U.S. patent application number 13/594150 was filed with the patent office on 2013-03-14 for compositions and methods for inhibiting viral replication.
The applicant listed for this patent is Matthias John, Roland Kreutzer, Stefan Limmer, Hans-Peter Vornlocher. Invention is credited to Matthias John, Roland Kreutzer, Stefan Limmer, Hans-Peter Vornlocher.
Application Number | 20130064879 13/594150 |
Document ID | / |
Family ID | 39686363 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130064879 |
Kind Code |
A1 |
John; Matthias ; et
al. |
March 14, 2013 |
Compositions and Methods for Inhibiting Viral Replication
Abstract
The present invention relates to a double-stranded ribonucleic
acid (dsRNA) having a nucleotide sequence which is less that 30
nucleotides in length and which is substantially identical to at
least a part of a 3'-untranslated region (3'-UTR) of a (+) strand
RNA virus, such as HCV, as well as pharmaceutical compositions
comprising the dsRNA, together with a pharmaceutically acceptable
carrier. The pharmaceutical compositions are useful for treating
infections and diseases caused by the replication or activity of
the (+) strand RNA virus, as well as methods for inhibiting viral
replication.
Inventors: |
John; Matthias; (Hallstadt,
DE) ; Limmer; Stefan; (Neudrossenfeld, DE) ;
Vornlocher; Hans-Peter; (Bayreuth, DE) ; Kreutzer;
Roland; (Weidenberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
John; Matthias
Limmer; Stefan
Vornlocher; Hans-Peter
Kreutzer; Roland |
Hallstadt
Neudrossenfeld
Bayreuth
Weidenberg |
|
DE
DE
DE
DE |
|
|
Family ID: |
39686363 |
Appl. No.: |
13/594150 |
Filed: |
August 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12631689 |
Dec 4, 2009 |
8273868 |
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13594150 |
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11959936 |
Dec 19, 2007 |
7745418 |
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12631689 |
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10384512 |
Mar 7, 2003 |
7348314 |
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11959936 |
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PCT/EP02/11432 |
Oct 11, 2002 |
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10384512 |
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Current U.S.
Class: |
424/450 ;
514/44A; 536/24.5 |
Current CPC
Class: |
C12N 15/1131 20130101;
A61P 31/14 20180101; C12N 2310/53 20130101; A61P 35/00 20180101;
C12N 2310/14 20130101 |
Class at
Publication: |
424/450 ;
536/24.5; 514/44.A |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61K 9/127 20060101 A61K009/127; A61P 31/14 20060101
A61P031/14; C07H 21/02 20060101 C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 12, 2001 |
DE |
10150187.0 |
Oct 26, 2001 |
DE |
10155280.7 |
Nov 29, 2001 |
DE |
10158411.3 |
Dec 7, 2001 |
DE |
10160151.4 |
Dec 20, 2001 |
DE |
10163098.0 |
Jan 9, 2002 |
EP |
PCT/EP02/00151 |
Jan 9, 2002 |
EP |
PCT/EP02/00152 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the
replication of a (+) strand RNA virus, wherein the dsRNA comprises
a sense RNA strand comprising a nucleotide sequence which is
substantially identical to at least a part of a 3'-untranslated
region (3'-UTR) of the (+) strand RNA virus, and wherein the dsRNA
comprises an antisense RNA strand which is complementary to at
least a part of the sense RNA strand, and wherein each strand of
the dsRNA is 21 to 24 nucleotides in length.
2. The dsRNA of claim 1, wherein the dsRNA comprises a blunt
end.
3. The dsRNA of claim 1, wherein the dsRNA comprises two blunt
ends.
4. The dsRNA of claim 1, wherein the antisense RNA strand and the
sense RNA strand comprise a 3'-terminus and a 5'-terminus, and
wherein at least one of said RNA strands comprises a nucleotide
overhang of 1 to 3 nucleotides in length.
5. The dsRNA of claim 4, wherein the nucleotide overhang is two
nucleotides in length.
6. The dsRNA of claim 4, wherein the nucleotide overhang is on the
3'-terminus of the antisense RNA strand.
7. The dsRNA of claim 4, wherein the dsRNA further comprises a
first end and a second end, wherein the first end comprises the
3'-terminus of the antisense RNA strand and the 5'-terminus of the
sense RNA strand, and wherein the second end comprises the
5'-terminus of the antisense RNA strand and the 3'-terminus of the
sense RNA strand, wherein the first end comprises a nucleotide
overhang on the 3'-terminus of the antisense RNA strand, and
wherein the second end is blunt.
8. The dsRNA of claim 4, wherein the antisense RNA strand is 23
nucleotides in length and comprises a 2-nucleotide overhang at the
3'-terminus, wherein the sense RNA strand is 23 nucleotides in
length and comprises a 2-nucleotide overhang at the 3'-terminus,
and wherein the dsRNA has a 21-nucleotide double-stranded
region.
9. A pharmaceutical composition for inhibiting the replication of a
(+) strand RNA virus in an organism, comprising a dsRNA and a
pharmaceutically acceptable carrier, wherein the dsRNA comprises a
sense RNA strand comprising a nucleotide sequence which is
substantially identical to at least a part of a 3'-untranslated
region (3'-UTR) of the (+) strand RNA virus, and wherein the dsRNA
comprises an antisense RNA strand which is complementary to at
least a part of the sense RNA strand, and wherein each strand of
the dsRNA is 21 to 24 nucleotides in length.
10. The pharmaceutical composition of claim 9, wherein the dsRNA
comprises a blunt end.
11. The pharmaceutical composition of claim 9, wherein the dsRNA
comprises two blunt ends.
12. The pharmaceutical composition of claim 9, wherein the
antisense RNA strand and the sense RNA strand comprise a
3'-terminus and a 5'-terminus, and wherein at least one of said RNA
strands comprise a nucleotide overhang of 1 to 3 nucleotides in
length.
13. The pharmaceutical composition of claim 12, wherein the
nucleotide overhang is two nucleotides in length.
14. The pharmaceutical composition of claim 12, wherein the
nucleotide overhang is on the 3'-terminus of the antisense RNA
strand.
15. The pharmaceutical composition of claim 12, wherein the dsRNA
further comprises a first end and a second end, wherein the first
end comprises the 3'-terminus of the antisense RNA strand and the
5'-terminus of the sense RNA strand, and wherein the second end
comprises the 5'-terminus of the antisense RNA strand and the
3'-terminus of the sense RNA strand, wherein the first end
comprises a nucleotide overhang on the 3'-terminus of the antisense
RNA strand, and wherein the second end is blunt.
16. The pharmaceutical composition of claim 12, wherein the
antisense RNA strand is 23 nucleotides in length and comprises a
2-nucleotide overhang at the 3'-terminus, wherein the sense RNA
strand is 23 nucleotides in length and comprises a 2-nucleotide
overhang at the 3'-terminus, and wherein the dsRNA has a
21-nucleotide double-stranded region.
17. The pharmaceutical composition of claim 9, wherein a dosage
unit of dsRNA is less than 5 milligram (mg) of dsRNA per kg body
weight of the mammal.
18. The pharmaceutical composition of claim 9, wherein a dosage
unit of dsRNA is in a range of 0.01 to 2.5 milligrams (mg), 0.1 to
200 micrograms (.mu.g), 0.1 to 100 .mu.g per kilogram body weight
of the mammal.
19. The pharmaceutical composition of claim 9, wherein a dosage
unit of dsRNA is less than 25 .mu.g per kilogram body weight of the
mammal.
20. The pharmaceutical composition of claim 9, wherein the
pharmaceutically acceptable carrier is an aqueous solution.
21. The pharmaceutical composition of claim 20, wherein the aqueous
solution is phosphate buffered saline.
22. The pharmaceutical composition of claim 9, wherein the
pharmaceutically acceptable carrier comprises a micellar structure
selected from the group consisting of a liposome, capsid, capsoid,
polymeric nanocapsule, and polymeric microcapsule.
23. The pharmaceutical composition of claim 9, which is formulated
to be administered by inhalation, infusion, injection, or
orally.
24. The pharmaceutical composition of claim 9, which is formulated
to be administered by intravenous or intraperitoneal injection.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/631,689 (pending), filed Dec. 4, 2009,
which is a continuation of U.S. patent application Ser. No.
11/959,936 (now issued as U.S. Pat. No. 7,745,418), filed Dec. 19,
2007, which is a divisional of U.S. patent application Ser. No.
10/384,512 (now issued as U.S. Pat. No. 7,348,314), filed Mar. 7,
2003, which is a continuation-in-part of International Application
No. PCT/EP02/11432, which designated the United States and was
filed on Oct. 11, 2002, which claims the benefit of German Patent
Application No. 101 50 187.0, filed on Oct. 12, 2001, German Patent
Application No. 101 55 280.7, filed on Oct. 26, 2001, German Patent
Application No. 101 58 411.3, filed on Nov. 29, 2001, German Patent
Application No. 101 60 151.4, filed on Dec. 7, 2001, German Patent
Application No. 101 63 098.0, filed on Dec. 20, 2001, International
Application No. PCT/EP02/00151, filed on Jan. 9, 2002, and
International Application No. PCT/EP02/00152, filed on Jan. 9,
2002. The entire teachings and contents of the above applications
are incorporated herein by reference, including any appendices or
attachments thereof, for all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to double-stranded ribonucleic acid
(dsRNA), and its use for inhibiting the replication of (+) strand
RNA viruses, such as Hepatitis C virus, as well as treating
viral-associated diseases.
BACKGROUND OF THE INVENTION
[0003] Positive or plus-strand RNA viruses share many similarities
in genomic organization and structure, most notably a
single-stranded coding RNA of positive polarity. Representative (+)
strand RNA viruses include the picornaviruses, flaviviruses,
togaviruses, coronaviruses, and caliciviruses. One clinically
significant representative of the flavivirus family is the
hepatitis C virus (HCV), the causative agent for hepatitis C.
Hepatitis C is an often chronic inflammatory disease of the liver
which typically results in fibrosis and liver cancer (Choo, et al.,
Science (1989) 244:359). Infection by HCV typically results from
contact with contaminated blood or blood products.
[0004] During HCV replication, a replicative (minus) RNA strand is
produced which serves as a template for generation of several
coding (+) RNA strands. The HCV genome, which contains
approximately 9600 nucleotides, is translated into a polyprotein
consisting of approximately 3000 amino acids (Leinbach, et al.,
Virology (1994) 204:163-169; Kato, et al., FEBS Letters (1991)
280:325-328). This polyprotein subsequently undergoes
post-translational cleavage, producing several proteins. Due to
high genetic variability and mutation rates, the HCV comprises
several distinct HCV genotypes that share approximately 70%
sequence identity (Simmonds, et al., J. Gen. Virol., (1994)
75:1053-1061). Despite this hypervariability, there are three
regions of the HCV genome that are highly conserved, including the
5'- and 3'-non-coding regions, known as the 5'-untranslated region
(5'-UTR) and 3'-untranslated region (3'-UTR), respectively. These
regions are thought to be vital for HCV RNA replication as well as
translation of the HCV polyprotein. In general, treatment of HCV is
complicated by its high mutation rate, as well as the mode of
transmission and possibility of simultaneous infection with
multiple HCV genotypes.
[0005] Hepatitis C has several clinical phases. The first phase
(i.e., acute phase) begins with infection by HCV. During this early
phase, it is possible to detect HCV-RNA in the serum of patients
using polymerase chain reaction (PCR). However, because only about
25% of patients exhibit jaundice during this phase, most cases
(75%) go undetected in the early stages. The inflammatory process,
characterized by an increase in serum liver enzyme concentrations,
begins approximately four weeks post infection. Although acute HCV
infection is not malignant, the majority of patients (approximately
80%) develop chronic liver disease, characterized by a permanent
elevation in the serum alanine aminotransferase level. Cirrhosis of
the liver develops in more than 20% of patients with chronic HCV
disease, which frequently leads to malignant hepatoma. Life
expectancy following diagnosis of the malignant hepatoma is
generally 12 months.
[0006] Current therapies to treat HCV infections have met with
limited success, with only a minority of patients experiencing
long-term improvement. The most prevalent treatment today involves
specific cytokines known as interferons, particularly
interferon-.alpha. (IFN-alpha) which reduces serum alanine
aminotransferase levels in approximately 50% of patients.
Unfortunately, serum levels of alanine aminotransferase usually
return to elevated levels following termination of treatment,
producing a number of adverse side effects (Dusheiko, et al., J.
Viral Hepatitis (1994) 1:3). Despite these problems, IFN-alpha is
commonly used to reduce the risk of cirrhosis of the liver and
malignant hepatoma. There is no currently available vaccine for
HCV.
[0007] Although IFN-alpha remains the conventional approach,
virologists have evaluated a number of potential alternative
therapies, including the use of specific ribozymes to inhibit
translation of viral protein. Welch et al. disclose a two
vector-expressed hairpin ribozyme directed against HCV (Welch, et
al., Gene Therapy (1996), 3(11):994). Lieber et al. report the
removal of HCV-RNA in infected human hepatocytes through
adenovirus-mediated expression of specific hammerhead ribozymes
(Lieber, et al., Virology (1996), 70 (12):8782). WO 99/55847
reports the degradation of 5'- and 3'-UTL regions of HCV-RNA, as
well as the 5'-coding region for the nucleoprotein, using
ribozymes. U.S. Pat. No. 5,610,054 discloses enzymatic nucleic acid
molecules that can inhibit replication of HCV. Despite these
efforts, the therapeutic value of ribozymes for treating HCV
infections remains questionable, particularly in view of their low
enzymatic activity.
[0008] More recently, double-stranded RNA molecules (dsRNA) have
been shown to block gene expression in a highly conserved
regulatory mechanism known as RNA interference (RNAi). Briefly, the
RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA)
of approximately 22 nucleotides, which serve as guide sequences to
induce target-specific mRNA cleavage by an RNA-induced silencing
complex RISC (Hammond, S. M., et al., Nature (2000), 404:293-296).
When administered to a cell or organism, exogenous dsRNA has been
shown to direct the sequence-specific degradation of endogenous
messenger RNA (mRNA) through RNAi. WO 99/32619 (Fires et al.)
discloses the use of a dsRNA of at least 25 nucleotides in length
to inhibit the expression of a target gene in C. elegans. dsRNA has
also been shown to degrade target RNA in other organisms, including
plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631,
Heifetz et al.); Drosophila (see, e.g., Yang, D., et al., Curr.
Biol. (2000) 10:1191-1200); and mammals (WO 00/44895, Limmer).
[0009] Despite significant advances in the field, there remains a
need for an agent that can inhibit the replication of a virus in a
host cell using the cell's own RNAi machinery. More specifically,
an agent that has high biological activity and can provide
long-term, effective inhibition of viral replication at a low dose
would be highly desirable. Compositions comprising such agents
would be useful for treating a variety of viral infections,
including HCV.
SUMMARY OF THE INVENTION
[0010] The present invention discloses double-stranded ribonucleic
acid (dsRNA), as well as compositions and methods for inhibiting
the replication of a (+) strand RNA virus, such as a Hepatitis C
Virus (HCV). In particular, the invention relates to a dsRNA having
an RNA strand (the complementary strand) comprising a region which
is complementary to at least a portion of a 3'-untranslated region
(3'-UTR) of a (+) strand RNA virus. The present invention also
discloses compositions and methods for treating hepatitis C or
HCV-associated diseases.
[0011] In one aspect, the invention relates to a dsRNA. The dsRNA
comprises a sense RNA strand comprising a nucleotide sequence which
is substantially identical to at least a part of a 3'-untranslated
region (3'-UTR) of a (+) strand RNA virus, and the dsRNA is less
than 30 nucleotides in length. The (+) strand RNA may be a
hepatitis C virus. The dsRNA may further comprise a complementary
RNA strand, wherein the complementary RNA strand comprises a
complementary nucleotide sequence which is less than 30 nucleotides
in length and is complementary to at least a portion of the 3'-UTR
of the virus. In a preferred embodiment, the nucleotide sequence is
within a highly conserved region of the 3'-UTR. The complementary
nucleotide sequence is preferably less than 25 nucleotides in
length, more preferably 21 to 24 nucleotides in length, and most
preferably 23 nucleotides in length. The dsRNA may comprise one or
two blunt ends. The complementary RNA strand and the sense RNA
strand may comprise a 3'-terminus and a 5'-terminus, and at least
one of the RNA strands may comprise a nucleotide overhang of 1 to 3
nucleotides in length, preferably two nucleotides in length. The
dsRNA may further comprise two ends, wherein one end comprises the
3'-terminus of the complementary RNA strand and the 5'-terminus of
the sense RNA strand, and the other end comprises the 5'-terminus
of the complementary RNA strand and the 3'-terminus of the sense
RNA strand. In one embodiment, one end of the dsRNA end has a
nucleotide overhang, preferably on the 3'-terminus of the
complementary RNA strand, and the second end is blunt. In another
embodiment, the complementary RNA strand is 24 nucleotides in
length and the sense RNA strand is 22 nucleotides in length, the
3'-end of the complementary RNA strand has a 2-nucleotide overhang,
and the other end of the dsRNA is blunt. In a particular
embodiment, the complementary RNA strand comprises the nucleotide
sequence of SEQ ID NO:5 and the sense RNA strand comprises the
nucleotide sequence of SEQ ID NO:4.
[0012] In another aspect, the invention relates to a pharmaceutical
composition for inhibiting the replication of a (+) strand RNA
virus in an organism, such as a mammal (e.g., human). The
pharmaceutical composition comprises the dsRNA as described above,
together with a pharmaceutically acceptable carrier. The dosage
unit of dsRNA in the composition may be less than 5 milligram (mg)
of dsRNA per kg body weight, preferably 0.01 to 2.5 milligrams
(mg), more preferably 0.1 to 200 micrograms (.mu.g), and most
preferably 0.1 to 100 .mu.g per kilogram body weight. In one
embodiment, the pharmaceutically acceptable carrier is an aqueous
solution, such as phosphate buffered saline. In another embodiment,
the pharmaceutically acceptable carrier comprises a micellar
structure, such a liposome, capsid, capsoid, polymeric nanocapsule,
or polymeric microcapsule. The pharmaceutical composition may be
formulated to be administered by inhalation, infusion, injection,
or orally. In one embodiment, the pharmaceutical composition is
formulated to be administered by intravenous or intraperitoneal
injection.
[0013] In yet another aspect, the invention relates to a method for
inhibiting the replication of a (+) strand RNA virus comprising a
3'-untranslated region (3'-UTR) in a cell. The method comprises
introducing a double-stranded ribonucleic acid (dsRNA), as
described above, into the cell. The dsRNA comprises a nucleotide
sequence which is substantially identical to at least a part of the
3'-UTR, and the dsRNA is less than 30 nucleotides in length, more
preferably less than 25 nucleotides, more preferably 21 to 24
nucleotides, and most preferably 23 nucleotides in length.
[0014] In still another aspect, the invention relates to a method
for treating a disease associated with infection of a (+) strand
RNA virus in an organism. The method comprises administering a
pharmaceutical composition to the organism, wherein the
pharmaceutical composition comprises a double-stranded ribonucleic
acid (dsRNA), as described above, together with a pharmaceutically
acceptable carrier. The dsRNA comprises a nucleotide sequence which
is substantially identical to at least a part of the 3'-UTR of the
(+) strand RNA virus, and the dsRNA is less than 30 nucleotides in
length.
[0015] The details of once or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the relevant sequence region from the p2
plasmid (SEQ ID NO 2) and the N-terminal amino acid sequence of the
corresponding reporter protein (SEQ ID NO 2).
[0017] FIG. 2 shows the relevant sequence region from the p3
plasmid (SEQ ID NO 3) and the N-terminal amino acid sequence of the
corresponding reporter protein (SEQ ID NO 3).
[0018] FIG. 3 shows the HCV1-2 dsRNA (SEQ ID NO 4, SEQ ID NO 5) in
contrast to the HCV sequence of an mRNA (SEQ ID NO 15) formed by
means of the p2 and p3 plasmids.
[0019] FIG. 4 shows the GAL1-2 dsRNA (SEQ ID NO 6, SEQ ID NO 7) in
contrast to the mRNA sequence (SEQ ID NO 16) corresponding to
.beta.-gal gene (positive control).
[0020] FIG. 5 shows the HCV3-4 the dsRNA (SEQ ID NO 8, SEQ ID NO
9), that exhibits no relation to the expressed genes (negative
control).
[0021] FIG. 6 shows the K22 dsRNA (SEQ ID NO 10, SEQ ID NO 11),
that exhibits no relation to the expressed genes (negative
control).
[0022] FIG. 7 shows the antisense oligonucleotides HCVPT01 (SEQ ID
NO 12), HCVPTO2 (SEQ ID NO 13), and HCVPTO3 (SEQ ID NO 14), in
comparison to the HCV sequence of mRNA (SEQ ID NO 15) formed by the
p3 plasmid.
[0023] FIG. 8 shows the effect of various concentrations of HCV
1-2, GAL1-2, and HCV3-4 dsRNAs on the activity of
.beta.-galactosidase expressed by means of the p2 plasmid.
[0024] FIG. 9 shows the effect of various concentrations of HCV
1-2, GAL-2, and HCV3-4 dsRNAs on the activity of
.beta.-galactosidase expressed by means of the p3 plasmid.
[0025] FIG. 10 shows the effect of the antisense oligonucleotides
HCVPTO1, HCVPTO2, and HCVPTO3 of dsRNAs HCV1-2, GAL1-2, and HCV3-4
on the activity of .beta.-galactosidase expressed by means of the
p3 plasmid.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention discloses double-stranded ribonucleic
acid (dsRNA), as well as compositions and methods for inhibiting
the replication of a (+) strand RNA virus, such as a Hepatitis C
Virus (HCV), using the dsRNA. The present invention also discloses
compositions and methods for treating diseases in organisms caused
by infection with HCV or HCV-associated diseases. dsRNA directs the
sequence-specific degradation of mRNA through a process known as
RNA interference (RNAi). The process occurs in a wide variety of
organisms, including mammals and other vertebrates. The dsRNA of
the invention comprises an RNA strand (the complementary strand)
having a region that is complementary to at least a portion of a
3'-untranslated region (3'-UTR) of a (+) strand RNA virus. Using a
cell-based assay, the present inventors have demonstrated that very
low dosages of these dsRNA can specifically and efficiently mediate
RNAi in mammalian cells, resulting in a significant reduction in
the activity or level of RNA encoded by the HCV genome as compared
to untreated control cells. The present invention encompasses these
dsRNAs and compositions comprising dsRNA and their use for
specifically inhibiting the activity or replication of a (+) strand
RNA virus such as HCV. The use of these dsRNAs enables the targeted
degradation of mRNAs of genes that are implicated in (+) RNA strand
viral infections, including Hepatitis C. Thus, the methods and
compositions of the present invention comprising these dsRNAs are
useful for treating HCV and HCV-associated diseases.
[0027] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
activity or replication of a (+) strand RNA virus, as well as
compositions and methods for treating viral diseases. The
pharmaceutical compositions of the present invention comprise a
dsRNA having a complementary nucleotide sequence of less than 30
nucleotides in length, preferably less than 25 nucleotides in
length, and most preferably 21 to 24 nucleotides in length, and
which is substantially identical to at least a part of a 3'-UTR of
a (+) strand RNA virus, together with a pharmaceutically acceptable
carrier. The dsRNA is less than 30 nucleotides in length,
preferably less than 25 nucleotides in length, and most preferably
21 to 24 nucleotides in length. The dsRNA may be blunt ended, or
one end, preferably the 3'-end of the complementary (antisense)
strand, may have a single-stranded nucleotide overhang of 1 to 3
nucleotides, preferably 2 nucleotides in length.
[0028] Accordingly, certain aspects of the present invention relate
to pharmaceutical compositions comprising the dsRNA of the present
invention together with a pharmaceutically acceptable carrier,
methods of using the compositions to inhibit the activity or
replication of (+) strand RNA viruses such as HCV, and methods of
using the pharmaceutical compositions to treat Hepatitis C and
HCV-associated diseases.
I. DEFINITIONS
[0029] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below.
[0030] As used herein, the terms "3'-untranslated region" and
"3'-UTR" refer to the conserved, non-coding region at the 3'-end of
a viral genome. The 3'-UTR can be the entire non-coding region or a
fragment thereof. As used herein, the term "highly conserved
region" refers to a region of the viral genome that remains
evolutionarily constant, i.e., a genomic region that has a very low
mutation rate and thus shares significant sequence identity
(>99%) between distinct viral genotypes.
[0031] The term "complementary RNA strand" (also referred to herein
as the "antisense strand") refers to the strand of a dsRNA which is
complementary to a 3'-UTR of a (+) strand RNA virus. As used
herein, the term "complementary nucleotide sequence" refers to the
region on the complementary RNA strand that is complementary to the
3'-UTR. "dsRNA" refers to a ribonucleic acid molecule having a
duplex structure comprising two complementary and anti-parallel
nucleic acid strands. Not all nucleotides of a dsRNA must exhibit
Watson-Crick base pairs; the two RNA strands may be substantially
complementary (i.e., having no more than one or two nucleotide
mismatches). The maximum number of base pairs is the number of
nucleotides in the shortest strand of the dsRNA. The RNA strands
may have the same or a different number of nucleotides. Similarly,
the complementary nucleotide sequence is less than 30, preferably
less than 25, and most preferably 21 to 24 nucleotides in length.
The dsRNA is also preferably less than 30, more preferably less
than 25, and most preferably 21 to 24 nucleotides in length. Thus,
the length of the dsRNA preferably corresponds to the length of the
complementary nucleotide sequence. "Introducing into" means uptake
or absorption in the cell, as is understood by those skilled in the
art. Absorption or uptake of dsRNA can occur through cellular
processes, or by auxiliary agents or devices. For example, for in
vivo delivery, dsRNA can be injected into a tissue site or
administered systemically. In vitro delivery includes methods known
in the art such as electroporation and lipofection.
[0032] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure when a 3'-end of one RNA strand extends beyond the 5'-end
of the other complementary strand, or vice versa. "Blunt" or "blunt
end" means that the lengths of the two RNA strand are the same at
that end of the dsRNA, and hence there is no nucleotide(s)
protrusion (i.e., no nucleotide overhang).
[0033] As used herein and as known in the art, the term "identity"
is the relationship between two or more polynucleotide sequences,
as determined by comparing the sequences. Identity also means the
degree of sequence relatedness between polynucleotide sequences, as
determined by the match between strings of such sequences. Identity
can be readily calculated (see, e.g., Computation Molecular
Biology, Lesk, A.M., eds., Oxford University Press, New York
(1998), and Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York (1993), both of which are
incorporated by reference herein). While there exist a number of
methods to measure identity between two polynucleotide sequences,
the term is well known to skilled artisans (see, e.g., Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press
(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux,
J., eds., M. Stockton Press, New York (1991)). Methods commonly
employed to determine identity between sequences include, for
example, those disclosed in Carillo, H., and Lipman, D., SIAM J.
Applied Math. (1988) 48:1073. "Substantially identical," as used
herein, means there is a very high degree of homology (preferably
100% sequence identity) between the sense strand of the dsRNA and
the corresponding part of the target 3'-UTR of the viral genome.
However, dsRNA having greater than 90%, or 95% sequence identity
may be used in the present invention, and thus sequence variations
that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence can be tolerated. Although
100% identity is preferred, the dsRNA may contain single or
multiple base-pair random mismatches between the RNA and the target
3'-UTR.
[0034] As used herein, the term "treatment" refers to the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disorder,
e.g., a disease or condition, a symptom of disease, or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease, the symptoms of disease, or the predisposition toward
disease.
[0035] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0036] The term "pharmaceutically acceptable carrier" refers to a
carrier or diluent for administration of a therapeutic agent.
Pharmaceutically acceptable carriers 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, ed. 1985), which is hereby incorporated by reference
herein. Such carriers include, but are not limited to, saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The term specifically excludes cell culture
medium. For drugs administered orally, pharmaceutically acceptable
carriers include, but are not limited to pharmaceutically
acceptable excipients such as inert diluents, disintegrating
agents, binding agents, lubricating agents, sweetening agents,
flavoring agents, coloring agents and preservatives. Suitable inert
diluents include sodium and calcium carbonate, sodium and calcium
phosphate, and lactose, while corn starch and alginic acid are
suitable disintegrating agents. Binding agents may include starch
and gelatin, while the lubricating agent, if present, will
generally be magnesium stearate, stearic acid or talc. If desired,
the tablets may be coated with a material such as glyceryl
monostearate or glyceryl distearate, to delay absorption in the
gastrointestinal tract.
[0037] As used herein, a "transformed cell" is a cell into which a
dsRNA molecule has been introduced by means of recombinant DNA
techniques.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)
[0038] In one embodiment, the invention relates to a
double-stranded ribonucleic acid (dsRNA) having a nucleotide
sequence which is substantially identical to at least a portion of
a target 3'-UTR of a (+) strand RNA virus. The dsRNA comprises two
RNA strands that are sufficiently complementary to hybridize to
form the duplex structure.
[0039] One strand of the dsRNA comprises the nucleotide sequence
that is substantially identical to a portion of the target 3'-UTR
(the "sense" strand), and the other strand (the "complementary" or
"antisense" strand) comprises a sequence that is complementary to
the 3'-UTR. Because of this complementarity, the complementary RNA
strand is able to base-pair with the complementary region of the
3'-UTR, thus inducing a structural change within the target 3'-UTR.
For example, the complementary region of the 3'-UTR may be cleaved
(through RNA interference) and/or ligated to other nucleic acid
molecules, thus resulting in degradation and/or insertion or
deletion mutations. Binding between the complementary RNA strand
and the target 3'-UTR can also induce a structural change in the
secondary and/or tertiary structure of the 3'-UTR. Because this
region is vital for viral replication, such structural changes can
block or significantly inhibit replication. Moreover, due to the
high sequence variability of the genome of (+) strand RNA viruses,
particularly HCV, sdRNAs that target conserved regions of the 3'UTR
may have a significant impact over a wide range of viral genotypes.
Thus, not only is the efficiency of inhibition of viral replication
increased by targeting a highly conserved region of the 3'-UTR, but
targeting such regions also enables the treatment of diverse
patient populations.
[0040] The sequence that is complementary to the target 3'-UTR
(i.e., the complementary nucleotide sequence) is less than 30
nucleotides, preferably less than 25 nucleotides, and most
preferably 21 to 24 nucleotides in length. Similarly, the dsRNA may
have less than 30 nucleotides, preferably less than 25 nucleotides,
and most preferably 21 to 24 nucleotides in length. The dsRNA can
be synthesized by standard methods known in the art, e.g., by use
of an automated DNA synthesizer, such as are commercially available
from Biosearch, Applied Biosystems, Inc. In specific embodiments,
the dsRNA can comprise the sequence set forth in SEQ ID NOS:12 or
13, or a complement thereof. In a particular embodiment, the
antisense (complementary) RNA strand comprises the sequence set
forth in SEQ ID NO:5, and the sense RNA strand comprises the
sequence set forth in SEQ ID NO:4.
[0041] In one embodiment, at least one end of the dsRNA is blunt.
dsRNA with at least one blunt end show improved stability as
compared to dsRNA having two nucleotide overhangs. dsRNA with at
least one blunt end shows greater in vivo stability (i.e., is more
resistant to degradation in the blood, plasma, and cells). However,
dsRNAs having at least one nucleotide overhang have unexpectedly
superior inhibitory properties than their blunt-ended counterparts.
Moreover, the present inventors have discovered that the presence
of only one nucleotide overhang strengthens the interference
activity of the dsRNA, without affecting its overall stability. The
stability, particularly plasma stability, can thus be adjusted in
accordance with needs of the particular application. dsRNA having
only one overhang has proven particularly effective in vivo (as
well as in a variety of cells, and cell culture mediums), and are
more stable than dsRNA having two blunt ends. The single-stranded
nucleotide overhang may be 1 to 3, preferably two, nucleotides in
length. Preferably, the single-stranded overhang is located at the
3'-end of the complementary (antisense) RNA strand. Such dsRNAs
have improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Preferably, the complementary strand of
the dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end
is blunt.
III. PHARMACEUTICAL COMPOSITIONS COMPRISING dsRNA
[0042] In one embodiment, the invention relates to a pharmaceutical
composition comprising a dsRNA, as described in the preceding
section, and a pharmaceutically acceptable carrier, as described
below. The pharmaceutical composition comprising the dsRNA is
useful for treating an infection or disease associated with the
activity or replication of a (+) strand RNA virus.
[0043] The pharmaceutical compositions of the present invention are
administered in dosages sufficient to inhibit the activity or
replication of a (+) strand RNA virus, such as HCV. The present
inventors have found that compositions comprising the dsRNA of the
invention can be administered at surprisingly low dosages. A
maximum dosage of 5 mg dsRNA per kilogram body weight per day is
sufficient to inhibit or completely suppress the activity or
replication of the target virus.
[0044] In general, a suitable dose of dsRNA will be in the range of
0.01 to 5.0 milligrams per kilogram body weight of the recipient
per day, preferably in the range of 0.1 to 2.5 milligrams per
kilogram body weight of the recipient per day, more preferably in
the range of 0.1 to 200 micrograms per kilogram body weight per
day, and most preferably in the range of 0.1 to 100 micrograms per
kilogram body weight per day. The pharmaceutical composition may be
administered once daily, or the dsRNA may be administered as two,
three, four, five, six or more sub-doses at appropriate intervals
throughout the day. In that case, the dsRNA contained in each
sub-dose must be correspondingly smaller in order to achieve the
total daily dosage. The dosage unit can also be compounded for
delivery over several days, e.g., using a conventional sustained
release formulation which provides sustained release of the dsRNA
over a several day period. Sustained release formulations are well
known in the art. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0045] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the infection
or disease, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
dsRNAs encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0046] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases. For example, mouse
repositories can be found at The Jackson Laboratory, Charles River
Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource
Centers (MMRRC) National Network and at the European Mouse Mutant
Archive. Such models may be used for in vivo testing of dsRNA, as
well as for determining a therapeutically effective dose.
[0047] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), rectal, vaginal and topical (including buccal and
sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection.
[0048] For oral administration, the dsRNAs useful in the invention
will generally be provided in the form of tablets or capsules, as a
powder or granules, or as an aqueous solution or suspension.
[0049] Tablets for oral use may include the active ingredients
mixed with pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0050] Capsules for oral use include hard gelatin capsules in which
the active ingredient is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredients is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil.
[0051] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, the pharmaceutical compositions of the invention
will generally be provided in sterile aqueous solutions or
suspensions, buffered to an appropriate pH and isotonicity.
Suitable aqueous vehicles include Ringer's solution and isotonic
sodium chloride. In a preferred embodiment, the carrier consists
exclusively of an aqueous buffer. In this context, "exclusively"
means no auxiliary agents or encapsulating substances are present
which might affect or mediate uptake of dsRNA in the cells that
harbor the virus. Such substances include, for example, micellar
structures, such as liposomes or capsids, as described below.
Surprisingly, the present inventors have discovered that
compositions containing only naked dsRNA and a physiologically
acceptable solvent are taken up by cells, where the dsRNA
effectively inhibits replication of the virus. Although
microinjection, lipofection, viruses, viroids, capsids, capsoids,
or other auxiliary agents are required to introduce dsRNA into cell
cultures, surprisingly these methods and agents are not necessary
for uptake of dsRNA in vivo. Aqueous suspensions according to the
invention may include suspending agents such as cellulose
derivatives, sodium alginate, polyvinyl-pyrrolidone and gum
tragacanth, and a wetting agent such as lecithin. Suitable
preservatives for aqueous suspensions include ethyl and n-propyl
p-hydxoxybenzoate.
[0052] The pharmaceutical compositions useful according to the
invention also include encapsulated formulations to protect the
dsRNA against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811; PCT publication
WO 91/06309; and European patent publication EP-A-43075, which are
incorporated by reference herein.
[0053] Toxicity and therapeutic efficacy of dsRNAs can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred.
[0054] The data obtained from cell culture assays and animal
studies can be used in formulation a range of dosage for use in
humans. The dosage of compositions of the invention lies preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
of the compound or, when appropriate, of the polypeptide product of
a target sequence (e.g., achieving a decreased concentration of the
polypeptide) that includes the IC50 (i.e., the concentration of the
test compound which achieves a half-maximal inhibition of symptoms)
as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0055] In addition to their administration individually or as a
plurality, as discussed above, the dsRNAs useful according to the
invention can be administered in combination with other known
agents effective in treating viral infections and diseases. In any
event, the administering physician can adjust the amount and timing
of dsRNA administration on the basis of results observed using
standard measures of efficacy known in the art or described
herein.
[0056] For oral administration, the dsRNAs useful in the invention
will generally be provided in the form of tablets or capsules, as a
powder or granules, or as an aqueous solution or suspension.
IV. METHODS FOR TREATING VIRAL INFECTIONS AND DISEASES
[0057] In one embodiment, the invention relates to a method for
treating a subject having an infection or a disease associated with
the replication or activity of a (+) strand RNA virus having a
3'-UTR, such as HCV. In this embodiment, the dsRNA can act as novel
therapeutic agents for inhibiting replication of the virus. The
method comprises administering a pharmaceutical composition of the
invention to the patient (e.g., human), such that viral replication
is inhibited. Because of their high specificity, the dsRNAs of the
present invention specifically target (+) strand RNA viruses having
a 3'-UTR, as described above, and at surprisingly low dosages.
[0058] Examples of (+) strand RNA viruses which can be targeted for
inhibition include, without limitation, picornaviruses,
caliciviruses, nodaviruses, coronaviruses, arteriviruses,
flaviviruses, and togaviruses. Examples of picornaviruses include
enterovirus (poliovirus 1), rhinovirus (human rhinovirus 1A),
hepatovirus (hepatitis A virus), cardiovirus (encephalomyocarditis
virus), aphthovirus (foot-and-mouth disease virus 0), and
parechovirus (human echovirus 22). Examples of caliciviruses
include vesiculovirus (swine vesicular exanthema virus), lagovirus
(rabbit hemorrhagic disease virus), "Norwalk-like viruses" (Norwalk
virus), "Sapporo-like viruses" (Sapporo virus), and "hepatitis
E-like viruses" (hepatitis E virus). Betanodavirus (striped jack
nervous necrosis virus) is the representative nodavirus.
Coronaviruses include coronavirus (avian infections bronchitis
virus) and torovirus (Berne virus). Arterivirus (equine arteritis
virus) is the representative arteriviridus. Togaviruses include
alphavirus (Sindbis virus) and rubivirus (Rubella virus). Finally,
the flaviviruses include flavivirus (Yellow fever virus),
pestivirus (bovine diarrhea virus), and hepacivirus (hepatitis C
virus). In a preferred embodiment, the virus is hepacivirus, the
hepatitis C virus. Although the foregoing list exemplifies
vertebrate viruses, the present invention encompasses the
compositions and methods for treating infections and diseases
caused by any (+) strand RNA virus having a 3'-UTR, regardless of
the host. For example, the invention encompasses the treatment of
plant diseases caused by sequiviruses, comoviruses, potyviruses,
sobemovirus, luteoviruses, tombusviruses, tobavirus, tobravirus,
bromoviruses, and closteroviruses.
[0059] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), rectal, vaginal and topical (including buccal and
sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection.
V. METHODS FOR INHIBITING EXPRESSION OF A TARGET GENE
[0060] In yet another aspect, the invention relates to a method for
inhibiting the replication or activity of a (+) strand RNA virus,
such as HCV. The method comprises administering a composition of
the invention to the host organism such that replication of the
target virus is inhibited. The organism may be an animal or a
plant. Because of their high specificity, the dsRNAs of the present
invention specifically target (+) strand RNA viruses having a
3'-UTR, and at surprisingly low dosages. Compositions and methods
for inhibiting the replication of a target virus using dsRNAs can
be performed as described elsewhere herein.
[0061] In one embodiment, the method comprises administering a
composition comprising a dsRNA, wherein the dsRNA comprises a
nucleotide sequence which is complementary to at least a part of a
3'-UTR of a (+) strand RNA virus. When the organism to be treated
is a mammal, such as a human, the composition may be administered
by any means known in the art including, but not limited to oral or
parenteral routes, including intravenous, intramuscular,
intraperitoneal, subcutaneous, transdermal, airway (aerosol),
rectal, vaginal and topical (including buccal and sublingual)
administration. In preferred embodiments, the compositions are
administered by intravenous or intraparenteral infusion or
injection.
[0062] The methods for inhibiting viral replication can be applied
to any (+) strand RNA virus, such as those described above.
[0063] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Inhibition of the 3'-UTR of HCV
[0064] To study RNA interference and the action of antisense
oligonucleotides in a non-pathogenic assay, Sequence No. 1 in the
sequence protocol was cloned in front of a gene that codes for E.
coli .beta.-galactosidase. Sequence No. 1 corresponds to a sequence
from a highly conserved region of the 3'-UTR of the HCV genome that
is 24 nucleotides in length. After transfection of the 3'-UTR
plasmid in human HuH-7 liver cells, the sequence was transcribed as
a part of an mRNA that codes for 13-galactosidase. The mRNA
sequence that corresponds to the 3'UTR is therefore identical to
the HCV genome sequence and was subsequently used as the target
sequence.
Generation of p2 and p3 Reporter Plasmids
[0065] The E. coli .beta.-galactosidase (.beta.-gal) gene was
isolated from the commercially available expression vector
p.beta.-Gal control (BD Biosciences Clontech, Tullastr. 4, 69126
Heidelberg, Germany, Gene Accession No. U13186, Nucleotide
280-3429).
[0066] The HCV sequence is part of a fusion gene in the p2 plasmid.
The HCV sequence is part of the open reading frame of the sequence
that codes for .beta.-galactosidase, so that the HCV sequence is
also expressed as part of a fusion protein. FIG. 1 shows the
relevant sequence segments of the p2 plasmid (Sequence No. 2 of the
sequence protocol). The HCV sequence is shown in italics. The
beginning of the .beta.-Gal gene (including 6 nucleotides of the
Kozak sequence in front of the ATG codon) is underlined. The
N-terminal amino acid sequence of the HCV .beta.-galactosidase
fusion protein is listed under the DNA sequence.
[0067] The HCV sequence is also part of a fusion gene in the p3
plasmid. However, the HCV sequence is located outside of the open
reading frame of the sequence that codes for .beta.-galactosidase,
so that the HCV sequence is not expressed as part of a fusion
protein. FIG. 2 shows the relevant sequence segment of the p3
plasmid (Sequence No. 3 in the sequence protocol). The HCV sequence
is shown in italics. The beginning of the .beta.-Gal gene
(including 6 nucleotides of the Kozak sequence in front of the ATG
codon) is underlined. The N-terminal amino acid sequence of the
expressed .beta.-galactosidase is listed under the DNA
sequence.
[0068] The fusion genes that were generated in this way were cloned
into the commercially available pcDNA3.1 (+) expression plasmid
(Invitrogen, Life Technologies, Karlsruhe Technology Part, Emmy
Noether Str. 10, 76131 Karlsruhe, Germany; Catalogue No. V790-20).
This plasmid contains a neomycin resistance gene and thus confers
on the HuH-7 cells that are transfected with it resistance to the
G418. HuH-7 cells selected in the presence of G418 therefore harbor
a reporter plasmid that stably integrated into the cell's genome.
The commercially available pGL3-ctrl plasmid (Promega GmbH, High
Tech Park, Schildkrotstr. 15, 68199 Mannheim, Germany; Gene
Accession No. U47296 was used as the control plasmid. It codes and
expresses the "firefly luciferase" gene.
Synthesis and Preparation of dsRNAs
[0069] Oligoribonucleotides are synthesized with an RNA synthesizer
(Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and
purified by High Pressure Liquid Chromatography (HPLC) using
NucleoPac PA-100 columns, 9.times.250 mm (Dionex Corp.; low salt
buffer: 20 mM Tris, 10 mM NaClO.sub.4, pH 6.8, 10% acetonitrile;
the high-salt buffer was: 20 mM Tris, 400 mM NaC104, pH 6.8, 10%
acetonitrile. flow rate: 3 ml/min). Formation of double stranded
dsRNAs is then achieved by heating a stoichiometric mixture of the
individual complementary strands (10 M) in 10 mM sodium phosphate
buffer, pH 6.8, 100 mM NaCl, to 80-90.degree. C., with subsequent
slow cooling to room temperature over 6 hours.
[0070] In addition, dsRNA molecules with linkers may be produced by
solid phase synthesis and addition of hexaethylene glycol as a
non-nucleotide linker (D. Jeremy Williams, Kathleen B. Hall,
Biochem (1996) 35:14665-14670). A Hexaethylene glycol linker
phosphoramidite (Chruachem Ltd, Todd Campus, West of Scotland
Science Park, Acre Road, Glasgow, G20 OUA, Scotland, UK) is coupled
to the support bound oligoribonucleotide employing the same
synthetic cycle as for standard nucleoside phosphoramidites
(Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany) but
with prolonged coupling times. Incorporation of linker
phosphoramidite is comparable to the incorporation of nucleoside
phosphoramidites.
DsRNA Oligonucleotides
[0071] Three short double-stranded ribonucleic acids (dsRNA) were
used for the RNA interference. These dsRNAs each consist of 2 short
oligoribonucleotides that are complementary to each other over
almost the entire sequence region. Two nucleotides have no base
pairing at either of the 3'-ends of the oligoribonucleotides, and
therefore form dsRNA overhangs.
[0072] The sequence of one of the oligoribonucleotides is identical
to the mRNA target sequence. This oligoribonucleotide is therefore
called the sense strand. The sequence of the other
oligoribonucleotide is complementary to the mRNA target sequence.
This oligoribonucleotide is therefore called the antisense
strand.
[0073] The double-stranded oligoribonucleotide designated as HCV1-2
is shown in FIG. 3 and compared to the HCV sequence of the mRNA
formed by means of the p2 and p3 plasmids. The nucleotides shown in
capital letters correspond to the HCV sequence in the p2 and p3
plasmids. HCV1-2 consists of the HCV 1 sense strand and the HCV 2
antisense strand, whereby two nucleotides in each exhibit no base
pairing at the 3'-ends of the strands. The sense strand (HCV 1)
depicted in Sequence No. 4 in the sequence protocol exhibits almost
the same nucleotide sequence as the HCV sequence of an mRNA formed
by means of the p2 and p3 plasmids, respectively. Three nucleotides
of the HCV sequence are missing at the 5'-end, and two nucleotides
are present at the 3'-end that are not a component of the HCV
sequence. The antisense strand (HCV 2) depicted in Sequence No. 5
in the sequence protocol is, except for the two nucleotides at the
3'-end, complementary to HCV 1, and therefore also to the HCV
sequence of an mRNA formed by means of the p2 and p3 plasmids,
respectively. The HCV sequence corresponds to a 3'-untranslated
region of the HCV genome.
[0074] A dsRNA designated as GAL 1-2 was used as the positive
control. It is shown in FIG. 4 in contrast to an mRNA sequence
(designated as mRNA in FIG. 4) that corresponds to the .beta.-Gal
gene of the p2 and p3 plasmids. GAL1-2 consists of the Gal 1 sense
strand and the Gal 2 antisense strand, whereas two nucleotides in
each exhibit no base pairing at the 3'-ends of the strands. The
sense strand (Gal 1) shown in Sequence No. 6 in the sequence
protocol exhibits almost the same nucleotide sequence as the mRNA
sequence that corresponds to the .beta.-Gal gene. The antisense
strand (Gal 2) shown in Sequence No. 7 in the sequence protocol is,
except for the two nucleotides at the 3'-end, complementary to Gal
1, and therefore also to the mRNA sequence that corresponds to the
.beta.-Gal gene.
[0075] In one part of the experiment, a dsRNA designated as HCV3-4,
which has no relationship to the genes expressed here, was used as
the negative control (FIG. 5). HCV3-4 consists of the HCV 3 sense
strand and the HCV 4 antisense strand, whereby two nucleotides in
each exhibit no base pairing at the 3'-ends of the strands. The
sense strand (HCV 3) shown in Sequence No. 8 of the sequence
protocol exhibits almost no similarity to the mRNA formed by means
of the p2 and p3 plasmids, and therefore has no relationship to the
expressed genes. The antisense strand (HCV 4) shown in Sequence No.
9 in the sequence protocol is, except for the two nucleotides at
the 3'-end, complementary to HCV 3 and therefore also has no
relationship to the mRNA that is formed.
[0076] In another part of the experiment, a dsRNA designated as K22
was used as the negative control. It also exhibits no relationship
to the gene expressed here (FIG. 6). The sequences of both
oligoribonucleotides that form the dsRNA are shown in Sequence Nos.
10 and 11 in the sequence protocol.
[0077] Three 21-nucleotide-long DNA antisense oligoribonucleotides
were used as phosphothioates in the experiments on antisense
oligoribonucleotides. The oligoribonucleotides were obtained from
Metabion GmbH, Lena-Christ Str. 44, 82152 Martinsried, Germany.
They are here designated as HCVPTOI, HCVPTO2, and HCVPTO3. HCVPTO1
and HCVPTO2 are complementary to different regions of the HCV-mRNA
sequence formed by means of the p3 plasmid. HCVPTO3 is the negative
control without relationship to the target sequence. HCVPTO1,
HCVPTO2, and HCVPTO3 are shown in FIG. 7 in contrast to the
HCV-mRNA sequence. RNA interference assays were tested on the HuH-7
type liver cell line (Nakabayashi et al. 1982 which can be infected
by HCV and is used routinely to culture these viruses. The cells
were cultured in DMEM (Dulbecco's Modified Eagle Medium) with 10%
fetal calf serum (FCS).
a) Experiments Relating to RNA Interference
Transfection
[0078] Prior to transfection, 2.times.10.sup.4 cells per well of a
96-well cell culture plate were seeded. 3 .mu.g p2 plasmid and p3
plasmid, respectively, were mixed with 1 .mu.g pGL3-ctrl plasmid.
0.25 .mu.g of this plasmid mixture was placed in each well for
transfection. Approximately 24 hours after seeding the cells, the
p2/pGL3-ctrl and p3/pGL3-ctrl reporter plasmids were transfected
together with dsRNA in HuH-7. The quantity of transfected DNA per
well was constant.
[0079] The dsRNA was added to the plasmid mixtures in decreasing
concentrations of 400 nmol/l to 12.5 nmol/l (in relation to 110
.mu.l total transfection volume). The initial concentration of the
HCV1-2, GALL-2, and nonspecific HCV3-4 dsRNAs in each stock.
solution was 20 .mu.mol/l. The dsRNAs were diluted by mixing them
stepwise with the same volume of annealing buffer (AB, 100
mmol/lNaCl, 20 mmol/l sodium phosphate, pH 6.8) to arrive at the
end concentration.
[0080] For an end concentration of 400 nmol/1, 2.2 .mu.l stock
solution was used for a transfection volume of 110 .mu.l per well,
and 6.6 .mu.l stock solution was used for a transfection volume of
330 .mu.l per well, respectively. The dilution steps were produced
as shown in Table 1.
TABLE-US-00001 TABLE 1 Production of dsRNA dilution steps
Concentration Quantity of Quantity End Solu- of Initial Initial of
Concen- tion Initial Solution Solution Added AB tration * No.
Solution (.mu.mol/l) (.mu.l) (.mu.l) (nmol/l) 1 Stock 20 14.0 400
solution 2 Solution 1 10 7.0 7.0 200 3 Solution 2 5 7.0 7.0 100 4
Solution 3 2.5 7.0 7.0 50 5 Solution 4 1.25 7.0 7.0 25 6 Solution 5
0.62 7.0 7.0 12.5 * End concentration, using 6.6 .mu.l of each
solution to a transfection volume of 330 .mu.l.
[0081] Plasmids and dsRNA were cotransfected. Gene Porter 2
(PeQLab, Carl Thiersch Str. 2B, 91052 Erlangen, Germany; Catalogue
No. 13-T202007) was used as the transfection agent. Each
cotransfection was repeated three times.
[0082] For 3 wells of the 96-well plates a mixture was made that
consisted of 2.0 .mu.l of a plasma mixture consisting of the p2
plasmid and the pGL3 control plasmid (0.3875 .mu.g/.mu.l; 3:1),
6.6p1 dsRNA (20, 10, 5, 2.5, 12.5, and 0.62 .mu.mol/l,
respectively), and 16.4 .mu.l DNA diluent B (supplied together with
Gene Porter 2, PeQLab). This mixture was mixed with a mixture
consisting of 6.0 .mu.l Gene Porter 2 and 19 .mu.l serum-free
medium. The total volume of the resultant mixture was 50 .mu.l, of
which 16.5 .mu.l was added to each of 2.times.10.sup.4 HuH-7 in 100
.mu.l of medium. Then a mixture was made that consisted of 2.0
.mu.l of a plasmid mixture consisting of the p3 plasmid and the
pGL3 control plasmid (0.3875 .mu.g/.mu.l); 3:1), 6.6 .mu.l dsRNA
(20, 10, 5, 2.5, 12.5, and 0.62 .mu.mol/l respectively), and 16.4
.mu.l DNA diluent B. This mixture was mixed with a mixture
consisting of 6.0 .mu.l Gene Porter 2 and 19 .mu.l serum-free
medium. The total volume of the resultant mixture was 50 .mu.l, of
which 16.5 .mu.l was added to each of 2.times.10.sup.4 HuH-7 in 100
.mu.l of medium. The transfected cells were incubated at 37.degree.
C. and 5% CO2. 35 .mu.l of fresh medium was added to each well, and
the cells were incubated for another 24 hours. The cells were then
trypsinied.
Detection Methods Used
[0083] The effect of dsRNA on the expression of the reporter genes
was determined by quantifying the .mu.l-galactosidase and
luciferase activity by means of chemoluminescence. For this,
lysates were made using the Tropix Lysebuffer (Applied Biosystems,
850 Lincoln Centre Drive, Foster City, Calif. 944404; Catalogue No.
BD100LP) in accordance with manufacturer's instructions.
[0084] To quantify .beta.-galactosidase activity, 2 p. 1 lysate was
used per analysis, as well as the substrate Galacto Star (Applied
Biosystems, Tropix; Catalogue No. BM100S), in accordance with
manufacturer instructions. To quantify luciferase activity, 5 gl
lysate was used per analysis, as well as the substrate Luciferin
(Applied Biosystems, Tropix; Catalogue No. BM100L) in accordance
with manufacturer instructions. Luminescence was measured in each
case using the Berthold Sirius luminometer (Berthold Detection
Systems GmbH, Bleichstr. 56-58, 75173 Pforzheim, Germany).
Results
[0085] For each transfection assay, three 96-well plates were
analyzed, such that in each case both .beta.-galactosidase and
luciferase were measured. The quotient of the relative light units
(RLU) of .beta.-galactosidase and the relative light units of
luciferase were calculated. An average was determined for these
three values. The average for p2/pGL3- and p3/pGL3 transfected
cells without dsRNA, respectively, was arbitrarily defined as 1.0.
The values that changed under the influence of dsRNA were recorded
as a ratio to 1.0 (see FIGS. 8 and 9), i.e., a value of 0.6
corresponds to a 40% inhibition of .beta.-galactosidase activity in
comparison with untreated cells. In FIG. 8, cotransfection of
sequence-specific dsRNA with the p2 plasmid resulted in a reduction
in .beta.-galactosidase activity. The HCV1-2 and GALL-2 dsRNAs
inhibit .beta.-galactosidase with comparable effectiveness. At
transfection volumes of 400 nmol/l and 200 mmol/1 of dsRNA,
.beta.-galactosidase activity decreases to 40% as compared to
untreated cells. The inhibitory effect decreased with decreasing
dsRNA concentration. The HCV3-4 control dsRNA leads to no decrease
in .beta.-galactosidase activity in lysate over the entire
concentration range. A reduction in f3-galactosidase expression is
also detectable with cotransfection of the sequence-specific HCV1-2
dsRNA with the p3 plasmid (FIG. 9). HCV1-2 and GAL1-2 inhibit
.beta.-galactosidase activity with comparable effectiveness. At
transfection volumes of 400=Oland 200 mol/l of dsRNA,
f3-galactosidase activity decreases to approximately 20% as
compared to untreated cells. The inhibitory effect decreased with
decreasing dsRNA concentration. The HCV3-4 control dsRNA showed a
weak inhibition of .beta.-galactosidase activity to approximately
70% as compared to untreated cells. In the presence of the HCV1-2
dsRNA, both the p2 and p3 plasmids showed a marked decrease in
.beta.-galactosidase activity. Comparable effects were seen with
the GAL1-2 dsRNA (positive control). The second control dsRNA,
HCV3-4, led to no and markedly less inhibition of
.beta.-galactosidase activity, respectively. Expression and/or
stability of RNA was markedly decreased by dsRNA in the experiments
described. This was also true for HCV target sequences outside the
open reading frame, which corresponds to the situation with the
natural 3'-UTR region of HCV.
b) Experiments with Antisense DNA Oligonucleotides
[0086] To prepare for the experiments, p3 was stably transfected
into HuH-7 cells using LipofectaminePLUS (GIBCO BRL Life
Technologies, Karlsruhe Technology Park, Emmy Noether Str. 10,
76131 Karlsruhe, Germany). For this, 2.times.10.sup.4 cells were
seeded per well of a 96-well cell culture plate. After 24 hours,
the medium was replaced with 50 .mu.l serum-free medium (DMEM). The
transfection mixture consisted of 0.2 .mu.g p3, 16.7 .mu.l DMEM, 2
.mu.l PLUS reagent, and 1 .mu.l Lipofectamine reagent. Cells were
transfected in accordance with manufacturer's instructions. After
three hours, the transfection medium was replaced with 150 .mu.l
complete medium (DMEM+10% fetal calf serum). After 48 hours, the
cells were transferred to wells in a 12-well cell culture plate,
and cultured with 400 .mu.g/ml G418 (Amersham Biosciences,
Munzinger Str. 9, 79111 Freiberg, Germany). Colonies were collected
and transferred to new wells in a 12-well cell culture plate. From
these, the cells that grew in the new wells after 14-21 days were
culled manually and cultured with 400 .mu.g/ml G418 until the
selection was complete. After approximately three manual
selections, .beta.-galactosidase activity was determined as
described below by means of enzyme measurements. Then the number of
cells that expressed galactosidase was determined using X-Gal
staining. For this, the medium was aspirated and the cells were
stained in the wells of a 96-well cell culture plate overnight in
100 .mu.l X-Gal solution (10 mmol/l sodium phosphate, pH 7.0; 1
mmol/lMgCL.sub.2; 150 mmol/l NaCl; 3.3
mmol/lK.sub.4Fe(CN).sub.6*3H.sub.2O; 3.3 mmol/lK.sub.4Fe(CN).sub.6;
0.2% X-Gal) (X-Gal from PeQLab, Erlangen, Germany; all other
chemicals from SIGMA, Griinwalcier Weg 30, 82024 Taufldrchen,
Germany). The best clone was designated "HuH-7 blue" and used for
the experiments.
Transfection with Dsrna and Antisense DNA Oligonucleotides
[0087] To prepare for a transfection, 2.times.10.sup.4 cells of
HuH-7 blue was seeded in 100 .mu.l DMEM+10% FCS per well of a
96-well cell culture plate. After 24 hours, the dsRNA and the
antisense DNA oligonucleotides were transfected. Fugene 6 (Roche
Applied Sciences, Sandhofer Str. 116, 68305 Mannheim, Germany;
Catalogue No. 1814443) was used for these transfections. Every
fifth well containing HuH-7 blue cells was not treated. Stock
solutions with a concentration of 20 .mu.mol/l were made from the
HCV 1-2, GAL1-2, and K22 dsRNAs. 1.6 .mu.l of this stock solution
was in each case mixed with 0.9 .mu.l Fugene 6 and 108 .mu.l DMEM.
The dsRNA was therefore present at a concentration of 15 nmol/l.
Each of 5 wells of a 96-well cell culture plate was transfected
with 20 .mu.l of this assay. Stock solutions were made with each of
the antisense DNA oligonucleotides HCVPTO1, HCVPTO2, and HCVPTO3,
and a concentration of 100 .mu.mol/l. 1.2 .mu.l of this stock
solution was in each case mixed with 2.4 .mu.l Fugene 6 and 108
.mu.l DMEM. The dsRNA was therefore present in a concentration of
200 nmol/l. Each of 5 wells of a 96-well cell culture plate was
transfected with 20 .mu.l of this mixture.
Detection Methods
[0088] The effect of dsRNA oligonucleotides and antisense DNA
oligonucleotides on the expression of reporter genes was determined
by quantifying the .beta.-galactosidase activity by means of
chemoluminescence. For this, lysates were made using the Tropix
Lysebuffer (Applied Biosystems, 850 Lincoln Centre Drive, Foster
City, Calif. 944404; Catalogue No. BD100LP) in accordance with
manufacturer's instructions. Chemoluminescence measurements were
quantified as follows:
[0089] 5 .mu.l of lysate were placed in each reagent vessel and
filled to 30 .mu.l with (3-Gal assay buffer (1 ml 1 mol/l sodium
phosphate buffer, pH 8.0; 10 .mu.l 1 mol/l MgCl.sub.2, 10 .mu.l
1.25 mg/ml Galakton [Tropix GCO20, Applied Biosystems]; 9 ml
deiodized water). Ml .beta.-Gal stop mix (1 ml 2 mol/lNa0H, 250
.mu.l 2.5% Emerald Enhancer [Applied Biosystems, Tropix, LAY250],
8.75 ml deionized water), mixed thoroughly, and immediately
measured in the luminometer. If not otherwise noted, all reagents
were supplied by SIGMA. Luminescence was measured in each case
using the Berthold Sirius luminometer (Berthold Detection Systems
GmbH, Bleichstr. 56-58, 75173 Pforzheim, Germany). 5 wells of a
96-well cell culture plate were analyzed per transfection assay.
.beta.-galactosidase activity was determined in each case, and the
average of the 5 individual values was established. The average
value for untransfected cells is arbitrarily defined as 1.0. The
average values for transfected cells are then expressed as a ratio
with the average for untransfected cells. For example, a value of
0.6 corresponds to an inhibition of P-galactosidase activity by 40%
in comparison to untreated cells. The results are shown in FIG.
10.
Results
[0090] With transfection of sequence-specific antisense
oligonucleotides (200 nmol/l) and dsRNA oligonucleotides (50
nmol/l) in the HuH-7 blue cell line, a reduction in
.beta.-galactosidase activity was detectable. HCVPTO1 reduced the
activity of .beta.-galactosidase by 35%, and HCVPTO2 by 40%. The
HCVPTO3 oligonucleotide used as the negative control increased the
activity by 40% as compared to untreated cells. The HCV1-2 and
GAL1-2 dsRNAs inhibited .beta.-galactosidase activity with
comparable effectiveness. .beta.-galactosidase activity decreased
by 37% in each case, as compared with untreated cells. The K22
nonspecific control increased activity by 15% in comparison with
untreated cells.
Example 2
Treatment of a HCV Infected Patient with dsRNA
[0091] In this Example, HCV specific double stranded dsRNAs are
injected into HCV infected patients and shown to specifically
inhibit HCV gene expression.
dsRNA Administration and Dosage
[0092] The present example provides for pharmaceutical compositions
for the treatment of human HCV infected patients comprising a
therapeutically effective amount of a HCV specific dsRNA as
disclosed herein, in combination with a pharmaceutically acceptable
carrier or excipient. DsRNAs useful according to the invention may
be formulated for oral or parenteral administration. The
pharmaceutical compositions may be administered in any effective,
convenient manner including, for instance, administration by
topical, oral, anal, vaginal, intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal or intradermal routes,
among others. One of skill in the art can readily prepare dsRNAs
for injection using such carriers that include, but are not limited
to, saline, buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. Additional examples of suitable carriers
are found in standard pharmaceutical texts, e.g. "Remington's
Pharmaceutical Sciences", 16th edition, Mack Publishing Company,
Easton, Pa., 1980.
Example 3
RNA Purification and Analysis
[0093] Efficacy of the dsRNA treatment is determined at defined
intervals after the initiation of treatment using real time PCR on
total RNA extracted from peripheral blood. Cytoplasmic RNA from
whole blood, taken prior to and during treatment, is purified with
the help of the RNeasy Kit (Qiagen, Hilden) and HCV mRNA levels are
quantitated by real time RT-PCR as described previously (Eder, M.,
et al., Leukemia (1999) 13:13831389; Scherr M et al.,
BioTechniques. (2001) 31:520-526).
Example 4
HCV-Specific Dsrna Expression Vectors
[0094] HCV-specific dsRNA molecules that interact with HCV target
RNA molecules and modulate HCV gene expression activity are
expressed from transcription units inserted into DNA or RNA vectors
(see, for example, Couture et A, 1996, TIG., 12, 5 1 0, Skillem et
A, International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299). These transgenes can be introduced as a linear
construct, a circular plasmid, or a viral vector, which can be
incorporated and inherited as a transgene integrated into the host
genome. The transgene can also be constructed to permit it to be
inherited as an extrachromosomal plasmid (Gassmann et al., 1995,
Proc. Natl. Acad. Sci. USA 92:1292).
[0095] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and cotransfected into
a target cell. Alternatively each individual strand of the dsRNA
can be transcribed by promoters both of which are located on the
same expression plasmid. In a preferred embodiment, the dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0096] The recombinant dsRNA expression vectors are preferably DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka et al. (1992, Curr. Topics in Micro.
and Immunol. 158:97-129)), adenovirus (see, for example, Berkner et
al. (1988, BioTechniques 6:616), Rosenfeld et al. (1991, Science
252:431-434), and Rosenfeld et al. (1992, Cell 68:143-155)), or
alphaviras as well as others known in the art. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, in vitro and/or in vivo (see for
example Eglitis, et al., 1985, Science 230:1395-1398; Danos and
Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et
al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991,
Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad.
Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy
3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). Recombinant retroviral vectors
capable of transducing and expressing genes inserted into the
genome of a cell can be produced by transfecting the recombinant
retroviral genome into suitable packaging cell lines such as PA317
and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone
et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant
adenoviral vectors can be used to infect a wide variety of cells
and tissues in susceptible hosts (e.g., rat, hamster, dog, and
chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and
also have the advantage of not requiring mitotically active cells
for infection.
[0097] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector of the invention may be a eukaryotic RNA
polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g.
CMV early promoter or actin promoter or Ul snRNA promoter) or
preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter,
provided the expression plasmid also encodes T7 RNA polymerase
required for transcription from all promoter. The promoter can also
direct transgene expression to the liver e.g. albumin regulatory
sequence (Pinkert et al., 1987, Genes Dev. 1:268276).
[0098] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the dsRNA transgene.
[0099] Preferably, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells ex-planted from the patient followed by reintroduction
into the patient, or by any other means that allow for introduction
into a desired target cell.
[0100] DsRNA expression DNA plasmids are typically transfected into
target cells as a complex with cationic lipid carriers (e.g.
Oligofectamine) or non-cationic lipid-based carriers (e.g.
Transit-TKO.TM.). Multiple lipid transfections for dsRNA-mediated
knockdowns targeting different regions of a single target gene or
multiple target genes over a period of a week or more are also
contemplated by the present invention. Successful introduction of
the vectors of the invention into host cells can be monitored using
various known methods. For example, transient transfection can be
signaled with a reporter, such as a fluorescent marker, such as
Green Fluorescent Protein (GFP). Stable transfection of ex vivo
cells can be ensured using markers that provide the transfected
cell with resistance to specific environmental factors (e.g.,
antibiotics and drugs), such as hygromycin B resistance.
[0101] The nucleic acid molecules of the invention described above
can also be generally inserted into vectors and used as gene
therapy vectors for human patients infected with HCV. Gene therapy
vectors can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
Sequence CWU 1
1
18124DNAArtificial SequenceSynthetically generated oligonucleotide
1gtcacggcta gctgtgaaag gtcc 24257DNAArtificial
SequenceSynthetically generated oligonucleotide 2gtcacc atg tcg tca
cgg cta gct gtg aaa ggt cca gtc acc atg tcg 48 Met Ser Ser Arg Leu
Ala Val Lys Gly Pro Val Thr Met Ser 1 5 10ttt act ttg 57Phe Thr
Leu15357DNAArtificial SequenceSynthetically generated
oligonucleotide 3gtcaccttgt cgtcacggct agctgtgaaa ggtccagtca cc atg
tcg ttt act 54 Met Ser Phe Thr 1ttg 57Leu5423RNAArtificial
SequenceSynthetically generated oligonucleotide corresponding to
Hepatitis C Virus 4acggcuagcu gugaaagguc cgu 23523RNAArtificial
SequenceSynthetic dsRNA corresponding to Hepatitis C Virus
5ggaccuuuca cagcuagccg uga 23623RNAArtificial SequenceSynthetic
dsRNA corresponding to E. coli beta-galactosidase 6gugaaauuau
cgaugagcgu ggu 23723RNAArtificial SequenceSynthetic dsRNA
corresponding to E. coli beta-galactosidase 7cacgcucauc gauaauuuca
ccg 23821RNAArtificial SequenceSynthetically generated
oligonucleotide 8agacagucga cuucagccug g 21921RNAArtificial
SequenceSynthetically generated oligonucleotide 9aggcugaagu
cgacugucug g 211024RNAArtificial SequenceSynthetically generated
oligonucleotide 10gaugaggauc guuucgcaug auug 241124RNAArtificial
SequenceSynthetically generated oligonucleotide 11aucaugcgaa
acgauccuca uccu 241221DNAArtificial SequenceSynthetically generated
oligonucleotide 12ggacctttca cagctagccg t 211321DNAArtificial
SequenceSynthetically generated oligonucleotide 13cctttcacag
ctagccgtga c 211421DNAArtificial SequenceSynthetically generated
oligonucleotide 14tgccgatcga cactttccag g 211528RNAHepatitis C
virus 15ucgucacggc uagcugugaa agguccag 281625RNAArtificial
SequenceSynthetic mRNA corresponding to the E. coli B-Gal gene
16cggugaaauu aucgaugagc guggu 251717PRTArtificial
SequenceSynthetically generated peptide 17Met Ser Ser Arg Leu Ala
Val Lys Gly Pro Val Thr Met Ser Phe Thr 1 5 10
15Leu185PRTArtificial SequenceSynthetically generated peptide 18Met
Ser Phe Thr Leu 1 5
* * * * *