nucleotides; and combining the synthesized first and second set
of RNA sub-strands under conditions, wherein a double-stranded RNA
molecule is formed.
[0051] In another embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing one RNA strand
having a length from about 19 to about 25 nucleotides, (b)
synthesizing a second RNA strand and a third RNA strand, wherein
the combined length of the second RNA strand and the third RNA
strand is from about 19 to about 25 nucleotides; and combining the
synthesized RNA strands under conditions, wherein a double-stranded
RNA molecule is formed, wherein the double-stranded RNA molecule
consists of a single double stranded region and single stranded
regions of about 1 to about 5 nucleotides at the 3' ends of at
least one of the strands of the double-stranded RNA molecule.
[0052] In a further embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing a first set of
two or more RNA sub-strands, wherein the combined length of the
first set of sub-strands is 25 or fewer nucleotides, synthesizing a
second set of two or more RNA sub-strands, wherein the combined
length of the second set of sub-strands is 25 or fewer nucleotides;
and combining the synthesized first and second set of RNA
sub-strands under conditions, wherein a double-stranded RNA
molecule is formed
[0053] In another embodiment, a method of preparing a
double-stranded RNA molecule comprises synthesizing one RNA strand
having a length from 25 or fewer nucleotides, synthesizing a second
RNA strand and a third RNA strand, wherein the combined length of
the second RNA strand and the third RNA strand is 25 or fewer
nucleotides; and combining the synthesized RNA strands under
conditions, wherein a double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule consists of a single
double stranded region and single stranded regions of about 1 to
about 5 nucleotides at the 3' ends of at least one of the strands
of the double-stranded RNA molecule.
[0054] It is to be understood that in each of the foregoing
embodiments, a sub-strand is at least 2 nucleotides in length, and
may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, or 23 nucleotides in length.
[0055] In each of the foregoing methods, the RNA strands or
sub-strands may be chemically or enzymatically synthesized. The
double-stranded RNA molecule comprises a single double stranded
region and single stranded regions of about 1 to about 5
nucleotides at the 3' ends of at least one or both of the strands
of the double-stranded RNA molecule. For example, in preferred
embodiments, both strands of the double-stranded RNA molecule each
have a 3'-overhang from about 1 to about 3 nucleotides, preferably
about 2 nucleotides. In each of the foregoing methods, each strand
may have a length from 19 to about 25 nucleotides, preferably about
20-22 nucleotides.
[0056] The double-stranded RNA molecule may comprises at least one
sugar-modified nucleotide, wherein the 2'-OH group of said
sugar-modified nucleotide is replaced by a group selected from H,
OR, R, halo. SH, SR, NH.sub.2, NHR, N(R).sub.2 or, wherein R is
C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, C, Br or
I. The double-stranded RNA molecule may comprise at least one
backbone-modified nucleotide containing a phosphorothioate
group.
[0057] In another embodiment, a method of preparing a
double-stranded RNA molecule comprises selecting a target mammalian
mRNA or target gene sequence, synthesizing a first RNA strand
having a length from about 19 to about 25 nucleotides, wherein the
first RNA strand is complementary to contiguous nucleotides in the
target sequence, synthesizing a second RNA strand; synthesizing a
third RNA strand, wherein the second and third RNA strands are
complementary to about 16 to about 24 nucleotides from the first
RNA strand; combining the synthesized RNA strands under conditions
suitable to form a double stranded RNA molecule, wherein said
double stranded RNA molecule consists of a single double stranded
region of from about 16 to about 24 nucleotides in length and one
or two single stranded 3' overhang regions of about 1 to about 3
nucleotides in length each.
[0058] Methods of synthesizing RNA molecules are known in the art.
In this context, it is particularly referred to chemical synthesis
methods as described in Verma and Eckstein (1998). The
single-stranded RNAs can also be prepared by enzymatic
transcription from synthetic DNA templates or from DNA plasmids
isolated from recombinant bacteria. Typically, phage RNA
polymerases are used such as T7, T3 or SP6 RNA polymerase (Milligan
and Uhlembeck (1989)).
[0059] Alternatively, siRNA can also be expressed from recombinant
circular or linear DNA plasmids using any suitable promoter.
Suitable promoters for expressing siRNA of the invention from a
plasmid include, for example, the U6 or H1 RNA poi III promoter
sequences and the cytomegalovirus promoter. Selection of other
suitable promoters is within the skill in the art. The recombinant
plasmids of the invention can also comprise inducible or
regulatable promoters for expression of the siRNA in a particular
tissue or in a particular intracellular environment.
[0060] The siRNA expressed from recombinant plasmids can either be
isolated from cultured cell expression systems by standard
techniques, or can be expressed intracellularly at or near the area
of neovascularization in vivo. The use of recombinant plasmids to
deliver siRNA of the invention to cells in vivo is discussed in
more detail below.
[0061] siRNA of the invention can be expressed from a recombinant
plasmid either as two separate, complementary RNA molecules, or as
a single RNA molecule with two complementary regions.
[0062] Selection of plasmids suitable for expressing siRNA of the
invention, methods for inserting nucleic acid sequences for
expressing the siRNA into the plasmid, and methods of delivering
the recombinant plasmid to the cells of interest are within the
skill in the art. See, for example Tuschl, T. (2002), Nat.
Biotechnol, 20: 446-448; Brummelkamp T R et al. (2002), Science
296: 550-553; Miyagishi M et al. (2002), Nat. Biotechnol. 20:
497-500; Paddison P J et al. (2002), Genes Dev. 16: 948-958; Lee N
S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul C P et al.
(2002), Nat. Biotechnol. 20: 505-508, the entire disclosures of
which are herein incorporated by reference.
[0063] A plasmid comprising nucleic acid sequences for expressing
an siRNA of the invention is described in Example 7 below. That
plasmid, called pAAVsiRNA, comprises a sense RNA strand coding
sequence in operable connection with a polyT termination sequence
under the control of a human U6 RNA promoter, and an antisense RNA
strand coding sequence in operable connection with a polyT
termination sequence under the control of a human U6 RNA promoter.
The plasmid pAAVsiRNA is ultimately intended for use in producing
an recombinant adeno-associated viral vector comprising the same
nucleic acid sequences for expressing an siRNA of the
invention.
[0064] As used herein, "in operable connection with a polyT
termination sequence" means that the nucleic acid sequences
encoding the sense or antisense strands are immediately adjacent to
the polyT termination signal in the 5' direction. During
transcription of the sense or antisense sequences from the plasmid,
the polyT termination signals act to terminate transcription.
[0065] As used herein, "under the control" of a promoter means that
the nucleic acid sequences encoding the sense or antisense strands
are located 3' of the promoter, so that the promoter can initiate
transcription of the sense or antisense coding sequences.
[0066] The siRNA of the invention can also be expressed from
recombinant viral vectors intracellularly at or near the area of
neovascularization in vivo. The recombinant viral vectors of the
invention comprise sequences encoding the siRNA of the invention
and any suitable promoter for expressing the siRNA sequences.
Suitable promoters include, for example, the U6 or H1 RNA pol III
promoter sequences and the cytomegalovirus promoter. Selection of
other suitable promoters is within the skill in the art. The
recombinant viral vectors of the invention can also comprise
inducible or regulatable promoters for expression of the siRNA in a
particular tissue or in a particular intracellular environment. The
use of recombinant viral vectors to deliver siRNA of the invention
to cells in vivo is discussed in more detail below.
[0067] siRNA of the invention can be expressed from a recombinant
viral vector either as two separate, complementary RNA molecules,
or as a single RNA molecule with two complementary regions.
[0068] Any viral vector capable of accepting the coding sequences
for the siRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of the
viral vectors can also be modified by pseudotyping the vectors with
envelope proteins or other surface antigens from other viruses. For
example, an AAV vector of the invention can be pseudotyped with
surface proteins from vesicular stomatitis virus (VSV), rabies,
Ebola, Mokola, and the like.
[0069] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the siRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310;
Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990),
Hum Gene Therap. 1: 5-14; and Anderson W F (1998), Nature 392:
25-30, the entire disclosures of which are herein incorporated by
reference.
[0070] Preferred viral vectors are those derived from AV and AAV.
In a particularly preferred embodiment, the siRNA of the invention
is expressed as two separate, complementary single-stranded RNA
molecules from a recombinant AAV vector comprising, for example,
either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV)
promoter.
[0071] A suitable AV vector for expressing the siRNA of the
invention, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0072] Suitable AAV vectors for expressing the siRNA of the
invention, methods for constructing the recombinant AAV vector, and
methods for delivering the vectors into target cells are described
in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et
al. (1996) J. Virol., 70: 520-532; Samulski R et al. (1989), J.
Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No.
5,139,941; International Patent Application No. WO 94/13788; and
International Patent Application No. WO 93/24641, the entire
disclosures of which are herein incorporated by reference. An
exemplary method for generating a recombinant AAV vector of the
invention is described in Example 7 below.
[0073] The ability of an siRNA containing a given target sequence
to cause RNAi-mediated degradation of the target mRNA can be
evaluated using standard techniques for measuring the levels of RNA
or protein in cells. For example, siRNA can be delivered to
cultured cells, and the levels of target mRNA can be measured by
Northern blot or dot blotting techniques, or by quantitative
RT-PCR. Alternatively, the levels of the target mRNA or protein in
the cultured cells can be measured by ELISA or Western blot. A
suitable cell culture system for measuring the effect of the
present siRNA on target mRNA or protein levels is described in
Example 1 below.
[0074] RNAi-mediated degradation of target mRNA by an siRNA
containing a given target sequence can also be evaluated with
animal models. For example, areas of neovascularization in an ROP
or CNV mouse can be measured before and after administration of an
siRNA. A reduction in the areas of neovascularization in these
models upon administration of the siRNA indicates the
down-regulation of the target mRNA.
[0075] In one preferred embodiment, the siRNA target and cause the
RNAi-mediated degradation of, for example, VEGF, Flt-1 or Flk-1/KDR
mRNA, or alternative splice forms, mutants or cognates thereof.
Degradation of the target mRNA by the present siRNA reduces the
production of a functional gene product from the VEGF, Flt-1 or
Flk-1/KDR genes. Thus, the invention provides a method of
inhibiting expression of VEGF, Flt-1 or Flk-1/KDR in a subject,
comprising administering an effective amount of an siRNA of the
invention to the subject, such that the target mRNA is degraded. As
the products of the VEGF, Flt-1 and Flk-1/KDR genes are required
for initiating and maintaining angiogenesis, the invention also
provides a method of inhibiting angiogenesis in a subject by the
RNAi-mediated degradation of the target mRNA by the present
siRNA.
[0076] In another preferred embodiment, siRNA target and cause the
RNAi-mediated degradation of, for example, human HIF-1 alpha gene
or alternative splice forms, mutants or cognates thereof.
Degradation of the target mRNA by the present siRNA reduces the
production of a functional gene product from the HIF-1 alpha gene.
Thus, the invention provides a method of inhibiting expression of
HIF-1 alpha in a subject, comprising administering an effective
amount of an siRNA of the invention to the subject, such that the
target mRNA is degraded. As the products of the HIF-1 alpha gene
are required for initiating and maintaining angiogenesis, the
invention also provides a method of inhibiting angiogenesis in a
subject by the RNAi-mediated degradation of the target mRNA by the
present siRNA.
[0077] In another preferred embodiment, siRNA target and cause the
RNAi-mediated degradation of, for example, human ICAM-1 gene or
alternative splice forms. mutants or cognates thereof. Degradation
of the target mRNA by the present siRNA reduces the production of a
functional gene product from the ICAM-1 gene. Thus, the invention
provides a method of inhibiting expression of ICAM-1 in a subject,
comprising administering an effective amount of an siRNA of the
invention to the subject, such that the target mRNA is degraded. As
the products of the ICAM-1 gene are required for initiating and
maintaining angiogenesis, the invention also provides a method of
inhibiting angiogenesis in a subject by the RNAi-mediated
degradation of the target mRNA by the present siRNA.
[0078] In another preferred embodiment, siRNA target and cause the
RNAi-mediated degradation of, for example, human Ang1, Ang2 or Tie2
gene or alternative splice forms, mutants or cognates thereof.
Degradation of the target mRNA by the present siRNA reduces the
production of a functional gene product from the Ang1, Ang2 or Tie2
genes. Thus, the invention provides a method of inhibiting
expression of Ang1, Ang2 or Tie2 in a subject, comprising
administering an effective amount of an siRNA of the invention to
the subject, such that the target mRNA is degraded. As the products
of the Ang1, Ang2 or Tie2 genes are required for initiating and
maintaining angiogenesis, the invention also provides a method of
inhibiting angiogenesis in a subject by the RNAi-mediated
degradation of the target mRNA by the present siRNA.
[0079] In another preferred embodiment, siRNA target and cause the
RNAi-mediated degradation of, for example, human complement genes
or alternative splice forms, mutants or cognates thereof.
Degradation of the target mRNA by the present siRNA reduces the
production of a functional gene product from the complement genes.
Thus, the invention provides a method of inhibiting expression of
complement in a subject, comprising administering an effective
amount of an siRNA of the invention to the subject, such that the
target mRNA is degraded.
[0080] As used herein, a "subject" includes a human being or
non-human animal. Preferably, the subject is a human being.
[0081] As used herein, an "effective amount" of the siRNA is an
amount sufficient to cause RNAi-mediated degradation of the target
mRNA, or an amount sufficient to inhibit the progression of
angiogenesis in a subject.
[0082] RNAi-mediated degradation of the target mRNA can be detected
by measuring levels of the target mRNA or protein in the cells of a
subject, using standard techniques for isolating and quantifying
mRNA or protein as described above.
[0083] It is understood that the siRNA of the invention can degrade
the target mRNA in substoichiometric amounts. Without wishing to be
bound by any theory, it is believed that the siRNA of the invention
causes degradation of the target mRNA in a catalytic manner.
[0084] Generally, an effective amount of the siRNA of the invention
comprises an intercellular concentration at or near the
neovascularization site of from about 1 nanomolar (nM) to about 100
nM, preferably from about 2 nM to about 50 nM, more preferably from
about 2.5 nM to about 10 nM. It is contemplated that greater or
lesser amounts of siRNA can be administered.
[0085] In one embodiment, an effective amount of the siRNA of the
invention comprises about from about 0.1 mg to about 20 mg of
siRNA. In a further embodiment, the effective amount is from about
0.2 mg to about 10 mg of siRNA. In an additional embodiment,
preferably the effective amount of the siRNA is from about 0.5 to
about 5 mg of siRNA. Further preferred embodiments provide
effective amounts of about 1 mg to about 3 mg, including about 1.5
mg, 2.5 mg or about 3 mg of siRNA.
[0086] For treating diseases, the siRNA of the invention can
administered to a subject in combination with a pharmaceutical
agent which is different from the present siRNA. Alternatively, the
siRNA of the invention can be administered to a subject in
combination with another therapeutic method designed to treat the
subject disease. For example, the siRNA of the invention can be
administered in combination with therapeutic methods currently
employed for treating cancer or preventing tumor metastasis (e.g.,
radiation therapy, chemotherapy, and surgery). For treating tumors,
the siRNA of the invention is preferably administered to a subject
in combination with radiation therapy, or in combination with
chemotherapeutic agents such as cisplatin, carboplatin,
cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or
tamoxifen.
[0087] In certain embodiments, the siRNA can be administered to the
subject either as naked siRNA, in conjunction with a delivery
reagent, or as a recombinant plasmid or viral vector which
expresses the siRNA.
[0088] Suitable delivery reagents for administration in conjunction
with the present siRNA include the Minis Transit TKO lipophilic
reagent; lipofectin; lipofectamine; cellfectin; or polycations
(e.g., polylysine), or liposomes. A preferred delivery reagent is a
liposome.
[0089] Liposomes can aid in the delivery of the siRNA to a
particular tissue, such as retinal or tumor tissue, and can also
increase the blood half-life of the siRNA. Liposomes suitable for
use are formed from standard vesicle-forming lipids, which
generally include neutral or negatively charged phospholipids and a
sterol, such as cholesterol. The selection of lipids is generally
guided by consideration of factors such as the desired liposome
size and half-life of the liposomes in the blood stream. A variety
of methods are known for preparing liposomes, for example as
described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:
467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369, the entire disclosures of which are herein incorporated
by reference.
[0090] Preferably, the liposomes encapsulating the present siRNA
comprises a ligand molecule that can target the liposome to a
particular cell or tissue at or near the site of therapeutic need.
For example, ligands which bind to receptors prevalent in tumor or
vascular endothelial cells, such as monoclonal antibodies that bind
to tumor antigens or endothelial cell surface antigens, are
preferred.
[0091] Particularly preferably, the liposomes encapsulating the
siRNA are modified so as to avoid clearance by the mononuclear
macrophage and reticuloendothelial systems, for example by having
opsonization-inhibition moieties bound to the surface of the
structure. In one embodiment, a liposome of the invention can
comprise both opsonization-inhibition moieties and a ligand.
[0092] Opsonization-inhibiting moieties for use in preparing the
liposomes of the invention are typically large hydrophilic polymers
that are bound to the liposome membrane. As used herein, an
opsonization inhibiting moiety is "bound" to a liposome membrane
when it is chemically or physically attached to the membrane, e.g.,
by the intercalation of a lipid-soluble anchor into the membrane
itself, or by binding directly to active groups of membrane lipids.
These opsonization-inhibiting hydrophilic polymers form a
protective surface layer which significantly decreases the uptake
of the liposomes by the macrophage-monocyte system ("MMS") and
reticuloendothelial system ("RES"); e.g., as described in U.S. Pat.
No. 4,920,016, the entire disclosure of which is herein
incorporated by reference. Liposomes modified with
opsonization-inhibition moieties thus remain in the circulation
much longer than unmodified liposomes. For this reason, such
liposomes are sometimes called "stealth" liposomes.
[0093] Stealth liposomes are known to accumulate in tissues fed by
porous or "leaky" microvasculature. Thus, target tissue
characterized by such microvasculature defects, for example solid
tumors, will efficiently accumulate these liposomes; see Gabizon,
et al. (1988), P.N.A.S., USA, 18: 6949-53. In addition, the reduced
uptake by the RES lowers the toxicity of stealth liposomes by
preventing significant accumulation in the liver and spleen. Thus,
liposomes of the invention that are modified with
opsonization-inhibition moieties can deliver the present siRNA to
tumor cells.
[0094] Opsonization inhibiting moieties suitable for modifying
liposomes are preferably water-soluble polymers with a
number-average molecular weight from about 500 to about 40,000
daltons, and more preferably from about 2,000 to about 20,000
daltons. Such polymers include polyethylene glycol (PEG) or
polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG,
and PEG or PPG stearate; synthetic polymers such as polyacrylamide
or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric
polyamidoamines; polyacrylic acids; polyalcohols, e.g.,
polyvinylalcohol and polyxylitol to which carboxylic or amino
groups are chemically linked, as well as gangliosides, such as
ganglioside GM.sub.1. Copolymers of PEG, methoxy PEG, or methoxy
PPG, or derivatives thereof, are also suitable. In addition, the
opsonization inhibiting polymer can be a block copolymer of PEG and
either a polyamino acid, polysaccharide, polyamidoamine,
polyethyleneamine, or polynucleotide. The opsonization inhibiting
polymers can also be natural polysaccharides containing amino acids
or carboxylic acids, e.g., galacturonic acid, glucuronic acid,
mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid,
alginic acid, carrageenan; aminated polysaccharides or
oligosaccharides (linear or branched); or carboxylated
polysaccharides or oligosaccharides, e.g., reacted with derivatives
of carbonic acids with resultant linking of carboxylic groups.
[0095] Preferably, the opsonization-inhibiting moiety is a PEG,
PPG, or derivatives thereof. Liposomes modified with PEG or
PEG-derivatives are sometimes called "PEGylated liposomes."
[0096] The opsonization inhibiting moiety can be bound to the
liposome membrane by any one of numerous well-known techniques. For
example, an N-hydroxysuccinimide ester of PEG can be bound to a
phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a
membrane. Similarly, a dextran polymer can be derivatized with a
stearylamine lipid-soluble anchor via reductive amination using
Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water
in a 30:12 ratio at 60.degree. C.
[0097] Recombinant plasmids which express siRNA of the invention
are discussed above. Such recombinant plasmids can also be
administered directly or in conjunction with a suitable delivery
reagent, including the Mirus Transit LT1 lipophilic reagent;
lipofectin; lipofectamine; cellfectin; polycations (e.g.,
polylysine) or liposomes. Recombinant viral vectors which express
siRNA of the invention are also discussed above, and methods for
delivering such vectors to an area of neovascularization in a
patient are within the skill in the art.
[0098] The siRNA of the invention can be administered to the
subject by any means suitable for delivering the siRNA to the cells
of the tissue at or near the area of neovascularization. For
example, the siRNA can be administered by gene gun,
electroporation, or by other suitable parenteral or enteral
administration routes.
[0099] Suitable enteral administration routes include oral, rectal,
or intranasal delivery.
[0100] Suitable parenteral administration routes include
intravascular administration (e.g. intravenous bolus injection,
intravenous infusion, intra-arterial bolus injection,
intra-arterial infusion and catheter instillation into the
vasculature); peri- and intra-tissue administration (e.g.,
peri-tumoral and intra-tumoral injection, intravitreal injection,
intra-retinal injection or subretinal injection); subcutaneous
injection or deposition including subcutaneous infusion (such as by
osmotic pumps); direct (e.g., topical) application to the area at
or near the site of neovascularization, for example by a catheter
or other placement device (e.g., a corneal pellet or a suppository,
eye-dropper, or an implant comprising a porous, non-porous, or
gelatinous material); and inhalation. Suitable placement devices
include the ocular implants described in U.S. Pat. Nos. 5,902,598
and 6,375,972, and the biodegradable ocular implants described in
U.S. Pat. No. 6,331,313, the entire disclosures of which are herein
incorporated by reference. Such ocular implants are available from
Control Delivery Systems, Inc. (Watertown, Mass.) and Oculex
Pharmaceuticals, Inc. (Sunnyvale, Calif.).
[0101] In a preferred embodiment, injections or infusions of the
siRNA are given at or near the site of neovascularization. More
preferably, the siRNA is administered topically to the eye, e.g. in
liquid or gel form to the lower eye lid or conjunctival cul-de-sac,
as is within the skill in the art (see, e.g., Acheampong A A et al,
2002, Drug Metabol. and Disposition 30: 421-429, the entire
disclosure of which is herein incorporated by reference).
[0102] In such embodiments, the siRNA of the invention may be
administered topically to the eye in amounts of from about 5
microliters to about 75 microliters, for example from about 7
microliters to about 50 microliters, preferably from about 10
microliters to about 30 microliters. It is understood that topical
instillation in the eye of siRNA in volumes greater than 75
microliters can result in loss of siRNA from the eye through
spillage and drainage. Thus, it is preferable to administer a high
concentration of siRNA (e.g., 100-1000 nM) in as small a volume as
possible.
[0103] A particularly preferred parenteral administration route is
intraocular administration. It is understood that intraocular
administration of the present siRNA can be accomplished by
injection or direct (e.g., topical) administration to the eye, as
long as the administration route allows the siRNA to enter the eye.
In addition to the topical routes of administration to the eye
described above, suitable intraocular routes of administration
include intravitreal, intraretinal, subretinal, subtenon, peri- and
retro-orbital, trans-corneal and trans-scleral administration. Such
intraocular administration routes are within the skill in the art;
see, e.g., and Acheampong A A et al, 2002, supra; and Bennett et
al. (1996), Hum. Gene Ther. 7: 1763-1769 and Ambati J et al., 2002,
Progress in Retinal and Eye Res. 21: 145-151, the entire
disclosures of which are herein incorporated by reference.
[0104] The siRNA of the invention can be administered in a single
dose or in multiple doses. Where the administration of the siRNA of
the invention is by infusion, the infusion can be a single
sustained dose or can be delivered by multiple infusions. Injection
of the agent directly into the tissue is at or near the site of
neovascularization preferred. Multiple injections of the agent into
the tissue at or near the site of neovascularization are
particularly preferred.
[0105] The siRNA can be administered to the subject once, such as
by a single injection or deposition at or near the
neovascularization site. Alternatively, the siRNA can be
administered to a subject multiple times daily or weekly. For
example, the siRNA can be administered to a subject once weekly for
a period of from about three to about twenty-eight weeks, more
preferably from about seven to about ten weeks. In a preferred
dosage regimen, the siRNA is injected at or near the site of
neovascularization (e.g., intravitreally) once a week for seven
weeks. It is understood that periodic administrations of the siRNA
of the invention for an indefinite length of time may be necessary
for subjects suffering from a chronic neovascularization disease,
such as wet ARMD or diabetic retinopathy.
[0106] Where a dosage regimen comprises multiple administrations,
it is understood that the effective amount of siRNA administered to
the subject can comprise the total amount of siRNA administered
over the entire dosage regimen.
[0107] The siRNA of the invention are preferably formulated as
pharmaceutical compositions prior to administering to a subject,
according to techniques known in the art. Pharmaceutical
compositions of the present invention are characterized as being at
least sterile and pyrogen-free. As used herein, "pharmaceutical
formulations" include formulations for human and veterinary use.
Methods for preparing pharmaceutical compositions of the invention
are within the skill in the art, for example as described in
Remington Pharmaceutical Science, 17th ed., Mack Publishing
Company, Easton, Pa. (1985), the entire disclosure of which is
herein incorporated by reference.
[0108] The present pharmaceutical formulations comprise an siRNA of
the invention (e.g., 0.1 to 90% by weight), or a physiologically
acceptable salt thereof, mixed with a physiologically acceptable
carrier medium. Preferred physiologically acceptable carrier media
are water, buffered water, saline solutions (e.g., normal saline or
balanced saline solutions such as Hank's or Earle's balanced salt
solutions), 0.4% saline, 0.3% glycine, hyaluronic acid and the
like.
[0109] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0110] For topical administration to the eye, conventional
intraocular delivery reagents can be used. For example,
pharmaceutical compositions of the invention for topical
intraocular delivery can comprise saline solutions as described
above, corneal penetration enhancers, insoluble particles,
petrolatum or other gel-based ointments, polymers which undergo a
viscosity increase upon instillation in the eye, or mucoadhesive
polymers. Preferably, the intraocular delivery reagent increases
corneal penetration, or prolongs preocular retention of the siRNA
through viscosity effects or by establishing physicochemical
interactions with the mucin layer covering the corneal
epithelium.
[0111] Suitable insoluble particles for topical intraocular
delivery include the calcium phosphate particles described in U.S.
Pat. No. 6,355,271 of Bell et al., the entire disclosure of which
is herein incorporated by reference. Suitable polymers which
undergo a viscosity increase upon instillation in the eye include
polyethylenepolyoxypropylene block copolymers such as poloxamer 407
(e.g., at a concentration of 25%), cellulose acetophthalate (e.g.,
at a concentration of 30%), or a low-acetyl gellan gum such as
Gelrite.RTM. (available from CP Kelco.RTM., Wilmington, Del.).
Suitable mucoadhesive polymers include hydrocolloids with multiple
hydrophilic functional groups such as carboxyl, hydroxyl, amide
and/or sulfate groups; for example, hydroxypropylcellulose,
polyacrylic acid, high-molecular weight polyethylene glycols (e.g.
>200,000 number average molecular weight), dextrans, hyaluronic
acid, polygalacturonic acid, and xylocan. Suitable conical
penetration enhancers include cyclodextrins, benzalkonium chloride,
polyoxyethylene glycol lauryl ether (e.g., Brij.RTM. 35),
polyoxyethylene glycol stearyl ether (e.g., Brij.RTM. 78),
polyoxyethylene glycol oleyl ether (e.g., Brij.RTM. 98), ethylene
diamine tetraacetic acid (EDTA), digitonin, sodium taurocholate,
saponins and polyoxyethylated castor oil such as Cremaphor EL.
[0112] For solid compositions, conventional nontoxic solid carriers
can be used; for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0113] For example, a solid pharmaceutical composition for oral
administration can comprise any of the carriers and excipients
listed above and 10-95%, preferably 25%-75%, of one or more siRNA
of the invention. A pharmaceutical composition for aerosol
(inhalational) administration can comprise 0.01-20% by weight,
preferably 1%-10% by weight, of one or more siRNA of the invention
encapsulated in a liposome as described above, and propellant. A
carrier can also be included as desired; e.g., lecithin for
intranasal delivery.
Example 1
[0114] Two different methods of producing siRNA were evaluated and
the efficacy of the resulting siRNA were tested. Specifically,
Cand5 duplex was formed by each of the methods and the resulting
products were tested for their ability to inhibit VEGF in human
embryonic kidney 293 cells.
[0115] Three different RNA single-stranded oligonucleotides were
prepared. These included a 10 base pair (bp) oligonucleotides
encoding by 1-10 of the Cand5 sense strand; an 11 bp
oligonucleotides encoding by 11-21 of the Cand5 sense strand; and
an oligonucleotides encoding the entire Cand5 antisense strand. In
the first method, the oligonucleotides were combined in an
equimolar ratio, heated to 95.degree. C. for 2 minutes and then
cooled to room temperature. In the second method, the
oligonucleotides were also combined and heated as described;
however, T4 DNA ligase was added to the mixture post-cooling, and
the sample was incubated at room temperature for an hour and then
at 65.degree. C. for 15 minutes.
[0116] The ability of each method's resulting product to reduce
VEGF protein levels in cell culture was evaluated. Human embryonic
kidney 293 cells were transiently transfected with each of the
products. Hypoxia was induced in the cells with the addition of
desferrioxamine in order to induce VEGF transcription and
translation. Forty-eight hours post-transfection, media was
collected and an ELISA was performed to quantify the levels of
secreted VEGF protein. Both synthesis methods allowed for the
formation of the Candy duplex, and the resulting Cand5 caused a
dose-dependent decrease in VEGF protein.
[0117] Cell Culture. Human embryonic kidney 293 cells (ATCC,
Manassas, Va.) were cultured in Dulbecco's Modified Eagle Medium
(DMEM; Cellgro, Herndon, Va.) with 10% fetal bovine serum (FBS; JRH
Biosciences, Lenexa, Kans.) and an antibiotic-antimycotic reagent
used for the prevention of cell culture growth contaminants (Gibco.
Carlsbad, Calif.) under 5% CO2 at 37.degree. C.
[0118] siRNAs and Oligonucleotides. All siRNAs and oligonucleotides
were synthesized by Dharmacon (Lafayette, Colo.). The fully
synthesized Cand5 siRNA served as a positive control in the in
vitro efficacy studies. The sense strand sequence was 5'-ACC UCA
CCA AGG CCA GCA CdTdT-3' (SEQ ID NO. 6), while the antisense strand
sequence was 5'-G UGC UGC CCU UGG UGA GGUdTdT-3' (SEQ ID NO.
7).
[0119] An siRNA targeting enhanced green fluorescent protein (EGFP)
served as a negative control. The sense strand sequence for this
siRNA was 5'-GGC UAC GUC CAG GAG CGC AdTdT-3' (SEQ ID NO. 8), and
the antisense strand sequence was 5'-U GCG CUC CUG GAC GUA GCCdTdT
(SEQ ID NO. 9).
[0120] Three different single-strand RNA oligonucleotides were
synthesized: one encoding base pairs 1-10 of the Cand5 sense strand
5'-ACC UCA CCA A-3' (SEQ ID NO. 10); one encoding base pairs 11-21
of the Cand5 sense strand 5'-GG CCA GCA CdTdT-3' (SEQ ID NO. 11);
and one encoding the Cand5 antisense strand 5'-G UGC UGG CCU UGG
UGA GGUdTdT-3' (SEQ ID NO. 7).
[0121] Combining the Cand5 RNA Precursor Oligonucleotides. Each of
the three RNA oligonucleotides were combined in equimolar amounts.
They were then mixed, heated to 95.degree. C. for 2 minutes, and
allowed to cool down to room temperature. The resulting "Cand5
Mixture" was either used for in vitro studies directly (see below)
or further modified to facilitate ligation of the sense strand
components prior to use. The additional modification involved
combing 10 .mu.L of the "Cand5 Mixture" with 0.5 .mu.l, of T4
ligase (5 U/.mu.L, Ambion, Austin, Tex.), 1.5 .mu.l 10.times.
ligation buffer and 3 .mu.L of water. The sample was incubated at
room temperature for one hour and then incubated at 65.degree. C.
for an additional 15 minutes to inactivate the ligase. The
resulting product was termed "Cand5 Mixture-ligated".
[0122] siRNA Transfection and Hypoxia Induction in Vitro. 293 cells
were cultured in 24-well plates at 37.degree. C. with 5% CO.sub.2
and incubated overnight. After approximately 24 hours of
incubation, when the cells were estimated to be 70% confluent,
aliquots of Cand5 Mixture or Cand5 Mixture-ligated were added,
along with a calcium phosphate (CaPi) reagent. Cand5 duplex, "Cand5
Mixture", or "Cand5 Mixture-ligated" was added to 20 .mu.L of 250
mM CaCl.sub.2 solution. The siRNA/CaCl.sub.2 mixture was added
drop-wise to 20 .mu.L of 2.times.Hanks Balanced Salt Solution
(HBS), while mixing by vortex. The siRNA/CaCl.sub.2/HBS complex was
added directly to the medium (300 .mu.L/well). After 4 hours of
incubation at 37.degree. C., the medium was removed, and the cells
were further incubated with 10% DMSO-containing serum-free medium
(300 .mu.L/well at room temperature for 1-2 minutes). This medium
was then removed, and the cells were fed again with 500 .mu.L/well
growth medium. The final concentration of siRNA dilutions were 100
.mu.M, 1 nM, and 25 nM. Negative controls included the transfection
reagent lacking siRNA or the nonspecific siRNA (EGFP siRNA) at a 25
nM concentration. To stimulate VEGF transcription and translation
within the cells, hypoxia was induced with the addition of
desferrioxamine (Sigma, Saint Louis, Mo.) at a final concentration
of 130 .mu.M, which was introduced to the cultures 4 hours after
transfection was performed. The assay was performed on duplicate
plates with samples prepared in triplicate.
[0123] VEGF Protein Quantification. Approximately 48 hours
post-transfection, the cell supernatant was removed from each well
for analysis. The Quantikine human VEGF ELISA Kit (R&D Systems,
Minneapolis, Minn.) was used to quantify VEGF protein, following
the manufacturer's protocol. 100 .mu.L of each sample were
analyzed. The ELBA results were read at 450 nm using an AD340 plate
reader (Beckman-Coulter).
[0124] Results and Discussion. Human VEGF protein was upregulated
in 293 cells treated with desferrioxamine, as shown in FIG. 1. The
hypoxia-induced increase in hVEGF protein was significantly reduced
in cells treated with Cand5 (Dharmacon), "Cand5 Mixture" and "Cand5
Mixture-ligated". The effect was dose-dependent. Treatment with
non-specific siRNA (EGFP siRNA; 25 nM NC), or mock incubations
without siRNA, had no effect on hVEGF levels. Cand5 showed better
suppression than "Cand5 Mixture" and "Cand5 Mixture-ligated".
[0125] Both methods involved the synthesizes and combination of the
three RNA oligonucleotides, collectively encoding the sense and
antisense strands of Cand5, in equimolar ratios, however, the
methods contemplated by the present invention may involve the
synthesis and combination of multiple oligonucleotides in
non-equimolar ratios. In one method described above, the
oligonucleotides were joined by mixing, heating to 95.degree. C.
and cooling to room temperature. In the other method described
above, the ligation of the sense strand components was facilitated
by the subsequent addition of ligase to the mixture.
[0126] Both methods allowed for Cand5 duplex formation, which is
evident by the mixtures' abilities to suppress human VEGF protein
production in hypoxic 293 cells; however, neither mixture appeared
to be as effective as the Cand5 duplex. This is likely due to the
fact that products from the two methods were not enriched for the
Cand5 duplex. In addition, there was similar efficacy between the
mixture without ligase and that with ligase, suggesting that
addition of ligase does not contribute to duplex formation.
[0127] Although the present invention has been described in
considerable detail with reference to certain preferred embodiments
thereof, other versions are possible. Therefore the spirit and
scope of the appended claims should not be limited to the
description and the preferred versions contained within this
specification.
Sequence CWU 1
1
11119DNAHomo sapiens 1tcatcacgaa gtggtgaag 19221RNAHomo sapiens
2ucaucacgaa guggugaagu u 21321RNAHomo sapiens 3uuaguagugc
uucaccacuu c 21421DNAHomo sapiensAntisense strand 4ucaucacgaa
guggugaagt t 21521DNAHomo sapiensSense strand 5ttaguagugc
uucaccacuu c 21621DNAHomo sapiensSense strand 6accucaccaa
ggccagcact t 21721DNAHomo sapiensAntisense strand 7gugcuggccu
uggugaggut t 21821DNAHomo sapiensSense strand 8ggcuacgucc
aggagcgcat t 21921DNAHomo sapiensAntisense strand 9ugcgcuccug
gacguagcct t 211010RNAHomo sapiens 10accucaccaa 101111DNAHomo
sapiensAntisense strand 11ggccagcact t 11
* * * * *