U.S. patent application number 11/151976 was filed with the patent office on 2006-04-27 for rnai modulation of rsv, piv and other respiratory viruses and uses thereof.
Invention is credited to Sailen Barik.
Application Number | 20060089324 11/151976 |
Document ID | / |
Family ID | 36578352 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060089324 |
Kind Code |
A1 |
Barik; Sailen |
April 27, 2006 |
RNAi modulation of RSV, PIV and other respiratory viruses and uses
thereof
Abstract
The present invention is based on the in vivo demonstration that
RSV and PIV can be inhibited through intranasal administration of
RNAi agents as well as by parenteral administration of such agents.
Further, it is shown that effective viral reduction can be achieved
with more than one virus being treated concurrently. Based on these
findings, the present invention provides general and specific
compositions and methods that are useful in reducing RSV or PIV
mRNA levels, RSV or PIV protein levels and viral titers in a
subject, e.g., a mammal, such as a human. These findings can be
applied to other respiratory viruses.
Inventors: |
Barik; Sailen; (Mobile,
AL) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
36578352 |
Appl. No.: |
11/151976 |
Filed: |
June 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60621552 |
Oct 22, 2004 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
C12N 15/1131 20130101;
A61P 31/14 20180101; A61K 2121/00 20130101; C12N 2310/14 20130101;
A61P 11/00 20180101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of reducing the levels of a viral protein, viral mRNA
or viral titer in a cell in a subject comprising the step of
administering an iRNA agent to said subject, wherein the iRNA agent
comprising a sense strand having at least 15 contiguous nucleotides
complementary to gene from a first mammalian respiratory virus and
an antisense strand having at least 15 contiguous nucleotides
complementary to said sense strand.
2. The method of claim 1, wherein said mammalian respiratory virus
is selected from the group consisting of PIV and RSV.
3. The method of claim 2, wherein said gene from said RSV is the P
protein gene.
4. The method of claim 2 wherein said agent comprises 15
nucleotides selected from one of the agents of Table 1.
5. The method of claim 1, wherein said iRNA agent is administered
intranasally to a subject.
6. The method of claim 1, wherein said iRNA agent is administered
via inhalation or nebulization to a subject.
7. The method of claim 1, wherein said iRNA agent reduces the viral
titer in said subject.
8. The method of claim 1 further comprising co-administering a
second iRNA agent to said subject, wherein said second iRNA agent
comprising a sense strand having at least 15 contiguous nucleotides
complementary to gene from a second mammalian respiratory virus and
an antisense strand having at least 15 contiguous nucleotides
complementary to said sense strand.
9. The method of claim 1, wherein the subject is diagnosed as
having a viral infection.
10. The method of claim 1, wherein said first mammalian respiratory
virus is RSV and said second mammalian respiratory virus is
PIV.
11. The method of claim 9, wherein the subject is diagnosed as
having a viral infection with said first and said second mammalian
respiratory virus.
12. A method of reducing the levels of a viral protein from a first
and a second mammalian respiratory virus in a cell in a subject
comprising the step of co-administering a first and a second iRNA
agent to said subject, wherein said first iRNA agent comprising a
sense strand having at least 15 contiguous nucleotides
complementary to gene from a first mammalian respiratory virus and
an antisense strand having at least 15 contiguous nucleotides
complementary to said sense strand and said second iRNA agent
comprising a sense strand having at least 15 contiguous nucleotides
complementary to gene from a second mammalian respiratory virus and
an antisense strand having at least 15 contiguous nucleotides
complementary to said sense strand.
13. The method of claim 1, wherein said first mammalian respiratory
virus is RSV and said second mammalian respiratory virus is
PIV.
14. The method of claim 13, wherein said iRNA agents are
administered intranasally.
15. The method of claim 13, wherein said iRNA agents are
administered via inhalation or nebulization.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/621,552, filed Oct. 22, 2004, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to the field of respiratory viral
therapy and compositions and methods for modulating viral
replication, and more particularly to the down-regulation of a
gene(s) of a respiratory virus by oligonucleotides via RNA
interference which are administered locally to the lungs and nasal
passage via inhalation/intranasally or systemically via
injection/intravenous.
BACKGROUND
[0003] By virtue of its natural function the respiratory tract is
exposed to a slew of airborne pathogens that cause a variety of
respiratory ailments. Viral infection of the respiratory tract is
the most common cause of infantile hospitalization in the developed
world with an estimated 91,000 annual admissions in the US at a
cost of $300 M. Human respiratory syncytial virus (RSV) and
parainfluenza virus (PIV) are two major agents of respiratory
illness; together, they infect the upper and lower respiratory
tracts, leading to croup, pneumonia and bronchiolitis (Openshaw, P.
J. M. Respir. Res. 3 (Suppl 1), S15-S20 (2002), Easton, A. J., et
al., Clin. Microbiol. Rev. 17, 390-412 (2004)). RSV alone infects
up to 65% of all babies within the first year of life, and
essentially all within the first 2 years. It is a significant cause
of morbidity and mortality in the elderly as well. Immunity after
RSV infection is neither complete nor lasting, and therefore,
repeated infections occur in all age groups. Infants experiencing
RSV bronchiolitis are more likely to develop wheezing and asthma
later in life. Research for effective treatment and vaccine against
RSV has been ongoing for nearly four decades with few successes
(Openshaw, P. J. M. Respir. Res. 3 (Suppl 1), S15-S20 (2002),
Maggon, K. et al, Rev. Med. Virol. 14, 149-168 (2004)). Currently,
no vaccine is clinically approved for either RSV or PIV. Strains of
both viruses also exist for nonhuman animals such as the cattle,
goat, pig and sheep, causing loss to agriculture and the dairy and
meat industry (Easton, A. J., et al., Clin. Microbiol. Rev. 17,
390-412 (2004)).
[0004] Both RSV and PIV contain nonsegmented negative-strand RNA
genomes and belong to the Paramyxoviridae family. A number of
features of these viruses have contributed to the difficulties of
prevention and therapy. The viral genomes mutate at a high rate due
to the lack of a replicational proof-reading mechanism of the RNA
genomes, presenting a significant challenge in designing a reliable
vaccine or antiviral (Sullender, W. M. Clin. Microbiol. Rev. 13,
1-15 (2000)). Promising inhibitors of the RSV fusion protein (F)
were abandoned partly because the virus developed resistant
mutations that were mapped to the F gene (Razinkov, V., et. al.,
Antivir. Res. 55, 189-200 (2002), Morton, C. J. et al. Virology
311, 275-288 (2003)). Both viruses associate with cellular
proteins, adding to the difficulty of obtaining cell-free viral
material for vaccination (Burke, E., et al., Virology 252, 137-148
(1998), Burke, E., et al., J. Virol. 74, 669-675 (2000), Gupta, S.,
et al., J. Virol. 72, 2655-2662 (1998)). Finally, the immunology of
both, and especially that of RSV, is exquisitely complex (Peebles,
R. S., Jr., et al., Viral. Immunol. 16, 25-34 (2003), Haynes, L.
M., et al., J. Virol. 77, 9831-9844 (2003)). Use of denatured RSV
proteins as vaccines leads to "immunopotentiation" or
vaccine-enhanced disease (Polack, F. P. et al. J. Exp. Med. 196,
859-865 (2002)), and this phenomenon has been neither tested nor
ruled out for PIV. The overall problem is underscored by the recent
closure of a number of anti-RSV biopharma programs.
[0005] The RSV genome comprises a single strand of negative sense
RNA that is 15,222 nucleotides in length and yields eleven major
proteins. (Falsey, A. R., and E. E. Walsh, 2000, Clinical
Microbiological Reviews 13:371-84.) Two of these proteins, the F
(fusion) and G (attachment) glycoproteins, are the major surface
proteins and the most important for inducing protective immunity.
The SH (small hydrophobic) protein, the M (matrix) protein, and the
M2 (22 kDa) protein are associated with the viral envelope but do
not induce a protective immune response. The N (major nucleocapsid
associated protein), P (phosphoprotein), and L (major polymerase
protein) proteins are found associated with virion RNA. The two
non-structural proteins, NS1 and NS2, presumably participate in
host-virus interaction but are not present in infectious
virions.
[0006] Human RSV strains have been classified into two major
groups, A and B. The G glycoprotein has been shown to be the most
divergent among RSV proteins. Variability of the RSV G glycoprotein
between and within the two RSV groups is believed to be important
to the ability of RSV to cause yearly outbreaks of disease. The G
glycoprotein comprises 289-299 amino acids (depending on RSV
strain), and has an intracellular, transmembrane, and highly
glycosylated stalk structure of 90 kDa, as well as heparin-binding
domains. The glycoprotein exists in secreted and membrane-bound
forms.
[0007] Successful methods of treating RSV infection are currently
unavailable (Maggon and Barik, 2004, Reviews in Medical Virology
14:149-68). Infection of the lower respiratory tract with RSV is a
self-limiting condition in most cases. No definitive guidelines or
criteria exist on how to treat or when to admit or discharge
infants and children with the disease. Hypoxia, which can occur in
association with RSV infection, can be treated with oxygen via a
nasal cannula. Mechanical ventilation for children with respiratory
failure, shock, or recurrent apnea can lower mortality. Some
physicians prescribe steroids. However, several studies have shown
that steroid therapy does not affect the clinical course of infants
and children admitted to the hospital with bronchiolitis. Thus
corticosteroids, alone or in combination with bronchodilators, may
be useless in the management of bronchiolitis in otherwise healthy
unventilated patients. In infants and children with underlying
cardiopulmonary diseases, such as bronchopulmonary dysphasia and
asthma, steroids have also been used.
[0008] Ribavirin, a guanosine analogue with antiviral activity, has
been used to treat infants and children with RSV bronchiolitis
since the mid 1980s, but many studies evaluating its use have shown
conflicting results. In most centers, the use of ribavirin is now
restricted to immunocompromised patients and to those who are
severely ill.
[0009] The severity of RSV bronchiolitis has been associated with
low serum retinol concentrations, but trials in hospitalized
children with RSV bronchiolitis have shown that vitamin A
supplementation provides no beneficial effect. Therapeutic trials
of 1500 mg/kg intravenous RSV immune globulin or 100 mg/kg inhaled
immune globulin for RSV lower-respiratory-tract infection have also
failed to show substantial beneficial effects.
[0010] In developed countries, the treatment of RSV
lower-respiratory-tract infection is generally limited to
symptomatic therapy. Antiviral therapy is usually limited to
life-threatening situations due to its high cost and to the lack of
consensus on efficacy. In developing countries, oxygen is the main
therapy (when available), and the only way to lower mortality is
through prevention.
[0011] RNA interference or "RNAi" is a term initially coined by
Fire and co-workers to describe the observation that
double-stranded RNA (dsRNA) can block gene expression when it is
introduced into worms (Fire et al., Nature 391:806-811, 1998).
Short dsRNA directs gene-specific, post-transcriptional silencing
in many organisms, including vertebrates, and has provided a new
tool for studying gene function. RNAi has been suggested as a
method of developing a new class of therapeutic agents. However, to
date, these have remained mostly as suggestions with no demonstrate
proof that RNAi can be used therapeutically.
[0012] Therefore, there is a need for safe and effective vaccines
against RSV, especially for infants and children. There is also a
need for therapeutic agents and methods for treating RSV infection
at all ages and in immuno-compromised individuals. There is also a
need for scientific methods to characterize the protective immune
response to RSV so that the pathogenesis of the disease can be
studied, and screening for therapeutic agents and vaccines can be
facilitated. The present invention overcomes previous shortcomings
in the art by providing methods and compositions effective for
modulating or preventing RSV and PIV infection, which be expanded
to other respiratory viruses. Specifically, the present invention
advances the art by providing iRNA agents that have been shown to
reduce RSV and PIV levels in vivo and a showing of therapeutic
activity of this class of molecules. It is further demonstrated
that more than one virus can be treated concurrently.
SUMMARY
[0013] The present invention is based on the in vivo demonstration
that RSV and PIV can be inhibited through intranasal administration
of RNAi agents, as well as by parenteral administration of such
agents. Further, it is shown that effective viral titer reduction
can be achieved with more than one virus being treated concurrently
using two different iRNA agents. Based on these findings, the
present invention provides general and specific compositions and
methods that are useful in reducing RSV or PIV mRNA levels, RSV or
PIV protein levels and RSV and PIV viral titers in a subject, e.g.,
a mammal, such as a human. These findings can be applied to other
respiratory viruses.
[0014] The present invention specifically provides iRNA agents
consisting of or comprising at least 15 contiguous nucleotides of
one of the genes of RSV, PIV or other respiratory virus,
particularly the P gene of RSV or PIV and the N G, F, SH, M, and L
genes of RSV. The iRNA agent preferably comprises less than 30
nucleotides per strand, e.g., 21-23 nucleotides. The double
stranded iRNA agent can either have blunt ends or more preferably
have overhangs of 1-4 nucleotides from one or both 3' ends of the
agent.
[0015] Further, the iRNA agent can either contain only naturally
occurring ribonucleotide subunits, or can be synthesized so as to
contain one or more modifications to the sugar or base of one or
more of the ribonucleotide subunits that is included in the agent.
The iRNA agent can be further modified so as to be attached to a
ligand that is selected to improve stability, distribution or
cellular uptake of the agent, e.g. cholesterol. The iRNA agents can
further be in isolated form or can be part of a pharmaceutical
composition used for the methods described herein, particularly as
a pharmaceutical composition formulated for delivery to the lungs
or nasal passage or formulated for parental administration. The
pharmaceutical compositions can contain one or more iRNA agents,
and in some embodiments, will contain two or more iRNA agents, each
one directed to a different respiratory virus, such as RSV and
PIV.
[0016] The present invention further provides methods for reducing
the level of RSV, PIV or other respiratory viral mRNA in a cell.
The present methods utilize the cellular mechanisms involved in RNA
interference to selectively degrade the viral mRNA in a cell and
are comprised of the step of contacting a cell with one of the
antiviral iRNA agents of the present invention. Such methods can be
preformed directly on a cell or can be performed on a mammalian
subject by administering to a subject one of the iRNA
agents/pharmaceutical compositions of the present invention.
Reduction of viral mRNA in a cells results in a reduction in the
amount of viral protein produced, and in an organism, results in a
decrease in replicating viral titer. The Examples demonstrate this
with PIV and RSV and this can be extended to other respiratory
viruses.
[0017] The present invention further provides methods for reducing
the level of two or more respiratory viral mRNA in a cell, each one
coming from a different virus. The present methods utilize the
cellular mechanisms involved in RNA interference to selectively
degrade the viral mRNA from two different viruses in a cell and are
comprised of the step of contacting a cell with two of the
antiviral iRNA agents of the present invention. Such methods can be
preformed directly on a cell or can be performed on a mammalian
subject by administering to a subject two of the iRNA agents of the
present invention. Reduction of viral mRNA from two different
viruses in a cells results in a reduction in the amount of both
viral protein produced, and in an organism, results in a decrease
in replicating viral titer of both viruses. The Examples
demonstrate this with PIV and RSV and concurrent administration of
iRNA agents. This embodiment of the present invention can be
applied to any two respiratory viruses.
[0018] The methods and compositions of the invention, e.g., the
methods and iRNA compositions can be used with any dosage and/or
formulation described herein, as well as with any route of
administration described herein. Particularly important is the
showing herein of intranasal administration of an iRNA agent and
its ability to inhibit viral replication in respiratory tissues.
This finding can be applied to other respiratory virus, such as PIV
as shown in the Examples and to other routes of local delivery to
the lungs, e.g. via inhalation/nebulization.
[0019] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from this description, the drawings, and from the claims.
This application incorporates all cited references, patents, and
patent applications by references in their entirety for all
purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1. Titration of anti-viral siRNAs ex vivo. (a)
Immunoblot analysis of total proteins of RSV-infected A549 cells
(ex vivo) with profilin as the internal control. The numbers in the
box represent levels of target P mRNA following siRNA treatment,
expressed as percentage of untreated levels. In the following three
panels, virus was administered 4 hrs after siRNA. (b) Pulmonary
infectious virus in RSV-infected mice (n=8 for each data point).
(c) Pulmonary infectious virus in HPIV3-infected mice (n=8 for each
data point). (d) As in (b) except that naked siRNA was administered
without any transfection reagent. Asterisks indicate significant
inhibition (P<0.05). siRNAs are described in Table 1.
[0021] FIG. 2. Knockdown of viral antigens in siRNA-treated murine
lung without IFN activation. (a) Virus was administered 4 hrs after
siRNA, and viral antigens were detected by indirect immunohistology
of lungs at 4 days p.i. (green, RSV; red, HPIV3). (1)
sham-infected, probed with RSV P antibody; (2-6) RSV-infected,
probed with RSV P antibody; (7,8) HPIV3-infected, probed with HPIV3
antibody. The following siRNAs (5 nmoles, .about.70 ug)) were used:
(1,2) none; (3) siRNA#1 plus TransIT-TKO reagent; (4) siRNA#1, no
reagent; (5) negative control siRNA plus TransIT-TKO; (6) luc-siRNA
plus TransIT-TKO; (7) none; (8) siRNA#4 plus TransIT-TKO.
Representative lung tissues were at 5 days p.i. Bar=400 um. (b)
Antisense strand of siRNA#1 was detected by Northern analysis of
varying amounts of total lung RNA at 2 days after siRNA
administration using labeled RSV P DNA as probe. A probe against
RSV NS1 did not react, showing specificity of detection. (c) IN
siRNA (10 nmole or 140 ug per mouse) did not activate pulmonary IFN
of either type I (IFN-.alpha.) or type II (IFN-.gamma.) above the
threshold of detection (.about.10 pg/ml), whereas in control lungs,
RSV-infection activated type II and low levels of type I. Lanes: 1,
siRNA#1; 2, siRNA#4; 3, Luc siRNA; 4, no siRNA but RSV-infected
(with error bar shown). Lungs were obtained 2 days after siRNA
administration and 4 days after infection (n=4 for each graph). (d)
siRNA-mediated inhibition of dual infection by RSV and HPIV3
determined by indirect immunohistology (green, RSV; red, HPIV3).
(1, 5) no siRNA; (2, 6) siRNA#1, 5 nanomole (70 ug); (3, 7)
siRNA#4, 5 nanomole (70 ug); (4, 8) siRNA#1 and siRNA#4, 5 nanomole
each. (1-4) probed with RSV P antibody; (5-8) probed with HPIV3
antibody. Virus was administered 4 hrs after siRNA, and lung
tissues were examined at 4 days p.i. Bar=400 um.
[0022] FIG. 3. Competitive viral inhibition at high siRNA
concentration in dual infection by RSV and HPIV3. (a) Real-time PCR
(ex vivo); (b) immunoblot (ex vivo); (c) pulmonary immunoblot with
goat antiviral antibodies. The respective viral N protein band
intensity was quantified and expressed as percentage of
siRNA-untreated lung samples. Virus was administered 4 hrs after
siRNA, and lung tissues were at 5 days p.i. (n=4 for each data
point). Black bar, RSV; white bar, HPIV3. Standard errors are as
shown.
[0023] FIG. 4. Relief of lung pathology and reduction of an asthma
marker in siRNA#1-treated mice. (a) Respiratory rate; (b) Pulmonary
histopathology; (c) Leukotriene. P<0.002 in all assays; n=4 for
all data points; standard error bars are shown. Virus was
administered 4 hrs after siRNA (70 ug). Mice treated with negative
control siRNA were indistinguishable from siRNA-untreated (data not
shown).
[0024] FIG. 5. Therapeutic effect of siRNA in RSV disease. Changes
in (a) body weight and (b) pulmonary viral titer during RSV
infection in mice. Standard error bars are shown; n=6 for each data
point. The arrows indicate the day of siRNA (70 ug)
administration.
DETAILED DESCRIPTION
[0025] For ease of exposition the term "nucleotide" or
"ribonucleotide" is sometimes used herein in reference to one or
more monomeric subunits of an RNA agent. It will be understood that
the usage of the term "ribonucleotide" or "nucleotide" herein can,
in the case of a modified RNA or nucleotide surrogate, also refer
to a modified nucleotide, or surrogate replacement moiety, as
further described below, at one or more positions.
[0026] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogates, all of which are described
herein or are well known in the RNA synthetic art. While numerous
modified RNAs and nucleoside surrogates are described, preferred
examples include those which have greater resistance to nuclease
degradation than do unmodified RNAs. Preferred examples include
those that have a 2' sugar modification, a modification in a single
strand overhang, preferably a 3' single strand overhang, or,
particularly if single stranded, a 5'-modification which includes
one or more phosphate groups or one or more analogs of a phosphate
group.
[0027] An "iRNA agent" sometimes referred to as an "RNAi agent"
(abbreviation for "interfering RNA agent") as used herein, is an
RNA agent, which can down-regulate the expression of a target gene,
e.g., RSV. While not wishing to be bound by theory, an iRNA agent
may act by one or more of a number of mechanisms, including
post-transcriptional cleavage of a target mRNA sometimes referred
to in the art as RNAi, or pre-transcriptional or pre-translational
mechanisms. An iRNA agent can include a single strand or can
include more than one strands, e.g., it can be a double stranded
(ds) iRNA agent. If the iRNA agent is a single strand it is
particularly preferred that it include a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group.
[0028] A "single strand iRNA agent" as used herein, is an iRNA
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or panhandle structure. Single strand iRNA
agents are preferably antisense with regard to the target
molecule.
[0029] A "ds iRNA agent" (abbreviation for "double stranded iRNA
agent"), as used herein, is an iRNA agent which includes more than
one, and preferably two, strands in which interchain hybridization
can form a region of duplex structure.
[0030] The isolated iRNA agents described herein, including ds iRNA
agents and siRNA agents, can mediate silencing of a target gene,
e.g., by RNA degradation. For convenience, such RNA is also
referred to herein as the RNA to be silenced. Such a gene is also
referred to as a target gene. Preferably, the RNA to be silenced is
a gene product of an endogenous RSV gene.
[0031] As used herein, the phrase "mediates RNAi" refers to the
ability of an agent to silence, in a sequence specific manner, a
target gene. "Silencing a target gene" means the process whereby a
cell containing and/or secreting a certain product of the target
gene when not in contact with the agent, will contain and/or secret
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of
such gene product when contacted with the agent, as compared to a
similar cell which has not been contacted with the agent. Such
product of the target gene can, for example, be a messenger RNA
(mRNA), a protein, or a regulatory element.
[0032] In the anti viral uses of the present invention, silencing
of a target gene will result in a reduction in "viral titer" in the
cell. As used herein, "reduction in viral titer" refers to a
decrease in the number of viable virus produced by a cell or found
in an organism undergoing the silencing of a viral target gene.
Reduction in the cellular amount of virus produced will preferably
lead to a decrease in the amount of measurable virus produced in
the tissues of a subject undergoing treatment and a reduction in
the severity of the symptoms of the viral infection. iRNA agents of
the present invention are also referred to as "antiviral iRNA
agents".
[0033] As used herein, a "RSV gene" refers to any one of the genes
identified in the RSV virus genome (See Falsey, A. R., and E. E.
Walsh, 2000, Clinical Microbiological Reviews 13:371-84). These
genes are readily known in the art and include the F, G, SH, M, N,
P and L genes.
[0034] As used herein, a "PIV gene" refers to any one of the genes
identified in the PIV virus genome (See GenBank Accession #
NC.sub.--001796). These genes are readily known in the art and
include the N, P, C, D, V, M, F, HN, and L genes.
[0035] As used herein, "respiratory virus" refers to viruses that
replicate in cells of the respiratory system. Such viruses include,
but are not limited to RSV, PIV, influenza, metapneumovirus,
adenovirus, and coronavirus (such as SARS).
[0036] As used herein, the term "complementary" is used to indicate
a sufficient degree of complementarity such that stable and
specific binding occurs between a compound of the invention and a
target RNA molecule, e.g. an RSV, PIV or other respiratory viral
mRNA molecule. Specific binding requires a sufficient degree of
complementarity to avoid non-specific binding of the oligomeric
compound to non-target sequences under conditions in which specific
binding is desired, i.e., under physiological conditions in the
case of in vivo assays or therapeutic treatment, or in the case of
in vitro assays, under conditions in which the assays are
performed. The non-target sequences typically differ by at least 4
nucleotides.
[0037] As used herein, an iRNA agent is "sufficiently
complementary" to a target RNA, e.g., a target mRNA (e.g., a target
RSV or PIV mRNA) if the iRNA agent reduces the production of a
protein encoded by the target RNA in a cell. The iRNA agent may
also be "exactly complementary" (excluding the SRMS containing
subunit(s)) to the target RNA, e.g., the target RNA and the iRNA
agent anneal, preferably to form a hybrid made exclusively of
Watson-Crick base pairs in the region of exact complementarity. A
"sufficiently complementary" iRNA agent can include an internal
region (e.g., of at least 10 nucleotides) that is exactly
complementary to a target viral RNA. Moreover, in some embodiments,
the iRNA agent specifically discriminates a single-nucleotide
difference. In this case, the iRNA agent only mediates RNAi if
exact complementary is found in the region (e.g., within 7
nucleotides of) the single-nucleotide difference. Preferred iRNA
agents will be based on or consist or comprise the sense and
antisense sequences provided in the Examples.
[0038] As used herein, "essentially identical" when used referring
to a first nucleotide sequence in comparison to a second nucleotide
sequence means that the first nucleotide sequence is identical to
the second nucleotide sequence except for up to one, two or three
nucleotide substitutions (e.g. adenosine replaced by uracil).
[0039] As used herein, a "subject" refers to a mammalian organism
undergoing treatment for a disorder mediated by viral expression,
such as RSV or PIV infection or undergoing treatment
prophylactically to prevent viral infection. The subject can be any
mammal, such as a primate, cow, horse, mouse, rat, dog, pig, goat.
In the preferred embodiment, the subject is a human.
[0040] As used herein, treating RSV infection, PIV infection, or
other respiratory virus infection, refers to the amelioration of
any biological or pathological endpoints that 1) is mediated in
part by the presence of the virus in the subject and 2) whose
outcome can be affected by reducing the level of viral protein
present.
[0041] As used herein, "co-administration" refers to administering
to a subject two or more agents, and in particular two or more iRNA
agents. The agents can be contained in a single pharmaceutical
composition and be administered at the same time, or the agents can
be contained in separate formulation and administered serially to a
subject. So long as the two agents can be detected in the subject
at the same time, the two agents are said to be
co-administered.
[0042] Because iRNA agent mediated silencing can persist for
several days after administering the iRNA agent composition, in
many instances, it is possible to administer the composition with a
frequency of less than once per day, or, for some instances, only
once for the entire therapeutic regimen.
[0043] Design and Selection of iRNA Agents
[0044] The present invention is based on the demonstration of
target gene silencing of a respiratory viral gene in vivo following
local administration to the lungs and nasal passage of an iRNA
agent either via intranasal administration/inhalation or
systemically/parenterally via injection and the resulting treatment
of viral infection. The present invention is further extended to
the use of iRNA agents to more than one respiratory virus and the
treatment of both virus infections with co-administration of two or
more iRNA agents.
[0045] Based on these results, the invention specifically provides
an iRNA agent that can be used in treating viral infection,
particularly respiratory viruses and in particular RSV or PIV
infection, in isolated form and as a pharmaceutical composition
described below. Such agents will include a sense strand having at
least 15 contiguous nucleotides that are complementary to a viral
gene and an antisense strand having at least 15 contiguous
nucleotides that is complementary to the sense strand sequences.
Particularly useful are iRNA agents that comprise a nucleotide
sequence from the P protein gene of RSV or PIV. Other target genes
in RSV include the F, G, SH, M, N and L. Other genes in PIV include
N, P, C, D, V, M, F, HN, and L genes. Exemplified agents are
provided in Table 1.
[0046] Candidate iRNA agents can be designed by performing, for
example, a gene walk analysis of the viral genes that will serve as
the iRNA target. Overlapping, adjacent, or closely spaced candidate
agents corresponding to all or some of the transcribed region can
be generated and tested. Each of the iRNA agents can be tested and
evaluated for the ability to down regulate the target gene
expression (see below, "Evaluation of Candidate iRNA agents").
[0047] An iRNA agent can be rationally designed based on sequence
information and desired characteristics. For example, an iRNA agent
can be designed according to the relative melting temperature of
the candidate duplex. Generally, the duplex should have a lower
melting temperature at the 5' end of the antisense strand than at
the 3' end of the antisense strand.
[0048] Accordingly, the present invention provides iRNA agents
comprising a sense strand and antisense strand each comprising a
sequence of at least 15, 16, 17, 18, 19, 20, 21 or 23 nucleotides
which is essentially identical to, as defined above, to a portion
of a gene from a respiratory virus, particularly the P protein
genes of RSV or PIV. Exemplified iRNA agents include those that
comprise 15 contiguous nucleotides from one of the agents provided
in Table 1.
[0049] The antisense strand of an iRNA agent should be equal to or
at least, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in
length. It should be equal to or less than 50, 40, or 30,
nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to
23, and 19 to 21 nucleotides in length. In several embodiments, the
agent will comprise 15 nucleotides from one of the agents in Table
1.
[0050] The sense strand of an iRNA agent should be equal to or at
least 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length.
It should be equal to or less than 50, 40, or 30 nucleotides in
length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to
21 nucleotides in length. In several embodiments, the agent will
comprise 15 nucleotides from one of the agents in Table 1.
[0051] The double stranded portion of an iRNA agent should be equal
to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,
or 50 nucleotide pairs in length. It should be equal to or less
than 50, 40, or 30 nucleotides pairs in length. Preferred ranges
are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in
length.
[0052] The agents provided in Table 1 are 21 nucleotide in length
for each strand. The iRNA agents contain a 19 nucleotide double
stranded region with a 2 nucleotide overhang on each of the 3' ends
of the agent. These agents can be modified as described herein to
obtain equivalent agents comprising at least a portion of these
sequences and or modifications to the oligonucleotide bases and
linkages.
[0053] Generally, the iRNA agents of the instant invention include
a region of sufficient complementarity to the viral gene, e.g. the
P protein of RSV or PIV, and are of sufficient length in terms of
nucleotides, that the iRNA agent, or a fragment thereof, can
mediate down regulation of the specific viral gene. The antisense
strands of the iRNA agents of the present invention are preferably
fully complementary to the mRNA sequences of viral gene, as is
herein for the P proteins of RSV and PIV. However, it is not
necessary that there be perfect complementarity between the iRNA
agent and the target, but the correspondence must be sufficient to
enable the iRNA agent, or a cleavage product thereof, to direct
sequence specific silencing, e.g., by RNAi cleavage of an RSV
mRNA.
[0054] Therefore, the iRNA agents of the instant invention include
agents comprising a sense strand and antisense strand each
comprising a sequence of at least 16, 17 or 18 nucleotides which is
essentially identical, as defined below, to one of the sequences of
a viral gene, particularly the P protein of RSV or PIV, except that
not more than 1, 2 or 3 nucleotides per strand, respectively, have
been substituted by other nucleotides (e.g. adenosine replaced by
uracil), while essentially retaining the ability to inhibit RSV
expression in cultured human cells, as defined below. These agents
will therefore possess at least 15 nucleotides identical to one of
the sequences of a viral gene, particularly the P protein gene of
RSV or PIV, but 1, 2 or 3 base mismatches with respect to either
the target viral mRNA sequence or between the sense and antisense
strand are introduced. Mismatches to the target viral mRNA
sequence, particularly in the antisense strand, are most tolerated
in the terminal regions and if present are preferably in a terminal
region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5'
and/or 3' terminus, most preferably within 6, 5, 4, or 3
nucleotides of the 5'-terminus of the sense strand or the
3'-terminus of the antisense strand. The sense strand need only be
sufficiently complementary with the antisense strand to maintain
the overall double stranded character of the molecule.
[0055] It is preferred that the sense and antisense strands be
chosen such that the iRNA agent includes a single strand or
unpaired region at one or both ends of the molecule, such as those
exemplified in Table 1. Thus, an iRNA agent contains sense and
antisense strands, preferably paired to contain an overhang, e.g.,
one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3
nucleotides. Most embodiments will have a 3' overhang. Preferred
siRNA agents will have single-stranded overhangs, preferably 3'
overhangs, of 1 to 4, or preferably 2 or 3 nucleotides, in length
one or both ends of the iRNA agent. The overhangs can be the result
of one strand being longer than the other, or the result of two
strands of the same length being staggered. 5'-are preferably
phosphorylated.
[0056] Preferred lengths for the duplexed region is between 15 and
30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the siRNA agent range discussed above. Embodiments
in which the two strands of the siRNA agent are linked, e.g.,
covalently linked are also included. Hairpin, or other single
strand structures which provide the required double stranded
region, and preferably a 3' overhang are also within the
invention.
[0057] Evaluation of Candidate iRNA Agents
[0058] A candidate iRNA agent can be evaluated for its ability to
down regulate target gene expression. For example, a candidate iRNA
agent can be provided, and contacted with a cell, e.g. a human
cell, that has been infected with or will be infected with the
virus of interest, e.g., a virus containing the target gene,.
Alternatively, the cell can transfected with a construct from which
target viral gene is expressed, thus preventing the need for a
viral infectivity model. The level of target gene expression prior
to and following contact with the candidate iRNA agent can be
compared, e.g. on an mRNA, protein level or viral titer. If it is
determined that the amount of RNA, protein or virus expressed from
the target gene is lower following contact with the iRNA agent,
then it can be concluded that the iRNA agent down regulates target
gene expression. The level of target viral RNA or viral protein in
the cell or viral titer in a cell or tissue can be determined by
any method desired. For example, the level of target RNA can be
determined by Northern blot analysis, reverse transcription coupled
with polymerase chain reaction (RT-PCR), bDNA analysis, or RNAse
protection assay. The level of protein can be determined, for
example, by Western blot analysis or immuno-flouresence. Viral
titer can be detected through a plaque formation assay.
[0059] Stability Testing, Modification, and Retesting of iRNA
Agents
[0060] A candidate iRNA agent can be evaluated with respect to
stability, e.g., its susceptibility to cleavage by an endonuclease
or exonuclease, such as when the iRNA agent is introduced into the
body of a subject. Methods can be employed to identify sites that
are susceptible to modification, particularly cleavage, e.g.,
cleavage by a component found in the body of a subject.
[0061] When sites susceptible to cleavage are identified, a further
iRNA agent can be designed and/or synthesized wherein the potential
cleavage site is made resistant to cleavage, e.g. by introduction
of a 2'-modification on the site of cleavage, e.g. a 2'-O-mathyl
group. This further iRNA agent can be retested for stability, and
this process may be iterated until an iRNA agent is found
exhibiting the desired stability.
[0062] In Vivo Testing
[0063] An iRNA agent identified as being capable of inhibiting RSV
gene expression can be tested for functionality in vivo in an
animal model (e.g., in a mammal, such as in mouse or rat) as shown
in the examples. For example, the iRNA agent can be administered to
an animal, and the iRNA agent evaluated with respect to its
biodistribution, stability, and its ability to inhibit viral, e.g.
RSV or PIV, gene expression or reduce viral titer.
[0064] The iRNA agent can be administered directly to the target
tissue, such as by injection, or the iRNA agent can be administered
to the animal model in the same manner that it would be
administered to a human. As shown herein, the agent can be
preferably administered via inhalation as a means of treating viral
infection.
[0065] The iRNA agent can also be evaluated for its intracellular
distribution. The evaluation can include determining whether the
iRNA agent was taken up into the cell. The evaluation can also
include determining the stability (e.g., the half-life) of the iRNA
agent. Evaluation of an iRNA agent in vivo can be facilitated by
use of an iRNA agent conjugated to a traceable marker (e.g., a
fluorescent marker such as fluorescein; a radioactive label, such
as .sup.35S, .sup.32P, .sup.33P, or .sup.3H; gold particles; or
antigen particles for immunohistochemistry).
[0066] An iRNA agent useful for monitoring biodistribution can lack
gene silencing activity in vivo. For example, the iRNA agent can
target a gene not present in the animal (e.g., an iRNA agent
injected into mouse can target luciferase), or an iRNA agent can
have a non-sense sequence, which does not target any gene, e.g.,
any endogenous gene). Localization/biodistribution of the iRNA can
be monitored, e.g. by a traceable label attached to the iRNA agent,
such as a traceable agent described above
[0067] The iRNA agent can be evaluated with respect to its ability
to down regulate viral gene expression. Levels of viral gene
expression in vivo can be measured, for example, by in situ
hybridization, or by the isolation of RNA from tissue prior to and
following exposure to the iRNA agent. Where the animal needs to be
sacrificed in order to harvest the tissue, an untreated control
animal will serve for comparison. Target viral mRNA can be detected
by any desired method, including but not limited to RT-PCR,
Northern blot, branched-DNA assay, or RNAase protection assay.
Alternatively, or additionally, viral gene expression can be
monitored by performing Western blot analysis on tissue extracts
treated with the iRNA agent. Viral titer can be determined using a
pfu assy.
[0068] iRNA Chemistry
[0069] Described herein are isolated iRNA agents, e.g., RNA
molecules, (double-stranded; single-stranded) that mediate RNAi to
inhibit expression of a viral gene, e.g. the P protein of RSV or
PIV.
[0070] RNA agents discussed herein include otherwise unmodified RNA
as well as RNA which have been modified, e.g., to improve efficacy,
and polymers of nucleoside surrogates. Unmodified RNA refers to a
molecule in which the components of the nucleic acid, namely
sugars, bases, and phosphate moieties, are the same or essentially
the same as that which occur in nature, preferably as occur
naturally in the human body. The art has referred to rare or
unusual, but naturally occurring, RNAs as modified RNAs, see, e.g.,
Limbach et al., (1994) Nucleic Acids Res. 22: 2183-2196. Such rare
or unusual RNAs, often termed modified RNAs (apparently because
these are typically the result of a post-transcriptional
modification) are within the term unmodified RNA, as used herein.
Modified RNA as used herein refers to a molecule in which one or
more of the components of the nucleic acid, namely sugars, bases,
and phosphate moieties, are different from that which occurs in
nature, preferably different from that which occurs in the human
body. While they are referred to as modified "RNAs," they will of
course, because of the modification, include molecules which are
not RNAs. Nucleoside surrogates are molecules in which the
ribophosphate backbone is replaced with a non-ribophosphate
construct that allows the bases to the presented in the correct
spatial relationship such that hybridization is substantially
similar to what is seen with a ribophosphate backbone, e.g.,
non-charged mimics of the ribophosphate backbone. Examples of each
of the above are discussed herein.
[0071] Modifications described herein can be incorporated into any
double-stranded RNA and RNA-like molecule described herein, e.g.,
an iRNA agent. It may be desirable to modify one or both of the
antisense and sense strands of an iRNA agent. As nucleic acids are
polymers of subunits or monomers, many of the modifications
described below occur at a position which is repeated within a
nucleic acid, e.g., a modification of a base, or a phosphate
moiety, or the non-linking O of a phosphate moiety. In some cases
the modification will occur at all of the subject positions in the
nucleic acid but in many, and in fact in most, cases it will not.
By way of example, a modification may only occur at a 3' or 5'
terminal position, may only occur in a terminal region, e.g. at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand. A modification may occur in a double
strand region, a single strand region, or in both. E.g., a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, may only occur in a terminal regions,
e.g., at a position on a terminal nucleotide or in the last 2, 3,
4, 5, or 10 nucleotides of a strand, or may occur in double strand
and single strand regions, particularly at termini. Similarly, a
modification may occur on the sense strand, antisense strand, or
both. In some cases, the sense and antisense strand will have the
same modifications or the same class of modifications, but in other
cases the sense and antisense strand will have different
modifications, e.g., in some cases it may be desirable to modify
only one strand, e.g. the sense strand.
[0072] Two prime objectives for the introduction of modifications
into iRNA agents is their stabilization towards degradation in
biological environments and the improvement of pharmacological
properties, e.g. pharmacodynamic properties, which are further
discussed below. Other suitable modifications to a sugar, base, or
backbone of an iRNA agent are described in co-owned PCT Application
No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can
include a non-naturally occurring base, such as the bases described
in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16,
2004. An iRNA agent can include a non-naturally occurring sugar,
such as a non-carbohydrate cyclic carrier molecule. Exemplary
features of non-naturally occurring sugars for use in iRNA agents
are described in co-owned PCT Application No. PCT/US2004/11829
filed Apr. 16, 2003.
[0073] An iRNA agent can include an internucleotide linkage (e.g.,
the chiral phosphorothioate linkage) useful for increasing nuclease
resistance. In addition, or in the alternative, an iRNA agent can
include a ribose mimic for increased nuclease resistance. Exemplary
internucleotide linkages and ribose mimics for increased nuclease
resistance are described in co-owned PCT Application No.
PCT/US2004/07070 filed on Mar. 8, 2004.
[0074] An iRNA agent can include ligand-conjugated monomer subunits
and monomers for oligonucleotide synthesis. Exemplary monomers are
described in co-owned U.S. application Ser. No. 10/916,185, filed
on Aug. 10, 2004.
[0075] An iRNA agent can have a ZXY structure, such as is described
in co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0076] An iRNA agent can be complexed with an amphipathic moiety.
Exemplary amphipathic moieties for use with iRNA agents are
described in co-owned PCT Application No. PCT/US2004/07070 filed on
Mar. 8, 2004.
[0077] In another embodiment, the iRNA agent can be complexed to a
delivery agent that features a modular complex. The complex can
include a carrier agent linked to one or more of (preferably two or
more, more preferably all three of): (a) a condensing agent (e.g.,
an agent capable of attracting, e.g., binding, a nucleic acid,
e.g., through ionic or electrostatic interactions); (b) a fusogenic
agent (e.g., an agent capable of fusing and/or being transported
through a cell membrane); and (c) a targeting group, e.g., a cell
or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type.
iRNA agents complexed to a delivery agent are described in co-owned
PCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.
[0078] An iRNA agent can have non-canonical pairings, such as
between the sense and antisense sequences of the iRNA duplex.
Exemplary features of non-canonical iRNA agents are described in
co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0079] Enhanced Nuclease Resistance
[0080] An iRNA agent, e.g., an iRNA agent that targets RSV can have
enhanced resistance to nucleases. One way to increase resistance is
to identify cleavage sites and modify such sites to inhibit
cleavage. For example, the dinucleotides 5'-UA-3', 5'-UG-3',
5'-CA-3', 5'-UU-3', or 5'-CC-3' can serve as cleavage sites.
[0081] For increased nuclease resistance and/or binding affinity to
the target, an iRNA agent, e.g., the sense and/or antisense strands
of the iRNA agent, can include, for example, 2'-modified ribose
units and/or phosphorothioate linkages. E.g., the 2' hydroxyl group
(OH) can be modified or replaced with a number of different "oxy"
or "deoxy" substituents.
[0082] Examples of"oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0083] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino,or diheteroaryl amino), --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality. Preferred substitutents are 2'-methoxyethyl,
2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.
[0084] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0085] In certain embodiments, all the pyrimidines of an iRNA agent
carry a 2'-modification, and the iRNA agent therefore has enhanced
resistance to endonucleases. Enhanced nuclease resistance can also
be achieved by modifying the 5' nucleotide, resulting, for example,
in at least one 5'-uridine-adenine-3' (5'-UA-3') dinucleotide
wherein the uridine is a 2'-modified nucleotide; at least one
5'-uridine-guanine-3' (5'-UG-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide; at least one
5'-cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide; at least one
5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide; or at least one
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide. The iRNA agent can include
at least 2, at least 3, at least 4 or at least 5 of such
dinucleotides.
[0086] The inclusion of furanose sugars in the oligonucleotide
backbone can also decrease endonucleolytic cleavage. An iRNA agent
can be further modified by including a 3' cationic group, or by
inverting the nucleoside at the 3'-terminus with a 3'-3' linkage.
In another alternative, the 3'-terminus can be blocked with an
aminoalkyl group, e.g., a 3'C5-aminoalkyl dT. Other 3' conjugates
can inhibit 3'-5' exonucleolytic cleavage. While not being bound by
theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit
exonucleolytic cleavage by sterically blocking the exonuclease from
binding to the 3'-end of oligonucleotide. Even small alkyl chains,
aryl groups, or heterocyclic conjugates or modified sugars
(D-ribose, deoxyribose, glucose etc.) can block
3'-5'-exonucleases.
[0087] Similarly, 5' conjugates can inhibit 5'-3' exonucleolytic
cleavage. While not being bound by theory, a 5' conjugate, such as
naproxen or ibuprofen, may inhibit exonucleolytic cleavage by
sterically blocking the exonuclease from binding to the 5'-end of
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0088] An iRNA agent can have increased resistance to nucleases
when a duplexed iRNA agent includes a single-stranded nucleotide
overhang on at least one end. In preferred embodiments, the
nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired
nucleotides. In a preferred embodiment, the unpaired nucleotide of
the single-stranded overhang that is directly adjacent to the
terminal nucleotide pair contains a purine base, and the terminal
nucleotide pair is a G-C pair, or at least two of the last four
complementary nucleotide pairs are G-C pairs. In further
embodiments, the nucleotide overhang may have 1 or 2 unpaired
nucleotides, and in an exemplary embodiment the nucleotide overhang
is 5'-GC-3'. In preferred embodiments, the nucleotide overhang is
on the 3'-end of the antisense strand. In one embodiment, the iRNA
agent includes the motif 5'-CGC-3' on the 3'-end of the antisense
strand, such that a 2-nt overhang 5'-GC-3' is formed.
[0089] Thus, an iRNA agent can include monomers which have been
modified so as to inhibit degradation, e.g., by nucleases, e.g.,
endonucleases or exonucleases, found in the body of a subject.
These monomers are referred to herein as NRMs, or Nuclease
Resistance promoting Monomers or modifications. In many cases these
modifications will modulate other properties of the iRNA agent as
well, e.g., the ability to interact with a protein, e.g., a
transport protein, e.g., serum albumin, or a member of the RISC, or
the ability of the first and second sequences to form a duplex with
one another or to form a duplex with another sequence, e.g., a
target molecule.
[0090] Modifications that can be useful for producing iRNA agents
that meet the preferred nuclease resistance criteria delineated
above can include one or more of the following chemical and/or
stereochemical modifications of the sugar, base, and/or phosphate
backbone:
[0091] (i) chiral (S.sub.P) thioates. Thus, preferred NRMs include
nucleotide dimers with an enriched or pure for a particular chiral
form of a modified phosphate group containing a heteroatom at the
nonbridging position, e.g., Sp or Rp, at the position X, where this
is the position normally occupied by the oxygen. The atom at X can
also be S, Se, Nr.sub.2, or Br.sub.3. When X is S, enriched or
chirally pure Sp linkage is preferred. Enriched means at least 70,
80, 90, 95, or 99% of the preferred form. Such NRMs are discussed
in more detail below;
[0092] (ii) attachment of one or more cationic groups to the sugar,
base, and/or the phosphorus atom of a phosphate or modified
phosphate backbone moiety. Thus, preferred NRMs include monomers at
the terminal position derivatized at a cationic group. As the
5'-end of an antisense sequence should have a terminal --OH or
phosphate group this NRM is preferably not used at the 5'-end of an
anti-sense sequence. The group should be attached at a position on
the base which minimizes interference with H bond formation and
hybridization, e.g., away form the face which interacts with the
complementary base on the other strand, e.g, at the 5' position of
a pyrimidine or a 7-position of a purine. These are discussed in
more detail below;
[0093] (iii) nonphosphate linkages at the termini. Thus, preferred
NRMs include Non-phosphate linkages, e.g., a linkage of 4 atoms
which confers greater resistance to cleavage than does a phosphate
bond. Examples include 3' CH2-NCH.sub.3--O--CH.sub.2-5' and 3'
CH.sub.2--NH--(O.dbd.)--CH.sub.2-5'.;
[0094] (iv) 3'-bridging thiophosphates and 5'-bridging
thiophosphates. Thus, preferred NRM's can included these
structures;
[0095] (v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides.
Thus, other preferred NRM's include: L nucleosides and dimeric
nucleotides derived from L-nucleosides; 2'-5' phosphate,
non-phosphate and modified phosphate linkages (e.g.,
thiophosphates, phosphoramidates and boronophosphates); dimers
having inverted linkages, e.g., 3'-3' or 5'-5' linkages; monomers
having an alpha linkage at the 1' site on the sugar, e.g., the
structures described herein having an alpha linkage;
[0096] (vi) conjugate groups. Thus, preferred NRM's can include,
e.g., a targeting moiety or a conjugated ligand described herein
conjugated with the monomer, e.g., through the sugar, base, or
backbone;
[0097] (vi) a basic linkages. Thus, preferred NRM's can include an
abasic monomer, e.g., an abasic monomer as described herein (e.g.,
a nucleobaseless monomer); an aromatic or heterocyclic or
polyheterocyclic aromatic monomer as described herein.; and
[0098] (vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus,
preferred NRM's include monomers, preferably at the terminal
position, e.g., the 5' position, in which one or more atoms of the
phosphate group is derivatized with a protecting group, which
protecting group or groups, are removed as a result of the action
of a component in the subject's body, e.g, a carboxyesterase or an
enzyme present in the subject's body. E.g., a phosphate prodrug in
which a carboxy esterase cleaves the protected molecule resulting
in the production of a thioate anion which attacks a carbon
adjacent to the O of a phosphate and resulting in the production of
an unprotected phosphate.
[0099] One or more different NRM modifications can be introduced
into an iRNA agent or into a sequence of an iRNA agent. An NRM
modification can be used more than once in a sequence or in an iRNA
agent. As some NRMs interfere with hybridization, the total number
incorporated should be such that acceptable levels of iRNA agent
duplex formation are maintained.
[0100] In some embodiments NRM modifications are introduced into
the terminal cleavage site or in the cleavage region of a sequence
(a sense strand or sequence) which does not target a desired
sequence or gene in the subject. This can reduce off-target
silencing.
[0101] Nuclease resistant modifications include some which can be
placed only at the terminus and others which can go at any
position. Generally, modifications that can inhibit hybridization
are used only in terminal regions, and preferably not at the
cleavage site or in the cleavage region of a sequence which targets
a subject sequence or gene. They can be used anywhere in a sense
sequence, provided that sufficient hybridization between the two
sequences of the iRNA agent is maintained. In some embodiments it
is desirable to put the NRM at the cleavage site or in the cleavage
region of a sequence which does not target a subject sequence or
gene, as it can minimize off-target silencing.
[0102] In most cases, the nuclease-resistance promoting
modifications will be distributed differently depending on whether
the sequence will target a sequence in the subject (often referred
to as an anti-sense sequence) or will not target a sequence in the
subject (often referred to as a sense sequence). If a sequence is
to target a sequence in the subject, modifications which interfere
with or inhibit endonuclease cleavage should not be inserted in the
region which is subject to RISC mediated cleavage, e.g., the
cleavage site or the cleavage region (As described in Elbashir et
al., 2001, Genes and Dev. 15: 188, hereby incorporated by
reference). Cleavage of the target occurs about in the middle of a
20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of
the first nucleotide which is complementary to the guide sequence.
As used herein cleavage site refers to the nucleotide on either
side of the cleavage site, on the target or on the iRNA agent
strand which hybridizes to it. Cleavage region means a nucleotide
with 1, 2, or 3 nucleotides of the cleave site, in either
direction.)
[0103] Such modifications can be introduced into the terminal
regions, e.g., at the terminal position or with 2, 3, 4, or 5
positions of the terminus, of a sequence which targets or a
sequence which does not target a sequence in the subject.
[0104] Tethered Ligands
[0105] The properties of an iRNA agent, including its
pharmacological properties, can be influenced and tailored, for
example, by the introduction of ligands, e.g. tethered ligands.
[0106] A wide variety of entities, e.g., ligands, can be tethered
to an iRNA agent, e.g., to the carrier of a ligand-conjugated
monomer subunit. Examples are described below in the context of a
ligand-conjugated monomer subunit but that is only preferred,
entities can be coupled at other points to an iRNA agent.
[0107] Preferred moieties are ligands, which are coupled,
preferably covalently, either directly or indirectly via an
intervening tether, to the carrier. In preferred embodiments, the
ligand is attached to the carrier via an intervening tether. The
ligand or tethered ligand may be present on the ligand-conjugated
monomer\when the ligand-conjugated monomer is incorporated into the
growing strand. In some embodiments, the ligand may be incorporated
into a "precursor" ligand-conjugated monomer subunit after a
"precursor" ligand-conjugated monomer subunit has been incorporated
into the growing strand. For example, a monomer having, e.g., an
amino-terminated tether, e.g., TAP--(CH.sub.2).sub.nNH.sub.2 may be
incorporated into a growing sense or antisense strand. In a
subsequent operation, i.e., after incorporation of the precursor
monomer subunit into the strand, a ligand having an electrophilic
group, e.g., a pentafluorophenyl ester or aldehyde group, can
subsequently be attached to the precursor ligand-conjugated monomer
by coupling the electrophilic group of the ligand with the terminal
nucleophilic group of the precursor ligand-conjugated monomer
subunit tether.
[0108] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand.
[0109] Preferred ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0110] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents;
nuclease-resistance conferring moieties; and natural or unusual
nucleobases. General examples include lipophiles, lipids, steroids
(e.g.,uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes,
e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized
lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin,
pyridoxal), carbohydrates, proteins, protein binding agents,
integrin targeting molecules,polycationics, peptides, polyamines,
and peptide mimics.
[0111] Ligands can include a naturally occurring substance, (e.g.,
human serum albumin (HSA), low-density lipoprotein (LDL), or
globulin); carbohydrate (e.g., a dextran, pullulan, chitin,
chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or
a lipid. The ligand may also be a recombinant or synthetic
molecule, such as a synthetic polymer, e.g., a synthetic polyamino
acid. Examples of polyamino acids include polyamino acid is a
polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,
styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic
moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt
of a polyamine, or an alpha helical peptide.
[0112] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type
such as a liver cell or a cell of the jejunum. A targeting group
can be a thyrotropin, melanotropin, lectin, glycoprotein,
surfactant protein A, Mucin carbohydrate, multivalent lactose,
multivalent galactose, N-acetyl-galactosamine,
N-acetyl-gulucosamine multivalent mannose, multivalent fucose,
glycosylated polyaminoacids, multivalent galactose, transferrin,
bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol,
a steroid, bile acid, folate, vitamin B12, biotin, or an RGD
peptide or RGD peptide mimetic.
[0113] Ligands can be proteins, e.g., glycoproteins, or peptides,
e.g., molecules having a specific affinity for a co-ligand, or
antibodies e.g., an antibody, that binds to a specified cell type
such as a cancer cell, endothelial cell, or bone cell. Ligands may
also include hormones and hormone receptors. They can also include
non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
or multivalent fucose. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-.kappa.B.
[0114] The ligand can be a substance, e.g, a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0115] In one aspect, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., liver tissue, including parenchymal cells of the liver. Other
molecules that can bind HSA can also be used as ligands. For
example, neproxin or aspirin can be used. A lipid or lipid-based
ligand can (a) increase resistance to degradation of the conjugate,
(b) increase targeting or transport into a target cell or cell
membrane, and/or (c) can be used to adjust binding to a serum
protein, e.g., HSA.
[0116] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0117] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0118] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a target cell, e.g., a proliferating cell.
These are particularly useful for treating disorders characterized
by unwanted cell proliferation, e.g., of the malignant or
non-malignant type, e.g., cancer cells. Exemplary vitamins include
vitamin A, E, and K. Other exemplary vitamins include are B
vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or
other vitamins or nutrients taken up by cancer cells. Also included
are HSA and low density lipoprotein (LDL).
[0119] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0120] 5'-Phosphate Modifications
[0121] In preferred embodiments, iRNA agents are 5' phosphorylated
or include a phosphoryl analog at the 5' prime terminus.
5'-phosphate modifications of the antisense strand include those
which are compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO)2(O)P--O-5');
5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)--O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.
RP(OH)(O)--O-5'-).
[0122] The sense strand can be modified in order to inactivate the
sense strand and prevent formation of an active RISC, thereby
potentially reducing off-target effects. This can be accomplished
by a modification which prevents 5'-phosphorylation of the sense
strand, e.g., by modification with a 5'-O-methyl ribonucleotide
(see Nykanen et al., (2001) ATP requirements and small interfering
RNA structure in the RNA interference pathway. Cell 107, 309-321.)
Other modifications which prevent phosphorylation can also be used,
e.g., simply substituting the 5'-OH by H rather than O-Me.
Alternatively, a large bulky group may be added to the 5'-phosphate
turning it into a phosphodiester linkage.
[0123] Delivery of iRNA Agents to Tissues and Cells
[0124] Formulation
[0125] The iRNA agents described herein can be formulated for
administration to a subject, preferably for administration locally
to the lungs and nasal passage (respiratory tissues) via inhalation
or intranasally administration, or parenterally, e.g. via
injection.
[0126] For ease of exposition, the formulations, compositions, and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It should be understood, however, that
these formulations, compositions, and methods can be practiced with
other iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention.
[0127] A formulated iRNA composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In another example, the iRNA is
in an aqueous phase, e.g., in a solution that includes water, this
form being the preferred form for administration via
inhalation.
[0128] The aqueous phase or the crystalline compositions can be
incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase), or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the iRNA composition is formulated in a manner that is
compatible with the intended method of administration.
[0129] An iRNA preparation can be formulated in combination with
another agent, e.g., another therapeutic agent or an agent that
stabilizes an iRNA, e.g., a protein that complexes with iRNA to
form an iRNP. Still other agents include chelators, e.g., EDTA
(e.g., to remove divalent cations such as Mg.sup.2+), salts, RNAse
inhibitors (e.g., a broad specificity RNAse inhibitor such as
RNAsin) and so forth.
[0130] In one embodiment, the iRNA preparation includes another
iRNA agent, e.g., a second iRNA agent that can mediate RNAi with
respect to a second gene. Still other preparations can include at
least three, five, ten, twenty, fifty, or a hundred or more
different iRNA species. In some embodiments, the agents are
directed to the same virus but different target sequences. In
another embodiment, each iRNA agents is directed to a different
virus. As demonstrated in the Example, more than one virus can be
inhibited by co-administering two iRNA agents simultaneously, or at
closely time intervals, each one directed to one of the viruses
being treated.
[0131] Treatment Methods and Routes of Delivery
[0132] A composition that includes an iRNA agent of the present
invention, e.g., an iRNA agent that targets RSV or PIV, can be
delivered to a subject by a variety of routes. Exemplary routes
include inhalation, intrathecal, parenchymal, intravenous, nasal,
oral, and ocular delivery. The preferred means of administering the
iRNA agents of the present invention is through direct
administration to the lungs and nasal passage or systemically
through parental administration.
[0133] An iRNA agent can be incorporated into pharmaceutical
compositions suitable for administration. For example, compositions
can include one or more iRNA agents and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0134] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic, intranasal,
transdermal, intrapulmonary), oral or parenteral. Parenteral
administration includes intravenous drip, subcutaneous,
intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
[0135] In general, the delivery of the iRNA agents of the present
invention is done to achieve delivery into the subject to the site
of infection. The preferred means of achieving this is through
either a local administration to the lungs or nasal passage, e.g.
into the respiratory tissues via inhalation or intranasal
administration, or via systemic administration, e.g. parental
administration.
[0136] Formulations for inhalation or parenteral administration are
well known in the art. Such formulation may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives. For intravenous use, the total concentration of
solutes should be controlled to render the preparation
isotonic.
[0137] The active compounds disclosed herein are preferably
administered to the lung(s) or nasal passage of a subject by any
suitable means. Active compounds may be administered by
administering an aerosol suspension of respirable particles
comprised of the active compound or active compounds, which the
subject inhales. The active compound can be aerosolized in a
variety of forms, such as, but not limited to, dry powder
inhalants, metered dose inhalants, or liquid/liquid suspensions.
The respirable particles may be liquid or solid. The particles may
optionally contain other therapeutic ingredients such as amiloride,
benzamil or phenamil, with the selected compound included in an
amount effective to inhibit the reabsorption of water from airway
mucous secretions, as described in U.S. Pat. No. 4,501,729.
[0138] The particulate pharmaceutical composition may optionally be
combined with a carrier to aid in dispersion or transport. A
suitable carrier such as a sugar (i.e., lactose, sucrose,
trehalose, mannitol) may be blended with the active compound or
compounds in any suitable ratio (e.g., a 1 to 1 ratio by
weight).
[0139] Particles comprised of the active compound for practicing
the present invention should include particles of respirable size,
that is, particles of a size sufficiently small to pass through the
mouth or nose and larynx upon inhalation and into the bronchi and
alveoli of the lungs. In general, particles ranging from about 1 to
10 microns in size (more particularly, less than about 5 microns in
size) are respirable. Particles of non-respirable size which are
included in the aerosol tend to deposit in the throat and be
swallowed, and the quantity of non-respirable particles in the
aerosol is preferably minimized. For nasal administration, a
particle size in the range of 10-500 uM is preferred to ensure
retention in the nasal cavity.
[0140] Liquid pharmaceutical compositions of active compound for
producing an aerosol may be prepared by combining the active
compound with a suitable vehicle, such as sterile pyrogen free
water. The hypertonic saline solutions used to carry out the
present invention are preferably sterile, pyrogen-free solutions,
comprising from one to fifteen percent (by weight) of the
physiologically acceptable salt, and more preferably from three to
seven percent by weight of the physiologically acceptable salt.
[0141] Aerosols of liquid particles comprising the active compound
may be produced by any suitable means, such as with a
pressure-driven jet nebulizer or an ultrasonic nebulizer. See,
e.g., U.S. Pat. No. 4,501,729. Nebulizers are commercially
available devices which transform solutions or suspensions of the
active ingredient into a therapeutic aerosol mist either by means
of acceleration of compressed gas, typically air or oxygen, through
a narrow venturi orifice or by means of ultrasonic agitation.
[0142] Suitable formulations for use in nebulizers consist of the
active ingredient in a liquid carrier, the active ingredient
comprising up to 40% w/w of the formulation, but preferably less
than 20% w/w. The carrier is typically water (and most preferably
sterile, pyrogen-free water) or a dilute aqueous alcoholic
solution, preferably made isotonic, but may be hypertonic with body
fluids by the addition of, for example, sodium chloride. Optional
additives include preservatives if the formulation is not made
sterile, for example, methyl hydroxybenzoate, antioxidants,
flavoring agents, volatile oils, buffering agents and
surfactants.
[0143] Aerosols of solid particles comprising the active compound
may likewise be produced with any solid particulate therapeutic
aerosol generator. Aerosol generators for administering solid
particulate therapeutics to a subject produce particles which are
respirable and generate a volume of aerosol containing a
predetermined metered dose of a therapeutic at a rate suitable for
human administration. One illustrative type of solid particulate
aerosol generator is an insufflator. Suitable formulations for
administration by insufflation include finely comminuted powders
which may be delivered by means of an insufflator or taken into the
nasal cavity in the manner of a snuff. In the insufflator, the
powder (e.g., a metered dose thereof effective to carry out the
treatments described herein) is contained in capsules or
cartridges, typically made of gelatin or plastic, which are either
pierced or opened in situ and the powder delivered by air drawn
through the device upon inhalation or by means of a
manually-operated pump. The powder employed in the insufflator
consists either solely of the active ingredient or of a powder
blend comprising the active ingredient, a suitable powder diluent,
such as lactose, and an optional surfactant. The active ingredient
typically comprises from 0.1 to 100 w/w of the formulation.
[0144] A second type of illustrative aerosol generator comprises a
metered dose inhaler. Metered dose inhalers are pressurized aerosol
dispensers, typically containing a suspension or solution
formulation of the active ingredient in a liquefied propellant.
During use these devices discharge the formulation through a valve
adapted to deliver a metered volume, typically from 10 to 200 ul,
to produce a fine particle spray containing the active ingredient.
Suitable propellants include certain chlorofluorocarbon compounds,
for example, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane and mixtures thereof. The formulation may
additionally contain one or more co-solvents, for example, ethanol,
surfactants, such as oleic acid or sorbitan trioleate, antioxidant
and suitable flavoring agents.
[0145] Administration can be provided by the subject or by another
person, e.g., a caregiver. A caregiver can be any entity involved
with providing care to the human: for example, a hospital, hospice,
doctor's office, outpatient clinic; a healthcare worker such as a
doctor, nurse, or other practitioner; or a spouse or guardian, such
as a parent. The medication can be provided in measured doses or in
a dispenser which delivers a metered dose.
[0146] The term "therapeutically effective amount" is the amount
present in the composition that is needed to provide the desired
level of drug in the subject to be treated to give the anticipated
physiological response. In one embodiment, therapeutically
effective amounts of two or more iRNA agents, each one directed to
a different respiratory virus, e.g. RSV and PIV, are administered
concurrently to a subject.
[0147] The term "physiologically effective amount" is that amount
delivered to a subject to give the desired palliative or curative
effect.
[0148] The term "pharmaceutically acceptable carrier" means that
the carrier can be taken into the lungs with no significant adverse
toxicological effects on the lungs.
[0149] The types of pharmaceutical excipients that are useful as
carrier include stabilizers such as human serum albumin (HSA),
bulking agents such as carbohydrates, amino acids and polypeptides;
pH adjusters or buffers; salts such as sodium chloride; and the
like. These carriers may be in a crystalline or amorphous form or
may be a mixture of the two.
[0150] Bulking agents that are particularly valuable include
compatible carbohydrates, polypeptides, amino acids or combinations
thereof. Suitable carbohydrates include monosaccharides such as
galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and the like; cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; alditols, such as
mannitol, xylitol, and the like. A preferred group of carbohydrates
includes lactose, threhalose, raffinose maltodextrins, and
mannitol. Suitable polypeptides include aspartame. Amino acids
include alanine and glycine, with glycine being preferred.
[0151] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate is preferred.
[0152] Dosage. An iRNA agent can be administered at a unit dose
less than about 75 mg per kg of bodyweight, or less than about 70,
60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,
0.001, or 0.0005 mg per kg of bodyweight, and less than 200 nmole
of RNA agent (e.g., about 4.4.times.1016 copies) per kg of
bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5,
0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of
RNA agent per kg of bodyweight. The unit dose, for example, can be
administered by injection (e.g., intravenous or intramuscular,
intrathecally, or directly into an organ), an inhaled dose, or a
topical application.
[0153] Delivery of an iRNA agent directly to an organ (e.g.,
directly to the liver) can be at a dosage on the order of about
0.00001 mg to about 3 mg per organ, or preferably about
0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about
0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.
[0154] The dosage can be an amount effective to treat or prevent a
disease or disorder.
[0155] In one embodiment, the unit dose is administered less
frequently than once a day, e.g., less than every 2, 4, 8 or 30
days. In another embodiment, the unit dose is not administered with
a frequency (e.g., not a regular frequency). For example, the unit
dose may be administered a single time.
[0156] In one embodiment, the effective dose is administered with
other traditional therapeutic modalities.
[0157] In one embodiment, a subject is administered an initial
dose, and one or more maintenance doses of an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into an siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof).
The maintenance dose or doses are generally lower than the initial
dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses
ranging from 0.01 .mu.g to 75 mg/kg of body weight per day, e.g.,
70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,
0.001, or 0.0005 mg per kg of bodyweight per day. The maintenance
doses are preferably administered no more than once every 5, 10, or
30 days. Further, the treatment regimen may last for a period of
time which will vary depending upon the nature of the particular
disease, its severity and the overall condition of the patient. In
preferred embodiments the dosage may be delivered no more than once
per day, e.g., no more than once per 24, 36, 48, or more hours,
e.g., no more than once every 5 or 8 days. Following treatment, the
patient can be monitored for changes in his condition and for
alleviation of the symptoms of the disease state. The dosage of the
compound may either be increased in the event the patient does not
respond significantly to current dosage levels, or the dose may be
decreased if an alleviation of the symptoms of the disease state is
observed, if the disease state has been ablated, or if undesired
side-effects are observed.
[0158] The effective dose can be administered in a single dose or
in two or more doses, as desired or considered appropriate under
the specific circumstances. If desired to facilitate repeated or
frequent infusions, implantation of a delivery device, e.g., a
pump, semi-permanent stent (e.g., intravenous, intraperitoneal,
intracistemal or intracapsular), or reservoir may be advisable.
[0159] In one embodiment, the iRNA agent pharmaceutical composition
includes a plurality of iRNA agent species. The IRNA agent species
can have sequences that are non-overlapping and non-adjacent with
respect to a naturally occurring target sequence, e.g., a target
sequence of the RSV gene. In another embodiment, the plurality of
iRNA agent species is specific for different naturally occurring
target genes. For example, an iRNA agent that targets the P protein
gene of RSV can be present in the same pharmaceutical composition
as an iRNA agent that targets a different gene, for example the N
protein gene. In another embodiment, the iRNA agents are specific
for different viruses, e.g. RSV and PIV.
[0160] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the compound of the invention is
administered in maintenance doses, ranging from 0.01 .mu.g to 100 g
per kg of body weight (see U.S. Pat. No. 6,107,094).
[0161] The concentration of the iRNA agent composition is an amount
sufficient to be effective in treating or preventing a disorder or
to regulate a physiological condition in humans. The concentration
or amount of iRNA agent administered will depend on the parameters
determined for the agent and the method of administration, e.g.
nasal, buccal, or pulmonary. For example, nasal formulations tend
to require much lower concentrations of some ingredients in order
to avoid irritation or burning of the nasal passages. It is
sometimes desirable to dilute an oral formulation up to 10-100
times in order to provide a suitable nasal formulation.
[0162] Certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, 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 an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent (e.g., a precursor, e.g., a larger iRNA agent
which can be processed into an siRNA agent, or a DNA which encodes
an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent,
or precursor thereof) can include a single treatment or,
preferably, can include a series of treatments. It will also be
appreciated that the effective dosage of an iRNA agent such as an
siRNA agent used for treatment may increase or decrease over the
course of a particular treatment. Changes in dosage may result and
become apparent from the results of diagnostic assays as described
herein. For example, the subject can be monitored after
administering an iRNA agent composition. Based on information from
the monitoring, an additional amount of the iRNA agent composition
can be administered.
[0163] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC50s found to be effective in in vitro and in
vivo animal models. In some embodiments, the animal models include
transgenic animals that express a human gene, e.g., a gene that
produces a target RSV RNA. The transgenic animal can be deficient
for the corresponding endogenous RNA. In another embodiment, the
composition for testing includes an iRNA agent that is
complementary, at least in an internal region, to a sequence that
is conserved between the target RSV RNA in the animal model and the
target RSV RNA in a human.
[0164] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
[0165] Designing Antiviral siRNAs Against RSV and HPIV3
Phosphoprotein mRNA
[0166] siRNA against RSV P and siRNAs against HPIV3 P mRNA were
synthesized chemically (Bitko, V. & Barik, S. BMC Microbiol. 1,
34 (2001)) and their IC50 (concentration of siRNA producing 50%
reduction of target) ex vivo was determined (FIG. 1a). The siRNA
sequences and IC50 values are listed (Table 1). Two siRNAs against
RSV-P (#1, #2) and one against HPIV3 (#4) showed appreciable
inhibitory activities, and were selected for further study. The
correlation of target mRNA with protein, as exemplified for siRNA#1
(FIG. 1a), agreed with an RNAi mechanism, and as we shown below,
the knockdown activity of a siRNA ex vivo also matched with its
activity in the animal (in vivo). Thus, the ex vivo assay provides
a reliable, inexpensive, quick and convenient initial screening for
an antiviral siRNA drug.
[0167] Intranasal (IN) siRNAs Inhibit RSV and HPIV3 Replication in
Mouse Lung
[0168] To determine if the siRNAs that are active ex vivo would be
effective during an actual infection, an animal model was used. The
BALB/c mouse is a well-established laboratory model for RSV
infection to study the progression, pathology, and immunology of
the disease (Graham, B. S., et al., J. Med. Virol. 26, 153-162
(1988), van Schaik, S. M., et al., J. Infect. Dis. 177, 269-276
(1998), Haeberle, H. A. et al. J. Virol. 75, 878-890 (2001)). Mice
were treated with siRNA complexed with TransIT-TKO.RTM. reagent
intranasally, and 4 hr later challenged each animal with 10.sup.7
pfu of RSV or HPIV3, also intranasally. Maximal RSV growth in
murine lung could be observed around 5-6 days p.i and this time
point was used in further studies. siRNAs that were effective ex
vivo (FIG. 1a) were highly antiviral in the animal (FIG. 1b,c). At
a dose of 5 nmole IN siRNA (averaging .about.70 ug for
double-stranded siRNAs) per mouse, siRNA#1 and siRNA#4 respectively
reduced pulmonary RSV and HPIV3 titer by about 5,000- and 100-fold
in individual infections (FIG. 1b,c). Importantly, siRNAs, free of
transfection reagents, also significantly inhibited pulmonary viral
titers (FIG. 1d). This demonstrates that inhalation based
anti-viral therapy is possible with simple pharmaceutical
compositions comprising an iRNA agent. It is to be noted that HPIV3
does not infect the mouse as readily as RSV (Durbin, A. P., Elkins,
W. R. & Murphy, B. R. Vaccine 18, 2462-2469 (2000)), which is
the reason for the relatively lower HPIV3 replication in the mouse
lung (FIG. 1c). Use of sucrose-purified high-titer HPIV3 as
inoculum enabled the model to achieve measurable infections in the
mouse lung.
[0169] A (delete this A) Several additional features of the Example
are applicable to various embodiments of the present invention.
First, the results demonstrate the virus-specific effect of the
siRNAs (FIG. 1). Even the most potent anti-RSV siRNA (#1) only
inhibited RSV but not HPIV3, and vice versa, which alone argues
against a nonspecific antiviral effect of the IN siRNA. Second,
anti-luciferase siRNA (Elbashir, S. M. et al. Nature 411, 494-498
(2001)), even at the highest dose tested (50 nmole or 700 ug/mouse)
did not inhibit either virus (FIG. 1b-d). Finally, IN siRNAs with
or without the Transit-TKO reagent in uninfected mice caused no
obvious discomfort (as judged by normal coat, activity, appetite
and weight gain, and lack of any respiratory distress), suggesting
a favorable pharmacology for potential drug development.
[0170] It should be noted that in all experiments described herein,
the results of viral protein immunoblot always matched with viral
titer, and therefore, for a given experiment each can serve as a
redundant marker of the other and all complementary data is not
presented.
[0171] Specific Antiviral Effects of IN siRNA Prevent Lung
Infection
[0172] Although the results presented above documented inhibition
of viral replication, they did not directly prove abrogation of
infection of the lung tissue. We, therefore, probed various
sections of both lungs at 5 days p.i. using antibodies specific for
the appropriate virus. With 10.sup.7 pfu instilled per mouse, both
viruses produced robust pulmonary infection. In representative
results (FIG. 2a), infection was strongly abolished in mice
pre-treated with 5 nmole (70 ug) of anti-RSV siRNA#1 complexed with
TransIT-TKO. Similar reduction of HPIV3 infection was also seen
with 5 nmole anti-HPIV3 siRNA#4. As with viral titer, siRNA without
transfection reagent showed significant reduction of infection, as
represented for RSV. For the same amount of siRNA, we estimate that
the reagent-free siRNA was roughly 70-80% as effective as siRNA
complexed with TransIT-TKO. Although we have only presented data
for TransIT-TKO-complexed siRNA in the rest the paper, these
results point to the exciting prospect that IN delivery of pure
naked siRNA, free of other chemicals, may offer substantial
protection against respiratory pathogens. This is particularly
important as transfection reagents themselves may have
side-effects. We note here that polyethyleneimine (PEI) has been
successfully used as a carrier for intravenous (IV) as well as
intratracheal (IT) delivery of siRNA and DNA against influenza
virus (Ge, Q., et al., Proc. Natl. Acad. Sci. USA 101, 8676-8681
(2004)). However, in our experience, direct IN administration of
PEI, with or without siRNA, often resulted in overt sickness and/or
death of the mouse.
[0173] IN siRNAs Locate to the Lung and do not Activate
Interferon
[0174] To provide further evidence that the viral inhibition
observed in the lung was a direct and specific effect of the
nasally applied siRNA, two kinds of experiments were performed.
First, we were able to detect the antisense strand of the siRNA in
the lung tissue (FIG. 2b) by specific Northern analyses. Second,
the possibility that the antiviral effect of siRNAs was due to
activation of interferons (IFNs) was ruled out by the following.
Paramyxoviruses in general encode diverse mechanisms to counteract
IFNs; RSV, in particular, is largely resistant to type I IFNs
(IFN-.alpha./.beta.) although sensitive to type II IFN
(IFN-.gamma.) (Schlender, J., et al., J. Virol. 74, 8234-8242
(2000), Ramaswamy, M., et al., Am. J. Respir. Cell Mol. Biol. 30,
893-900 (2004)). Our early studies showed that the siRNAs were
active against RSV and HPIV3 in Vero cells that contain deletions
of type I IFN genes (data not shown). Nonetheless, we measured the
levels of IFN-.alpha. and IFN-.gamma. in murine lung tissues at
different days following treatment with various siRNAs, and found
no activation of either type of IFN (FIG. 2c).
[0175] siRNAs Competitively Protect Against RSV and HPIV3 in Mixed
Infection
[0176] Co-infection of the respiratory tract by multiple agents is
always a possibility, and in some studies joint infection by RSV
and HPIV3 has been diagnosed (Coiras, M. T., et al, J. Med. Virol.
72, 484-495 (2004)). In fact, chimeric viruses and recombinant
vaccines incorporating RSV as well HPIV3 antigens have been
constructed with the hope that they would offer simultaneous
protection against both viruses (Schmidt, A. C., et al., J. Virol.
75, 4594-4603 (2001), Bernhard, W. et al. Am. J. Respir. Cell Mol.
Biol. 25, 725-731 (2001)). The specific antiviral effect of siRNA#1
and siRNA#4 against RSV and HPIV3, respectively, prompted us to
test them together (5 nmole or 70 ug of each) in mixed infection of
the mice by both viruses. Control mice were treated with a single
kind of siRNA (either #1 or #4). As there is no easy way to
determine the pfu of each virus in a mixture of the two, we
resorted to immunofluorescence microscopy as described above, and
subjected the lung tissue sections to dual staining using a mixture
of anti-RSV and anti-HPIV3 antibodies. Mixed infection of the lung
tissue was indeed achieved by this criterion (FIG. 2d). In mice
pre-treated with either siRNA#1 or siRNA#4, RSV and HPIV3 infection
respectively was prevented, as seen by the loss of either green or
red fluorescence, but not both (FIG. 2d). Using a combination of
the two siRNAs (5 nmole, i.e., 70 ug of each) both types of
fluorescence disappeared, documenting the inhibition of both
viruses (FIG. 2d). As before, the same siRNA mix without any
transfection reagent was also highly active (data not shown).
[0177] Interestingly, when excessively high amounts of one siRNA
were used, the activity of the other siRNA was inhibited in a dual
infection assay (FIG. 3). By quantitative real-time RT-PCR, the
IC50 of siRNA#4 (against HPIV3 P) ex vivo increased through 15, 35
and 100 nM as the concentration of siRNA#1 (against RSV P) was
raised from 0 to 20 to 200 nM (FIG. 3a). These results were
validated by measurement of HPIV3 P protein in immunoblot (FIG.
3b). In dual infection of mice, immunoblot quantitation of N
protein of each virus produced an essentially identical conclusion:
whereas 5 nmole (70 ug) of each siRNA effectively inhibited both
viruses, 50 nmole of one siRNA reduced the effect of 5 nmole of the
other in a mutual manner (FIG. 3c).
[0178] IN siRNAs Prevent Pulmonary Pathology
[0179] Since the siRNAs prevented infection a logical query was
whether they prevented the development of pathological features as
well. Upon visual inspection the siRNA-treated RSV-exposed mice
acted and appeared essentially like uninfected mice with normal
activity, shiny coat and general well-being. We then measured the
respiratory rate, induction of leukotriene, and pulmonary
inflammation. Respiratory rate of BALB/c mice is known to increase
in response to RSV infection (Haeberle, H. A. et al. J. Virol. 75,
878-890 (2001), Volovitz, B., et al., Pediatr. Res. 24, 504-507
(1988)). Leukotrienes, a product of the lipoxygenase pathway, bind
to the leukotriene receptors present in bronchial smooth muscle and
are elevated in the respiratory secretions of asthmatic patients,
human infants with RSV infection, and mice infected with RSV
(Volovitz, B., et al., Pediatr. Res. 24, 504-507 (1988), Welliver,
R. C., 2nd, et al., J. Infect. Dis. 187, 1773-1779 (2003)). These
compounds provoke airway mucus secretion, bronchoconstriction and
airway infiltration by inflammatory cells, which are important
hallmarks of severe RSV disease. When we administered anti-RSV
siRNA#1 at the same time as (or prior to) RSV, a significant
reduction in respiratory rate, pulmonary histopathology and
leukotriene accumulation in bronchoalveolar fluid (BALF) was
observed (FIG. 4). These values remained near baseline and were
essentially comparable to those in sham-infected mice. All
parameters remained low at least 14 days post-infection,
demonstrating that the siRNA truly prevented illness and not just
postponed it. In fact, siRNA-treated mice showed no visible signs
of respiratory distress up to 6 weeks of observation. Negative
control RNA or luc-siRNA (Table 1) offered no relief in all
experiments (data not shown).
[0180] IN siRNAs are Effective Antivirals Post-Infection
[0181] Having shown that siRNAs can prevent respiratory viral
disease if administered prior to infection, we asked the question
whether they may have a curative effect once infection has
established, as this is an important goal in pediatric medicine. In
this series of experiments, we administered siRNA#1 at various days
after RSV infection and the mice were weighed daily. Mice are known
to lose weight up to about 8-10 days following RSV infection, after
which they either die or slowly regain weight depending on whether
the starting inoculum is too high or moderate-to-low (Haeberle, H.
A. et al. J. Virol. 75, 878-890 (2001)). In a similar cohort of
mice, the lungs were sampled on pre-determined days to assay for
infectious virus. As expected, the siRNA-untreated mice maintained
their body weight for about 4 days p.i., followed by a gradual loss
that continued at least up to 9 days (FIG. 5a). Mice treated with
siRNA prior to (data not shown) or at the same time as RSV (Day 0)
essentially appeared uninfected and continued to gain weight
without interruption. Most mice receiving siRNA on Day 1 were also
quite hard to distinguish from the sham-infected controls. Those
receiving siRNA at subsequent days (Day 1-4) showed gradually less
and less protection, although significant improvement of weight was
observed at all days for all treatments.
[0182] A similar picture emerged when pulmonary RSV titer was
determined in these mice on Day 2, 4, 6, 8, 10, and 16 (FIG. 5b).
In the siRNA-untreated mice, the titer rose till Day 4-5, and then
slowly dropped to undetectable levels by Day 16. siRNA treatment
before or concomitant to RSV infection held the titer 5,000-fold
down at all days tested. Administration of siRNA at later times in
infection was progressively less effective, but the viral titer was
always lower than the untreated controls on any day tested. It
appeared that the siRNA, no matter when it was administered, slowed
down the rate of virus replication, resulting in a lower peak
titer. Subsequently, the titer fell below detectable levels at
earlier and earlier times the sooner the siRNA was administered.
For example, whereas in the untreated infected mice pulmonary RSV
could be detected up to about 16 days p.i., it could not be
detected in the Day 1 siRNA-treated mice after 10 days p.i. As
before, negative control siRNA or luciferase siRNA (Table 1) had no
effect in all experiments (data not shown). Together, these results
showed that the RSV P siRNA had a curative effect even when
administered post-infection and that the mice were always less sick
and recovered quicker than their untreated cohorts.
DISCUSSION
[0183] The principal finding of this paper is that appropriately
designed siRNAs, applied intranasally, offer protection from
respiratory infection as well as provide significant therapy when
applied post-infection. siRNAs, delivered by small particle
aerosols in a simple hand-held inhaler, can be used to prevent or
treat pulmonary infections. While our manuscript was in
preparation, two reports appeared in which siRNAs inhibited
influenza virus, another major respiratory pathogen, in a murine
model (Ge, Q., et al., Proc. Natl. Acad. Sci. USA 101, 8676-8681
(2004), Tompkins, S. M., et al., Proc. Natl. Acad. Sci. USA 101,
8682-8686 (2004)). In one study (Ge, Q., et al., Proc. Natl. Acad.
Sci. USA 101, 8676-8681 (2004)), synthetic siRNA or plasmid DNA
expressing siRNA was administered via a combination of IV and IT
routes. In the other study (Tompkins, S. M., et al., Proc. Natl.
Acad. Sci. USA 101, 8682-8686 (2004)), siRNA was first delivered by
hydrodynamic IV delivery; 16-24 hr later mice were infected with
influenza virus and given a second dose of siRNA in a lipid
carrier, both via IN route. Presence of pulmonary virus was tested
2 days later and a 10-50 fold inhibition was observed with
different strains of influenza virus. Our studies against RSV and
PIV offer the following improvement and simplification over the
previous ones: (i) The delivery is solely intranasal and therefore,
relatively noninvasive and painless, making it amenable to an
inhaler or mist-based therapy. (ii) siRNA without any carrier is
significantly effective, thus reducing the potential risk of side
effects of the carrier. (iii) A single dose of about 5 nanomole
siRNA (70 ug of double-stranded RNA) appears to provide benefit
over the full duration of infection. A more comprehensive screening
of the target sequence (e.g., RSV P) and use of newer chemistry may
lead to siRNAs with significantly lower IC50 and better
pharmacokinetics, resulting in a lower dosage. siRNAs exhibit
various degrees of non-specific, off-target effects, especially at
high concentrations (Jackson, A. L. et al. Nat. Biotechnol. 21,
635-637 (2003), Sledz, C. A., et al., Nat. Cell Biol. 5, 834-839
(2003), Persengiev, S. P., et al., RNA 10, 12-18 (2004), Bridge, A.
J., et al., Nat. Genet. 34, 263-264 (2003)). This is an obvious
concern in therapy, and IV administration of siRNA may result in
systemic side-effects. In contrast, the intranasally delivered
siRNA is more likely to be concentrated--if not exclusively
localized--in the respiratory tissues, thus minimizing the side
effects. Lack of IFN activation by intranasally delivered synthetic
siRNA supports and extends previous finding that chemically
synthesized siRNAs, devoid of 5' phosphates, do not activate the
IFN pathway in cell culture (Kim, D. H. et al. Nat. Biotechnol. 22,
321-325 (2004)). Together, correlation of antiviral activity with
specific mRNA knockdown (FIG. 1), detection of the siRNA in the
target tissue (lung) (FIG. 2b), lack of IFN activation (FIG. 2c),
and virus-specific effect of the siRNAs (FIGS. 1-3)--all provide
evidence that the antiviral effect is specific, directed and is
RNAi-mediated. (iii) Respiratory viruses, such as RSV and PIV,
exhibit high selectivity in tissue tropism in infecting the
respiratory tissues. Thus, IN delivery ensures that the siRNA is
targeted to the site of infection--an ideal condition for
pharmacology.
[0184] Although RSV and HPIV sometimes co-infect, their
interactions have remained largely ignored. The exact reason behind
the observed inhibition of one siRNA by another needs further
study. The fact that it also happens ex vivo (cell culture) argues
against, although does not rule out, humoral factors and cytokines
in the animal, such as interferon. RSV actually inhibits interferon
activation (Schlender, J., et al., J. Virol. 74, 8234-8242 (2000),
Ramaswamy, M., et al., Am. J. Respir. Cell Mol. Biol. 30, 893-900
(2004)), and thus, should facilitate rather than inhibit PIV
growth. Another possibility is that growth of one virus inhibits
the other through other mechanisms, such as competition for
intracellular resources. On the other hand, it is known that the
RNAi machinery in a cell is saturable and thus two siRNAs could
potentially compete for a fixed pool of this machinery (Barik, S.
Virus Res. 102, 27-35 (2004), (Hutvagner, G., et al., PLoS Biol. 2,
E98 (2004)). It is to be noted that such competition was only
appreciable at relatively high doses of the siRNAs, i.e., with tens
or hundreds of nanomoles (FIG. 3). In contrast, only a few
nanomoles of our siRNAs offered nearly complete protection in mice.
Thus, the observed competition is not a matter of practical concern
with siRNAs of IC50 in the low nanomolar range.
[0185] When used as a prophylactic, the siRNA not only prevented
the infection but also inhibited the appearance of various aspects
of the disease process as measured by body weight, pulmonary
pathology, respiratory parameters and allergy markers (FIG. 4). The
kinetics of the disease process in mice and men are relatively
similar and in both species immunopathological changes occur
rapidly following RSV infection. When used as a treatment drug
after infection ensued, the siRNAs are not expected to correct the
pathology that has already occurred. Even then, however, inhibition
of further growth of the virus resulted in a quicker cure and
recovery (FIG. 5). Thus, it seems that the "window of opportunity"
of treatment exists at all times in the RSV-infected patient
although, as in any disease, earlier treatments should produce
better prognosis. The effectiveness of the naked siRNA remains to
be explained. It is possible that the respiratory tissue,
especially the lung, is naturally more receptive to the exchange of
small molecules, or perhaps becomes so when infected.
[0186] Lastly, depending on the stringency of siRNA-target pairing,
exposure to siRNA may cause selection of siRNA-resistant viruses,
and this has been demonstrated in HIV (Das, A.T. et al. J. virol.
78, 2601-2605 (2004)). We have not faced this problem so far with
the siRNAs tested here. The viruses that could be recovered from
the siRNA-treated murine lung were grown in A549 cell culture and
found to exhibit the same IC50 for the siRNA as the original
inoculum (data not shown). Moreover, sequencing of the siRNA region
of the P gene in six such independent plaque-purified RSV isolates
revealed the wild type parental sequence (data not shown). Even if
occasional resistance is encountered in the future, a second siRNA
with a low IC50 and targeting a different region of the P mRNA or a
different viral mRNA can be used in a multidrug regimen, thereby
reducing the odds of viral resistance.
[0187] Methods
[0188] Virus, siRNA and other reagents. RSV Long strain and human
PIV type 3 (HPIV3) JS strain were grown on HEp-2 monolayers as
described for RSV (Burke, E., et al., Virology 252, 137-148 (1998),
Burke, E., et al., J. Virol. 74, 669-675 (2000), Gupta, S., et al.,
J. Virol. 72, 2655-266 (1998)). The extracellular media containing
liberated progeny virus was collected at about 70 h for RSV and 50
h for HPIV3. The viruses were purified and concentrated by
precipitation with polyethylene glycol (MW 8,000) and sucrose
gradient centrifugation essentially as described for RSV (Ueba, O.
Acta. Med. Okayama 32, 265-272 (1978)). The final preparations had
infectious titers in the range of 10.sup.8-10.sup.9 pfu /ml and
were stored frozen at -80.degree. C. in small portions. All
infectious viral titers (pfu) were determined by agarose plaque
assay on HEp-2 monolayers with neutral red staining (Burke, E., et
al., Virology 252, 137-148 (1998), Burke, E., et al., J. Virol. 74,
669-675 (2000), Gupta, S., et al., J. Virol. 72, 2655-2662
(1998)).
[0189] siRNAs were purchased from Dharmacon and processed as
recommended by the 25 manufacturer (Bitko, V. & Barik, S. BMC
Microbiol. 1, 34 (2001). The TransIT-TKO.RTM. reagent was from
Mirus Bio Corp (Madison, Wis.). RSV-P antibody, raised in rabbit,
was used in all immunohistological staining (Bitko, V. & Barik,
S. BMC Microbiol. 1, 34 (2001). Polyclonal RSV and HPIV3 antibodies
were raised against purified virions in goat and purchased from
Chemicon (Temecula, Calif.) and BiosPacific (Emeryville, Calif.),
respectively; the nucleocapsid protein (N) is the major viral band
detected by these antibodies in immunoblot. Profilin antibody has
been described (Burke, E., et al., J. Virol. 74, 669-675 (2000),
Gupta, S., et al., J. Virol. 72, 2655-2662 (1998)).
[0190] Virus infection and siRNA treatment. Infection and siRNA
treatment of A549 cells grown in monolayers were carried out as
described (Bitko, V. & Barik, S. BMC Microbiol. 1, 34 (2001)).
Intranasal application of RSV in mice is an established procedure
and causes bronchiolitis. Pathogen-free 8-10 week old female BALB/c
mice, weighing between 16 and 20 g) were purchased from Charles
River Laboratories. Anesthesia for infection or siRNA
administration was achieved with intraperitoneal injection of 0.2
ml of nembutal (5 mg/ml). Euthanasia was carried out by cervical
dislocation following anesthesia with 0.3 ml nembutal. The siRNA
was appropriately diluted in the dilution buffer provided by the
manufacturer so that the desired amount is contained in 1 ul. This
was mixed with 5 ul of the TransIT-TKO.RTM. reagent and 35 ul of
Opti-MEM (Gibco Life Technologies, Invitrogen, Carlsbad, Calif.)
immediately before experiment to produce a total volume of 41 ul.
When siRNA was used without carrier, the 5 ul transfection reagent
was substituted with 5 ul of Opti-MEM. The sucrose-purified virus
was appropriately diluted in cold phosphate-buffered saline (PBS)
immediately prior to infection such that 10.sup.7 pfu virus was
contained in 30 .mu.l. Sham infection was performed with the same
volume of virus-free PBS. Both the siRNA mix and the virus were
equally divided into the two nostrils and applied with a
micropipette (i.e., each nostril received 35 ug siRNA in 20.5 ul
and 0.5.times.10.sup.7 pfu virus in 15 ul). No special equipment
was needed as the mice inhaled all fluid through natural breathing.
For dual infection, RSV and HPIV3 stocks were diluted such that
each mouse was given a mixture of 10.sup.7 pfu of each virus and a
mixture of 5 nmole (70 ug) each of siRNA#1 and siRNA#4 in the same
volumes as before. Animal experiments obeyed all prescribed
guidelines and were approved by the IACUC.
[0191] Pulmonary viral assay and clinical measurements. The animals
were checked daily and weighed. Standard RSV symptoms were noted,
including nasal mucus, increased respiratory rate due to congestion
and bronchiolitis, a dull coat, ruffled fur and/or loss of fur, and
a general lethargy and malaise. Respiratory rates (breaths per min)
were determined by video recording (Volovitz, B., et al., Pediatr.
Res. 24, 504-507 (1988)). Sneezing, sniffing and sighing were
excluded from counting. At various days post-infection (p.i.),
lungs were removed for RSV detection by infectious virus assay,
immunoblot analysis, or immunostaining, as described below.
[0192] To determine viral titer, the lung was homogenized in DMEM
supplemented with 2% FBS (2 ml DMEM per 100 mg tissue) in cold. The
extract was centrifuged at 2,000.times.g for 10 min, and serial
dilutions of the supernatant were assayed for pfu. For immunoblot
of viral proteins (Burke, E., et al., Virology 252, 137-148
(1998)), 10 ul of the homogenized sample (before centrifugation)
was added to 10 ul of 2.times. SDS-PAGE sample buffer, the mixture
heated at 98.degree. C. for 5 min, clarified by centrifugation in a
microfuge at room temperature, and 10 ul of the clear supernatant
analyzed by immunoblot using goat anti-RSV and anti-HPIV3
antibodies. To measure IFN (Durbin, J. E. et al. J. Immunol. 168,
2944-2952 (2002)), the lungs were homogenized in PBS, processed as
above, and serial dilutions were assayed by ELISA kits (R&D
Systems, Minneapolis, Minn.) having detection limits of 10
pg/ml.
[0193] For pulmonary histopathology, lungs were perfused and fixed
in 10% buffered formalin and embedded in paraffin. Multiple, 4
.mu.m thick sections were stained with haematoxylin & eosin and
scored for cellular inflammation under light microscopy by two
independent researchers. Inflammatory infiltrates were scored by
enumerating the layers of inflammatory cells surrounding the
vessels and bronchioles. Zero to three layers of inflammatory cells
were considered normal, whereas more than three layers of
inflammatory cells surrounding 50% or more of the circumference of
the vessel or bronchioles were considered abnormal. The number of
abnormal perivascular and peribronchial spaces divided by total
such spaces was the percentage reported as the pathology score. A
total of about 20 spaces per lung were counted for each animal.
With 10.sup.7 RSV (and no siRNA) (Haeberle, H. A. et al. J. Virol.
75, 878-890 (2001)), about 30-35% of perivascular and peribronchial
spaces could be found abnormal as early as Day 1 and peaked at
around Day 5.
[0194] For immunohistology (Haeberle, H. A. et al. J. Virol. 75,
878-890 (2001)), the lung tissue was embedded in 100% OCT compound,
and frozen at -80.degree. C. Sections were cut onto slides,
air-dried, fixed in acetone, were washed in PBS and permeabilized
with 0.2% Triton X-100 in PBS, blocked for 20 min with 10% goat
serum in PBS at room temperature. After multiple washes in PBS the
tissue was incubated for 2 h at room temperature with either
anti-RSV-P or anti-HPIV3 antibody diluted in PBS containing 1.5%
goat serum. The slides were again washed multiple times in PBS, and
the two antibodies were detected with FITC-conjugated anti-rabbit
and TRITC-conjugated anti-goat immunoglobulin G antibody. After 1 h
incubation at room temperature, the slides were given a final wash
in PBS, mounted with the DABCO-DAPI mounting media and viewed by
fluorescence microscopy (Bitko, V. & Barik, S. BMC Microbiol.
1, 34 (2001)).
[0195] Bronchoalveolar lavage fluid (BALF), was collected by
perfusing the bronchi and the lungs with 5.times.1.0 ml normal
saline (containing 10 ug indomethacin per ml) (Bernhard, W. et al.
Am. J Respir. Cell Mol. Biol. 25, 725-731 (2001)); total recovery
of BALF per mouse was 4.2-4.4 ml. Samples containing visible signs
of blood contamination were discarded. Cells were removed from BALF
by centrifugation at 5,000.times.g for 15 min at 4.degree. C., and
samples stored at -80.degree. C. until further analyses. The
concentration of cysteinyl leukotrienes conjugates in the BALF was
determined by an ELISA kit following the manufacturer's protocol
(R&D Systems, Minneapolis, Minn.). According to the product
insert, the cross-reactivity of the kit to the various leukotrienes
was: LTC4 100%, LTD4 115%, LTE4 63% and LTB4 1.2%.
[0196] For Real Time PCR experiments, RNA was isolated from
HPIV3-infected cells and the first-strand cDNA made using the
GeneAmp RNA PCR Core kit (Perkin-Elmer Applied Biosystems, Foster
City, Calif.). Primers are designed by the Beacon Designer software
v 2.13 from Premier Biosoft. The following primers were used to
amplify HPIV3 P mRNA: 5'-GGTCATCACACGAATGTACAAC-3' and
5'-CTTGGAACATCTGCAGATTGTC-3'. Real-time PCR was performed on the
iCycler iQ Quantitative PCR system from BioRad Laboratories
(Hercules, Calif.) using the iQ Sybr Green SuperMix. Gene
expression measurements were calculated using the manufacturer's
software and GAPDH as the internal control.
[0197] The antisense strand of siRNA in the lung was extracted and
detected by Northern hybridization essentially as described
(Reinhart, B. J., et al., Genes & Dev. 16, 1616-1626 (2002))
using complementary synthetic oligodeoxynucleotide terminally
labeled with .sup.32P.
[0198] Statistical analysis. Changes between treatment groups or
between sets of in vitro experiments were analyzed by one-way ANOVA
and then by Student's t test with Bonferroni correction. Increases
in leukotriene concentrations were determined by the Mann-Whitney
test. All numerical data were collected from at least 3 separate
experiments. Results were expressed as mean.+-.SEM (error bars in
graphs). Differences were considered to be significant at
P<0.05. TABLE-US-00001 TABLE 1 siRNA sequences IC50 Name Target
siRNA sequence (nM) siRNA#1 RSV-P 5'-CGAUAAUAUAACUGCAAGAdTdT-3' 18
3'-dTdTGCUAUUAUAUUGACGUUCU-5' siRNA#2 RSV-P
5'-CCCUACACCAAGUGAUAAUdTdT-3' 80 3'-dTdTGGGAUGUGGUUCACUAUUA-5'
siRNA#3 RSV-P 5'-GAUGCCAUGAUUGGUUUAAdTdT-3' >300
3'-dTdTCUACGGUACUAACCAAAUU-5' siRNA#4 HPIV3-P
5'-CGAGUUGUAUGUGUAGCAAdTdT-3' 15 3'-dTdTGCUCAACAUACACAUCGUU-5'
siRNA#5 HPIV3-P 5'-GAUAGACUUCCUAGCAGGAdTdT-3' >300
3'-dTdTCUAUCUGAAGGAUCGUCCU-5' Luc- Luci-
5'-CGUACGCGGAAUACUUCGAdTdT-3' -- siRNA ferase
3'-dTdTGCAUGCGCCUUAUGAAGCU-5' Negative
5'-UUCUCCGAACGUGUCACGUdTdT-3' -- 3'-dTdTAAGAGGCUUGCACAGUGCA-5'
[0199] The GenBank accession numbers for RSV-P, HPIV3-P and
luciferase sequences are M22644, Z11575 and X65324 respectively.
Note that the siRNA sequences were based on actual sequencing of
the viral strains in our laboratory; thus, siRNA#2 differs by one
nucleotide from the GenBank sequence (the underlined C is U in
M22644). Negative control siRNA sequence was from Qiagen (Valencia,
Calif.).
Sequence CWU 1
1
16 1 22 DNA Artificial Sequence Primer 1 ggtcatcaca cgaatgtaca ac
22 2 22 DNA Artificial Sequence Primer 2 cttggaacat ctgcagattg tc
22 3 21 DNA Artificial Sequence Synthetically generated
oligonucleotide 3 cgauaauaua acugcaagan n 21 4 21 DNA Artificial
Sequence Synthetically generated oligonucleotide 4 ucuugcaguu
auauuaucgn n 21 5 21 DNA Artificial Sequence Synthetically
generated oligonucleotide 5 cccuacacca agugauaaun n 21 6 21 DNA
Artificial Sequence Synthetically generated oligonucleotide 6
auuaucacuu gguguagggn n 21 7 21 DNA Artificial Sequence
Synthetically generated oligonucleotide 7 gaugccauga uugguuuaan n
21 8 21 DNA Artificial Sequence Synthetically generated
oligonucleotide 8 uuaaaccaau cauggcaucn n 21 9 21 DNA Artificial
Sequence Synthetically generated oligonucleotide 9 cgaguuguau
guguagcaan n 21 10 21 DNA Artificial Sequence Synthetically
generated oligonucleotide 10 uugcuacaca uacaacucgn n 21 11 21 DNA
Artificial Sequence Synthetically generated oligonucleotide 11
gauagacuuc cuagcaggan n 21 12 21 DNA Artificial Sequence
Synthetically generated oligonucleotide 12 uccugcuagg aagucuaucn n
21 13 21 DNA Artificial Sequence Synthetically generated
oligonucleotide 13 cguacgcgga auacuucgan n 21 14 21 DNA Artificial
Sequence Synthetically generated oligonucleotide 14 ucgaaguauu
ccgcguacgn n 21 15 21 DNA Artificial Sequence Synthetically
generated oligonucleotide 15 uucuccgaac gugucacgun n 21 16 21 DNA
Artificial Sequence Synthetically generated oligonucleotide 16
acgugacacg uucggagaan n 21
* * * * *