U.S. patent application number 11/978089 was filed with the patent office on 2009-06-25 for novel sirnas and methods of use thereof.
Invention is credited to Elena Feinstein, Igor Mett, Rami Skaliter.
Application Number | 20090162365 11/978089 |
Document ID | / |
Family ID | 39324997 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162365 |
Kind Code |
A1 |
Feinstein; Elena ; et
al. |
June 25, 2009 |
Novel siRNAS and methods of use thereof
Abstract
The invention relates to compounds, in particular siRNAs, which
inhibit the expression of specific human genes. The invention also
relates to pharmaceutical compositions comprising such compounds
and a pharmaceutically acceptable carrier. The present invention
also provides a method of treating and/or preventing the incidence
or severity of various diseases or conditions associated with the
genes and/or symptoms associated with such diseases or conditions
comprising administering to a subject in need of treatment for such
disease or condition and/or symptom the compound or the
pharmaceutical composition in a therapeutically effective dose so
as to thereby treat the subject. The invention also provides
antibodies which inhibit specified human polypeptides and
pharmaceutical compositions comprising one or more such
antibodies.
Inventors: |
Feinstein; Elena; (Rehovot,
IL) ; Skaliter; Rami; (Nes Ziona, IL) ; Mett;
Igor; (Rehovot, IL) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
30 Rockefeller Plaza, 20th Floor
NEW YORK
NY
10112
US
|
Family ID: |
39324997 |
Appl. No.: |
11/978089 |
Filed: |
October 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60854503 |
Oct 25, 2006 |
|
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60930493 |
May 15, 2007 |
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Current U.S.
Class: |
424/139.1 ;
514/44R; 536/24.5 |
Current CPC
Class: |
A61P 27/16 20180101;
A61P 27/02 20180101; A61P 19/02 20180101; C12Y 304/22055 20130101;
A61P 13/12 20180101; C12N 2310/11 20130101; A61P 27/06 20180101;
C12N 2310/321 20130101; A61K 31/70 20130101; A61P 11/00 20180101;
A61P 25/00 20180101; C12N 2310/315 20130101; C12N 15/1137 20130101;
A61P 9/00 20180101; A61P 11/08 20180101; A61P 37/06 20180101; C12N
15/1136 20130101; C12N 2310/343 20130101; A61P 43/00 20180101; A61P
17/00 20180101; C12N 2310/14 20130101; A61P 9/10 20180101; A61P
27/04 20180101; C12N 15/113 20130101; A61P 17/02 20180101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
424/139.1 ;
536/24.5; 514/44 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A compound having the structure: 5' (N).sub.x-Z 3' (antisense
strand) 3' Z'-(N').sub.y 5' (sense strand) wherein each of N and N'
is a ribonucleotide which may be modified or unmodified in its
sugar residue; wherein each of (N).sub.x and (N').sub.y is an
oligomer in which each consecutive N or N' is joined to the next N
or N' by a covalent bond; wherein each of x and y is an integer
between 19 and 40; wherein each of Z and Z' may be present or
absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein the sequence of (N').sub.y is present within an mRNA
whose sequence is set forth in one of SEQ ID NO:46, SEQ ID NO: 1-41
or SEQ ID NO:47-48.
2. The compound of claim 1, wherein the covalent bond joining each
consecutive N or N' is a phosphodiester bond.
3. The compound of claim 1, wherein x=y.
4. The compound of claim 3, wherein each of x and y is 19, 21 or
23.
5. The compound of claim 1, wherein Z and Z' are absent.
6. The compound of claim 1, wherein one of Z or Z' is present.
7. The compound of claim 1, wherein each of N or N' is unmodified
in its sugar residue.
8. The compound of claim 1, wherein at least one N or N' comprises
a modification in its sugar residue.
9. The compound of claim 8, wherein the modification comprises a
modification at the 2' position.
10. The compound of claim 9, wherein the modification at the 2'
position comprises the presence of an amino, a fluoro, an alkoxy or
an alkyl group.
11. The compound of claim 10 wherein the modification comprises the
presence of an alkoxy group.
12. The compound of claim 11, wherein the alkoxy group is methoxy
(2'-O-methyl) group.
13. The compound of claim 1, wherein alternating ribonucleotides in
(N).sub.x are modified and alternating ribonucleotides in
(N').sub.y are modified.
14. The compound of claim 13, wherein each N at the 5' and 3'
termini in (N).sub.x are modified in their sugar residues, and each
N' at the 5' and 3' termini of (N').sub.y are unmodified in their
sugar residues.
15. The compound of claim 14, wherein both (N).sub.x and the
(N').sub.y are non-phosphorylated at both their 3' and 5' termini
or wherein both (N).sub.x and (N').sub.y are phosphorylated at the
3' termini.
16. A compound having the structure: 5' (N).sub.x-Z 3' (antisense
strand) 3' Z'-(N').sub.y 5' (sense strand) wherein each of N and N'
is a ribonucleotide which may be modified or unmodified in its
sugar residue; wherein each of (N).sub.x and (N').sub.y is an
oligomer in which each consecutive N or N' is joined to the next N
or N' by a covalent bond; wherein each of x and y is an integer
between 19 and 40; wherein each of Z and Z' may be present or
absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein each of (N).sub.x and (N').sub.y is set forth in any
one of SEQ ID NOS: 97 to 68654.
17. A pharmaceutical composition comprising a compound of claim 1
or a vector capable of expressing such a compound in an amount
effective to inhibit the gene, and a pharmaceutically acceptable
carrier.
18. A method of treating a disease or condition selected from
hearing loss, acute renal failure, glaucoma, acute respiratory
distress syndrome, an acute lung injury, organ transplantation
rejection, ischemia-reperfusion injury, nephrotoxicity,
neurotoxicity, spinal cord injury, pressure sores, osteoarthritis,
dry eye and chronic obstructive pulmonary disease (COPD), in a
subject in need thereof, comprising administering to the subject an
oligonucleotide which inhibits expression of a gene whose mRNA
sequence is set forth in any one of SEQ ID NOS: 1-41 or 46-48 in an
amount effective to treat the disease or condition.
19. The method according to claim 18 wherein the oligonucleotide is
siRNA.
20. The method according to claim 19 wherein the siRNA comprises an
oligomer whose sequence is set forth in any one of SEQ ID NOS: 277
to 50970 and 50993-68654 (Table B).
21. The method according to claim 20 wherein the siRNA comprises an
oligomer whose sequence is set forth in any one of SEQ ID NOS:
97-276 (Tables C1, C2) and SEQ ID NOS: 50971-50992 (Table C3).
22. A method of treating acute renal failure in a subject in need
thereof, comprising administering to the subject an oligonucleotide
which inhibits expression of any one of TP53BP (whose mRNA sequence
is set forth in SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set
forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in
SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID
NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID
NO:15), or Rac1 (whose mRNA sequence is set forth in SEQ ID
NO:24-26) in an amount effective to treat the acute renal
failure.
23. A method of treating spinal-cord injury in a subject in need
thereof, comprising administering to the subject an oligonucleotide
which inhibits expression of any one of RHOA (whose mRNA sequence
is set forth in SEQ ID NO:46); TP53BP (whose mRNA sequence is set
forth in SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set forth
in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID
NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:
10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO: 15),
Rac1 (whose mRNA sequence is set forth in SEQ ID NO:24-26, CD38
(whose mRNA sequence is set forth in SEQ ID NO:32) or BMP2 (whose
mRNA sequence is set forth in SEQ ID NO:34) in an amount effective
to treat the spinal cord injury.
24. A method of treating a disease or condition selected from
hearing loss, acute renal failure, glaucoma, acute respiratory
distress syndrome, an acute lung injury, organ transplantation
rejection, ischemia-reperfusion injury, nephrotoxicity,
neurotoxicity, spinal cord injury, pressure sores, osteoarthritis
and chronic obstructive pulmonary disease (COPD), in a subject in
need thereof, comprising administering to the subject an antibody
which inhibits a polypeptide whose sequence is set forth in any one
of SEQ ID NOS: 90-93 in an amount effective to treat the disease or
condition.
25. A pharmaceutical composition comprising an antibody which
inhibits a polypeptide whose sequence is set forth in any one of
SEQ ID NOS: 90-93, in an amount effective to inhibit the
polypeptide, and a pharmaceutically acceptable carrier.
Description
[0001] This application claims the benefit of U.S. Provisional
patent application No. 60/854,503 filed Oct. 25, 2006, and of U.S.
Provisional patent application No. 60/930,493 filed May 15, 2007,
both of which are hereby incorporated by reference in their
entirety.
[0002] Throughout this application various patents and publications
are cited. The disclosures of these documents in their entireties
are hereby incorporated by reference into this application to more
fully describe the state of the art to which this invention
pertains.
FIELD OF THE INVENTION
[0003] The present invention relates to compounds, pharmaceutical
compositions comprising same and methods of use thereof for the
inhibition of certain genes, including pro-apoptotic genes. The
compounds and compositions are thus useful in the treatment of
subjects suffering from diseases or conditions and or symptoms
associated with such diseases or conditions in which gene
expression has adverse consequences. In particular embodiments, the
invention provides siRNA oligonucleotides, compositions comprising
same and methods of use thereof in the treatment of hearing loss
including acoustic trauma and presbycusis; acute renal failure
(ARF); glaucoma; acute respiratory distress syndrome (ARDS) and
other acute lung and respiratory injuries; ischemia-reperfusion
(I/R) injury following lung transplantation, organ transplantation
including lung, liver, heart, pancreas, and kidney transplantation;
nephro- and neurotoxicity; spinal cord injury; pressure sores;
age-related macular degeneration (AMD); dry eye syndrome; oral
mucositis, and chronic obstructive pulmonary disease (COPD).
BACKGROUND OF THE INVENTION
siRNAs and RNA Interference
[0004] RNA interference (RNAi) is a phenomenon involving
double-stranded (ds) RNA-dependent gene-specific
posttranscriptional silencing. Initial attempts to study this
phenomenon and to manipulate mammalian cells experimentally were
frustrated by an active, non-specific antiviral defense mechanism
which was activated in response to long dsRNA molecules (Gil et
al., Apoptosis, 2000. 5:107-114). Later, it was discovered that
synthetic duplexes of 21 nucleotide RNAs could mediate gene
specific RNAi in mammalian cells, without stimulating the generic
antiviral defense mechanisms Elbashir et al. Nature 2001,
411:494-498 and Caplen et al. PNAS 2001, 98:9742-9747). As a
result, small interfering RNAs (siRNAs), which are short
double-stranded RNAs, have been widely used to inhibit gene
expression and understand gene function.
[0005] RNA interference (RNAi) is mediated by small interfering
RNAs (siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs
(miRNAs) (Ambros V. Nature 2004, 431:350-355); and Bartel D P.
Cell. 2004 116(2):281-97). The corresponding process is commonly
referred to as specific post-transcriptional gene silencing when
observed in plants and as quelling when observed in fungi.
[0006] An siRNA is a double-stranded RNA which down-regulates or
silences (i.e. fully or partially inhibits) the expression of an
endogenous or exogenous gene/mRNA. RNA interference is based on the
ability of certain dsRNA species to enter a specific protein
complex, where they are then targeted to complementary cellular
RNAs and specifically degrades them. Thus, the RNA interference
response features an endonuclease complex containing an siRNA,
commonly referred to as an RNA-induced silencing complex (RISC),
which mediates cleavage of single-stranded RNA having a sequence
complementary to the antisense strand of the siRNA duplex. Cleavage
of the target RNA may take place in the middle of the region
complementary to the antisense strand of the siRNA duplex
(Elbashir, et al., Genes Dev., 2001, 15:188). In more detail,
longer dsRNAs are digested into short (17-29 bp) dsRNA fragments
(also referred to as short inhibitory RNAs or "siRNAs") by type III
RNAses (DICER, DROSHA, etc., (see Bernstein et al., Nature, 2001,
409:363-6 and Lee et al., Nature, 2003, 425:415-9). The RISC
protein complex recognizes these fragments and complementary mRNA.
The whole process is culminated by endonuclease cleavage of target
mRNA (McManus and Sharp, Nature Rev Genet, 2002, 3:737-47; Paddison
and Hannon, Curr Opin Mol. Ther. 2003, 5(3): 217-24). (For
additional information on these terms and proposed mechanisms, see
for example, Bernstein, et al., RNA. 2001, 7(11):1509-21;
Nishikura, Cell. 2001, 107(4):415-8 and PCT Publication No. WO
01/36646).
[0007] Studies have revealed that siRNA can be effective in vivo in
both mammals and humans. Specifically, Bitko et al., showed that
specific siRNAs directed against the respiratory syncytial virus
(RSV) nucleocapsid N gene are effective in treating mice when
administered intranasally (Bitko et al., Nat. Med. 2005,
11(1):50-55). For reviews of therapeutic applications of siRNAs see
Barik (Mol. Med. 2005, 83: 764-773) and Chakraborty (Current Drug
Targets 2007 8(3):469-82). In addition, clinical studies with short
siRNAs that target the VEGFR1 receptor in order to treat
age-related macular degeneration (AMD) have been conducted in human
patients. In studies such siRNA administered by intravitreal
(intraocular) injection was found effective and safe in 14 patients
tested (Kaiser, Am J Opthalmol. 2006 142(4):660-8).
[0008] Pro-Apoptotic Genes
[0009] Pro-apoptotic genes are generally defined as genes that play
a role in apoptotic cell death. A non-limiting list of
pro-apoptotic genes, useful in the present invention is as follows:
tumor protein p53 binding protein 2 (TP53BP2); leucine-rich repeats
and death domain containing (LRDD); cytochrome b-245, alpha
polypeptide (CYBA, p22phox); activating transcription factor 3
(ATF3); caspase 2, apoptosis-related cysteine peptidase (CASP2);
NADPH oxidase 3 (NOX3); harakiri, BCL2 interacting protein (HRK,
BID3); complement component 1, q subcomponent binding protein
(C1QBP); BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3);
mitogen-activated protein kinase 8 (MAPK8, JNK1); mitogen-activated
protein kinase 14 (MAPK14, p38); ras-related C3 botulinum toxin
substrate 1 (rho family, small GTP binding protein RAC1); glycogen
synthase kinase 3 beta (GSK3B); purinergic receptor P2X,
ligand-gated ion channel, 7 (P2RX7); transient receptor potential
cation channel, subfamily M, member 2 (TRPM2); poly (ADP-ribose)
glycohydrolase (PARG); CD38 molecule (CD38); STEAP family member 4
(STEAP4); bone morphogenetic protein 2 (BMP2); gap junction
protein, alpha 1, 43 kDa (connexin 43, GJA1); TYRO protein tyrosine
kinase binding protein (TYROBP); connective tissue growth factor
(CTGF); secreted phosphoprotein 1 (osteopontin, SPP1); reticulon 4
receptor (RTN4R); annexin A2 (ANXA2); ras homolog gene family,
member A (RHOA); and dual oxidase 1 (DUOX1).
[0010] Hearing Loss:
[0011] Chemical-Induced Ototoxicity
[0012] The ototoxic effects of various therapeutic drugs on
auditory cells and spiral ganglion neurons are often the factor
limiting their therapeutic usefulness. Commonly used ototoxic drugs
include the widely used chemotherapeutic agent cisplatin and its
analogs, aminoglycoside antibiotics, e.g. gentamycin, quinine and
its analogs, salicylate and its analogs, and loop-diuretics.
[0013] For example, antibacterial aminoglycosides such as
gentamycin, streptomycin, kanamycin, tobramycin, and the like are
known to have serious toxic side effects, particularly ototoxicity
and nephrotoxicity, which reduce their value as therapeutic agents
(see Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 6th ed., A. Goodman Gilman et al., eds; Macmillan
Publishing Co., Inc., 1980. NY, pp. 1169-71). Thus, ototoxicity is
a recognized dose-limiting side-effect of antibiotic
administration. Studies have shown that from 4 to 15% of patients
receiving one gram per day for greater than one week develop
measurable hearing loss, which gradually worsens and can lead to
permanent deafness if treatment continues.
[0014] Ototoxicity is also a serious dose-limiting side-effect for
cisplatin, a platinum coordination complex, that has proven
effective on a variety of human cancers including testicular,
ovarian, bladder, and head and neck cancer. Cisplatin
(Platinol.RTM.) damages auditory and vestibular systems.
Salicylates, such as aspirin, are the drugs most commonly used
because of their anti-inflammatory, analgesic, anti-pyretic and
anti-thrombotic effects. Unfortunately, they too have ototoxic side
effects including tinnitus ("ringing in the ears") and temporary
hearing loss. Moreover, if the drug is used at high doses for a
prolonged time, hearing impairment can become persistent and
irreversible.
[0015] Without being bound by theory, it is believed that cisplatin
drugs and other potentially ototoxic drugs induce the ototoxic
effects via apoptosis in inner ear tissue, particularly inner ear
hair cells (Zhang et al., Neuroscience 2003, 120(1):191-205; Wang
et al., J. Neuroscience, 2003, 23(24):8596-8607). In mammals,
auditory hair cells are produced only during embryonic development
and do not regenerate if lost during postnatal life. Therefore, a
loss of hair cells will result in profound and irreversible
deafness. Unfortunately, there are presently no effective therapies
to treat the cochlea and reverse this condition. Thus, an effective
therapy to prevent cell death of auditory hair cells would be of
great therapeutic value. U.S. patent application Ser. No.
11/655,610, assigned to the applicant of the present invention
relates to methods for treating hearing impairment in a subject
comprising administering to the subject a composition comprising an
effective amount of a p53 polynucleotide inhibitor, and optionally
an inhibitor of a pro-apoptotic gene.
[0016] Presbycusis
[0017] Another type of hearing loss is presbycusis, which is
hearing loss that gradually occurs in most individuals as they age.
About 30-35 percent of adults between the ages of 65 and 75 years
and 40-50 percent of people 75 and older experience hearing loss.
Accordingly, there exists a need for means to prevent, reduce or
treat the incidence and/or severity of inner ear disorders and
hearing impairments involving inner ear tissue, particularly inner
ear hair cells.
[0018] Acoustic Trauma
[0019] Acoustic trauma is a type of hearing loss that is caused by
prolonged exposure to loud noises. Without wishing to be bound to
theory, exposure to loud noise causes the hair cells on the cochlea
to become less sensitive. With more severe exposure, injury can
proceed from a loss of adjacent supporting cells to complete
disruption of the organ of Corti. Death of the sensory cell can
lead to progressive Wallerian degeneration and loss of primary
auditory nerve fibers.
[0020] Of particular interest are those adverse conditions arising
as a side-effect of therapeutic drugs including cisplatin and its
analogs, aminoglycoside antibiotics, salicylate and its analogs, or
loop diuretics. Thus, there exits a need for treatment methods
which will allow higher and thus more effective dosing, while
preventing or reducing ototoxic effects caused by these drugs.
Thus, compositions and methods are needed that provide a safe,
effective, and prolonged means for prophylactic or curative
treatment of hearing impairments related to inner ear tissue
damage, loss, or degeneration, particularly ototoxin-induced and
particularly involving inner ear hair cells. In mammals, auditory
hair cells are produced only during embryonic development and do
not regenerate if lost during postnatal life, therefore, a loss of
hair cells will result in profound and irreversible deafness.
Unfortunately, at present, there are no effective therapies to
treat the cochlea and reverse this condition. Thus, an effective
therapy to prevent cell death of auditory hair cells would be of
great therapeutic value.
[0021] Acute Renal Failure
[0022] Acute renal failure (ARF) is a clinical syndrome
characterized by rapid deterioration of renal function that occurs
within days. The principal feature of ARF is an abrupt decline in
glomerular filtration rate (GFR), resulting in the retention of
nitrogenous wastes (urea, creatinine). Worldwide, severe ARF occurs
in about 170-200 persons per million of population annually. Today,
there is no specific treatment for established ARF. Several drugs
have been found to ameliorate toxic and ischemic experimental ARF,
as manifested by lower serum creatinine levels, reduced
histological damage and faster recovery of renal function in animal
models. These include anti-oxidants, calcium channel blockers,
diuretics, vasoactive substances, growth factors, anti-inflammatory
agents and more. However, when these drugs were tested in clinical
trials no benefit was shown and their use for treating ARF has not
been approved.
[0023] In the majority of hospitalized ARF patients, ARF is caused
by acute tubular necrosis (ATN), which results from ischemic and/or
nephrotoxic insults. Renal hypoperfusion is caused by hypovolemic,
cardiogenic and septic shock, by administration of vasoconstrictive
drugs or renovascular injury. Nephrotoxins include exogenous toxins
such as contrast media and aminoglycosides as well as endogenous
toxin such as myoglobin. Recent studies suggest that apoptosis in
renal tissues is prominent in most human cases of ARF. The
principal site of apoptotic cell death is the distal nephron.
During the initial phase of ischemic injury, loss of integrity of
the actin cytoskeleton leads to flattening of the epithelium, with
loss of the brush border, loss of focal cell contacts, and
subsequent disengagement of the cell from the underlying
substratum. It has been suggested that apoptotic tubule cell death
may be more predictive of functional changes than necrotic cell
death (Komarov et al. Science. 1999, 285(5434):1733-7); Supavekin
et al. Kidney Int. 2003, 63(5):1714-24). In conclusion, there are
no currently satisfactory modes of therapy for the prevention
and/or treatment of acute renal failure, and there is a clear need
to develop novel compounds for this purpose.
[0024] Renal Transplant
[0025] Delayed Graft Function
[0026] Delayed graft function (DGF) is the most common complication
of the immediate postoperative period in renal transplantation and
results in poor graft outcome (Moreso et al. 1999. Nephrol. Dial.
Transplant. 14(4):930-35). Although the incidence and definition of
DGF vary among transplant centers, the consequences are invariable:
prolonged hospital stay, additional invasive procedures, and
additional cost to the patient and health-care system.
[0027] Acute Transplant Rejection
[0028] Graft rejection has been categorized into three subsets
depending on the onset of graft destruction: Hyperacute rejection
is the term applied to very early graft destruction, usually within
the first 48 hours. Acute rejection has an onset of several days
days to months or even years after transplantation and can involve
humoral and/or cellular mechanisms. Chronic rejection relates to
chronic alloreactive immune response.
[0029] Glaucoma
[0030] Glaucoma is one of the leading causes of blindness in the
world. It affects approximately 66.8 million people worldwide. At
least 12,000 Americans are blinded by this disease each year (Kahn
and Milton, Am J Epidemiol. 1980, 111(6):769-76). Glaucoma is
characterized by the degeneration of axons in the optic nerve head,
primarily due to elevated intraocular pressure (IOP). One of the
most common forms of glaucoma, known as primary open-angle glaucoma
(POAG), results from the increased resistance of aqueous humor
outflow in the trabecular meshwork (TM), causing IOP elevation and
eventual optic nerve damage. Mucke (IDrugs 2007, 10(1):37-41)
reviews current therapeutics, including siRNA to various targets
for the treatment of ocular diseases, for example, age-related
macular degeneration (AMD) and glaucoma.
[0031] Acute Respiratory Distress Syndrome
[0032] Acute respiratory distress syndrome (ARDS), also known as
respiratory distress syndrome (RDS) or adult respiratory distress
syndrome (in contrast with infant respiratory distress syndrome,
IRDS) is a serious reaction to various forms of injuries to the
lung. This is the most important disorder resulting in increased
permeability pulmonary edema.
[0033] ARDS is a severe lung disease caused by a variety of direct
and indirect insults. It is characterized by inflammation of the
lung parenchyma leading to impaired gas exchange with concomitant
systemic release of inflammatory mediators which cause
inflammation, hypoxemia and frequently result in failure of
multiple organs. This condition is life threatening and often
lethal, usually requiring mechanical ventilation and admission to
an intensive care unit. A less severe form is called acute lung
injury (ALI).
[0034] Acute Lung Transplant Rejection
[0035] Acute allograft rejection remains a significant problem in
lung transplantation despite advances in immunosuppressive
medication. Rejection, and ultimately early morbidity and mortality
may result from ischemia-reperfusion (I/R) injury and hypoxic
injury.
[0036] Spinal Cord Injury
[0037] Spinal cord injury or myelopathy, is a disturbance of the
spinal cord that results in loss of sensation and/or mobility. The
two most common types of spinal cord injury are due to trauma and
disease. Traumatic injuries are often due to automobile accidents,
falls, gunshots diving accidents, and the like. Diseases which can
affect the spinal cord include polio, spina bifida, tumors, and
Friedreich's ataxia.
[0038] Ischemia-Reperfusion Injury Following Organ
Transplantation
[0039] Ischemia reperfusion injury (IRI) is one of the leading
causes of death in organ allograft recipients. Significant IRI
occurs in every organ transplant from a deceased donor and in some
from live donors. It contributes to increased acute rejection and
impaired long-term allograft function. Lung transplantation, the
only definitive therapy for many patients with end stage lung
disease, has poor survival rates in all solid allograft
recipients.
[0040] Pressure Sores
[0041] Pressure sores, often known as bedsores or pressure ulcers,
are areas of damaged skin and tissue. With unrelieved pressure,
tissue ischemia can develop resulting in the accumulation of
metabolic waste in the interstitial tissue, resulting in anoxia and
cellular death. This pressure-induced ischemia also leads to
excessive tissue hypoxia, further promoting bacterial proliferation
and tissue destruction.
[0042] Age-Related Macular Degeneration
[0043] The most common cause of decreased best-corrected, vision in
individuals over 65 years of age in the United States is the
retinal disorder known as age-related macular degeneration (AMD).
The area of the eye affected by AMD is the macula, a small area in
the center of the retina, composed primarily of photoreceptor
cells. As AMD progresses, the disease is characterized by loss of
sharp, central vision. So-called "dry" AMD accounts for about
85%-90% of AMD patients and involves alterations in eye pigment
distribution, loss of photoreceptors and diminished retinal
function due to overall atrophy of cells. "Wet" AMD involves
proliferation of abnormal choroidal vessels leading to clots or
scars in the sub-retinal space. Thus, the onset of "wet" AMD occurs
because of the formation of an abnormal choroidal neovascular
network (choroidal neovascularization, CNV) beneath the neural
retina. The newly formed blood vessels are excessively leaky. This
leads to accumulation of subretinal fluid and blood leading to loss
of visual acuity. Eventually, there is total loss of functional
retina in the involved region, as a large disciform scar involving
choroids and retina forms. While dry AMD patients may retain vision
of decreased quality, wet AMD often results in blindness. (Hamdi
& Kenney, Frontiers in Bioscience, e305-314, May 2003).
[0044] Diabetic Retinopathy
[0045] Diabetic retinopathy (DR) is recognized as a retinal
vascular disorder exhibiting excess capillary permeability,
vascular closure, and proliferation of new vessels. DR occurs in
two stages: nonproliferative and proliferative. In the
nonproliferative stage the disease is characterized by a loss of
retinal capillary pericytes, thickening of the basement membrane
and development of microaneurysms, dot-blot hemorrhages, and hard
exudates. In the proliferative stage the disease is characterized
by extensive neovascularization, vessel intrusion into the
vitreous, bleeding and fibrosis with subsequent retinal traction,
which leads to severe vision impairment. U.S. Pat. No. 6,740,738
and related patents and applications to the assignee of the present
invention are directed to inhibition of RTP801 gene and protein,
involved in retinopathy.
[0046] Oral Mucositis
[0047] Oral mucositis, also referred to as a stomatitis, is a
common and debilitating side effect of chemotherapy and
radiotherapy regimens, which manifests itself as erythema and
painful ulcerative lesions of the mouth and throat. Routine
activities such as eating, drinking, swallowing, and talking may be
difficult or impossible for subjects with severe oral mucositis.
Palliative therapy includes administration of analgesics and
topical rinses.
[0048] Dry-Eye Syndrome
[0049] Dry eye syndrome is a common problem usually resulting from
a decrease in the production of tear film that lubricates the eyes.
Most patients with dry eye experience discomfort, and no vision
loss; although in severe cases, the cornea may become damaged or
infected. Wetting drops (artificial tears) may be used for
treatment while lubricating ointments may help more severe
cases.
[0050] More effective therapies to treat the above mentioned
diseases and disorders would be of great therapeutic value.
SUMMARY OF THE INVENTION
[0051] The present invention provides inhibitors of a pro-apoptotic
gene selected from the group consisting of TP53BP2, LRDD, CYBA,
ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B,
P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1,
RTN4R, ANXA2, RHOA, and DUOX1 (See Table A, infra, for genes'
details). In various embodiments the inhibitor is selected from the
group consisting of siRNA, shRNA, an aptamer, an antisense
molecule, miRNA, a ribozyme, and an antibody. In the presently
preferred embodiments the inhibitor is siRNA.
[0052] Accordingly, in one aspect the present invention provides
novel double stranded oligoribonucleotides that inhibit expression
of a pro-apoptotic gene selected from the group consisting of
TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8,
MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43,
TYROBP, CTGF, SPP1, RHOA, and DUOX1. In some embodiments the gene
is one of TP53BP2, CASP2, NOX3, RAC1, RHOA, or DUOX1. In other
embodiments the gene is one of LRDD, CYBA, HRK, BNIP3, CD38, BMP2,
or SPP1. The invention also provides pharmaceutical compositions
comprising one or more such oligoribonucleotides or a vector
capable of expressing the oligoribonucleotide. The present
invention further relates to methods for treating or preventing the
incidence or severity of various diseases or conditions in a
subject in need thereof wherein the disease or condition and/or
symptoms associated therewith is selected from the group consisting
of hearing loss, acute renal failure (ARF), glaucoma, acute
respiratory distress syndrome (ARDS) and other acute lung and
respiratory injuries, ischemia-reperfusion injury following lung
transplantation, organ transplantation including lung, liver,
heart, pancreas, and kidney transplantation, nephro- and
neurotoxicity, spinal cord injury, pressure sores, age-related
macular degeneration (AMD), dry eye syndrome, oral mucositis and
chronic obstructive pulmonary disease (COPD). Such methods involve
administering to a mammal in need of such treatment a
prophylactically or therapeutically effective amount of one or more
such compounds which inhibit or reduce expression or activity of at
least one such gene.
[0053] In one aspect the present invention provides a compound
having the structure: [0054] 5' (N).sub.x-Z 3' (antisense strand)
[0055] 3' Z'-(N').sub.y 5' (sense strand) wherein each of N and N'
is a nucleotide which may be modified or unmodified in its sugar
residue; wherein each of (N).sub.x and (N').sub.y is an
oligonucleotide in which each consecutive N or N' is joined to the
next N or N' by a covalent bond; wherein each of x and y is an
integer between 18 and 40; wherein each of Z and Z' may be present
or absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein the sequence of (N').sub.y is present within a mRNA
whose sequence is set forth in any one of SEQ ID NOS:1-48. In one
embodiment, the sequence of (N').sub.y is present within a mRNA
whose sequence is set forth in one of SEQ ID NO: 1-41 or SEQ ID
NO:46-48. The presently preferred genes are mammalian genes
selected from the group consisting of TP53BP2 (SEQ ID NOS:1-2),
LRDD (SEQ ID NOS:3-5), CYBA (SEQ ID NO:6), CASP2 (SEQ ID
NOS:10-11), NOX3 (SEQ ID NO:12), HRK (SEQ ID NO:13), BNIP3 (SEQ ID
NO:15), RAC1 (SEQ ID NOS:24-26), CD38 (SEQ ID NO:32), BMP2 (SEQ ID
NO:34), SPP1 (SEQ ID NOS:39-41), RHOA (SEQ ID NO:46), and DUOX1
(SEQ ID NOS:47-48). SEQ ID NOS represent the mRNA sequences of the
listed genes.
[0056] In some embodiments the covalent bond joining each
consecutive N or N' is a phosphodiester bond. In various
embodiments all the covalent bonds are phosphodiester bonds.
[0057] In various embodiments the compound comprises
ribonucleotides wherein x=y and each of x and y is 19, 20, 21, 22
or 23. In some embodiments x=y=23. In other embodiments x=y=19.
[0058] In some embodiments the compound is blunt ended, for example
wherein both Z and Z' are absent. In an alternative embodiment, the
compound comprises at least one 3' overhang, wherein at least one
of Z or Z' is present. Z and Z' can independently comprise one or
more covalently linked modified or non-modified nucleotides, for
example inverted dT or dA; dT, LNA, mirror nucleotide and the like.
In some embodiments each of Z and Z' are independently selected
from dT and dTdT.
[0059] In some embodiments N or N' comprises a modification in the
sugar residue of one or more ribonucleotides. In other embodiments
the compound comprises at least one ribonucleotide modified in the
sugar residue. In some embodiments the compound comprises a
modification at the 2' position of the sugar residue. In some
embodiments the modification in the 2' position comprises the
presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In
certain embodiments the 2' modification comprises methoxy moiety. A
presently preferred modification is a 2' methoxy of the sugar
residue (2'-O-methyl; 2'-O-Me; 2'-O--CH.sub.3).
[0060] In some embodiments the compound comprises modified
alternating ribonucleotides in one or both of the antisense and the
sense strands. In certain embodiments the compound comprises
modified alternating ribonucleotides in the antisense and the sense
strands. In other embodiments the compound comprises modified
alternating ribonucleotides in the antisense strand only. In
certain embodiments the middle ribonucleotide of the antisense
strand is not modified; e.g. ribonucleotide in position 10 in a
19-mer strand or position 12 in a 23-mer strand.
[0061] In additional embodiments the compound comprises modified
ribonucleotides in alternating positions wherein each N at the 5'
and 3' termini of (N).sub.x are modified in their sugar residues,
and each N' at the 5' and 3' termini of (N').sub.y are unmodified
in their sugar residues. In some embodiments, neither (N).sub.x nor
(N').sub.y are phosphorylated at the 3' and 5' termini. In other
embodiments either or both (N).sub.x and (N').sub.y are
phosphorylated at the 3' termini.
[0062] In various embodiments the compound comprises an antisense
sequence present in Tables B1-B76 (SEQ ID NOS:277 to 50970 and
50993-68654). In other embodiments the present invention provides a
mammalian expression vector comprising an antisense sequence
present in Tables B1-B76 (SEQ ID NOS:277 to 50970 and 50993-68654).
In certain embodiments N and N' are selected from the oligomers set
forth in any one of Tables C1, C2 or C3 (SEQ ID NOS: 97-276 and SEQ
ID NOS: 50971-50992).
[0063] In certain embodiments the present invention provides a
compound having the structure: [0064] 5' (N).sub.x-Z 3' (antisense
strand) [0065] 3' Z'-(N').sub.y 5' (sense strand) wherein each of N
and N' is a ribonucleotide which may be modified or unmodified in
its sugar residue; wherein each of (N).sub.x and (N').sub.y is an
oligomer in which each consecutive N or N' is joined to the next N
or N' by a covalent bond; wherein each of x and y is an integer
between 19 and 40; wherein each of Z and Z' may be present or
absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein the sequence of (N').sub.y is present within an mRNA
whose sequence is set forth in one of SEQ ID NO:46, SEQ ID NO:1-41
or SEQ ID NO:47-48.
[0066] In certain embodiments the present invention provides a
compound having the structure: [0067] 5' (N).sub.x-Z 3' (antisense
strand) [0068] 3' Z'-(N').sub.y 5' (sense strand) wherein each of N
and N' is a ribonucleotide which may be modified or unmodified in
its sugar residue; wherein each of (N).sub.x and (N').sub.y is an
oligomer in which each consecutive N or N' is joined to the next N
or N' by a covalent bond; wherein each of x and y is an integer
between 19 and 40; wherein each of Z and Z' may be present or
absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein the sequence of (N).sub.x and (N').sub.y is set forth
in any one of SEQ ID NOS: 277 to 50970 and 50993-68654.
[0069] In certain preferred embodiments, each of (N).sub.x and
(N').sub.y is set forth in any one of SEQ ID NOS: 97-276 (Tables
C1, C2) and SEQ ID NOS: 50971-50992 (Table C3).
[0070] In certain embodiments the present invention provides a
compound having the structure: [0071] 5' (N).sub.x 3' antisense
strand [0072] 3' (N').sub.y 5' sense strand wherein each of N and
N' is a nucleotide which may be modified or unmodified in its sugar
residue; wherein x=y=19 and the sequence of (N).sub.x and
(N').sub.y are fully complementary; wherein alternating
ribonucleotides in (N).sub.x and (N').sub.y are modified to result
in a 2'-O-methyl modification in the sugar residue of the
ribonucleotides; wherein the ribonucleotides at the 5' and 3'
termini of (N).sub.x are modified; wherein the ribonucleotides at
the 5' and 3' termini of (N').sub.y are unmodified; wherein
(N).sub.x and (N').sub.y are phosphorylated or non-phosphorylated
at the 3' and 5' termini; and wherein each of N and N' is selected
from the group of oligomers set forth in Tables B1-B25 or B76 (SEQ
ID NOS:277 to 15114 and SEQ ID NOS 68647-68654). In certain
embodiments the N and N' are selected from the oligomers set forth
in any one of Tables C1 and C3 (SEQ ID NOS: 97-266 (Table C1) and
SEQ ID NOS: 50971-50992 (Table C3)).
[0073] In certain embodiments the present invention provides a
compound having the structure
[0074] 5' (N)x 3' antisense strand
[0075] 3' (N')y 5' sense strand
wherein x=y=23 and the sequence of (N).sub.x and (N').sub.y are
fully complementary; wherein alternating ribonucleotides in
(N).sub.x and (N').sub.y are modified to result in a 2'-O-methyl
modification in the sugar residue of the ribonucleotides; wherein
the ribonucleotides at the 5' and 3' termini of (N).sub.x are
modified; wherein the ribonucleotides at the 5' and 3' termini of
(N').sub.y are unmodified; wherein (N).sub.x and (N').sub.y are
phosphorylated or non-phosphorylated at the 3' and 5' termini; and
wherein each of N and N' is selected from the group of oligomers
set forth in Tables B51-B75 (SEQ ID NOS:30939 to 68646). In certain
embodiments the N and N' are selected from the oligomers set forth
in Table C2 (SEQ ID NOS: 267-276).
[0076] In a second aspect the present invention provides a
pharmaceutical composition comprising one or more compounds of the
present invention, in an amount effective to inhibit human gene
expression wherein the gene is selected from the group consisting
of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3,
MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2,
CX43, TYROBP, CTGF, SPP1, RTN4R, ANXA2 RHOA, and DUOX1; and a
pharmaceutically acceptable carrier.
[0077] In another aspect, the present invention relates to a method
for the treatment of a subject in need of treatment for a disease
or disorder or symptoms associated with the disease or disorder,
associated with the expression of a gene selected from TP53BP2,
LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14,
RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP,
CTGF, SPP1, RTN4R, ANXA2, RHOA, and DUOX1, comprising administering
to the subject an amount of an siRNA which reduces or inhibits
expression of at least one of those pro-apoptotic genes.
[0078] More specifically, the present invention provides methods
and compositions useful in treating a subject suffering from acute
renal failure (ARF), hearing loss, glaucoma, acute respiratory
distress syndrome (ARDS) and other acute lung and respiratory
injuries, injury (e.g. ischemia-reperfusion injury) in organ
transplant including lung, kidney, bone marrow, heart, pancreas,
cornea or liver transplantation, nephrotoxicity, spinal cord
injury, pressure sores, dry eye syndrome, oral mucositis and
chronic obstructive pulmonary disease (COPD). The methods of the
invention comprise administering to the subject one or more
inhibitory compounds which reduces, inhibits or down-regulate
expression of a gene selected from the group consisting of TP53BP2,
LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14,
RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP,
CTGF, SPP1, RTN4R, ANXA2, RHOA, and DUOX1; and in particular siRNA
in a therapeutically effective dose so as to thereby treat the
patient.
[0079] In one embodiment, the present invention provides methods of
treating a disease or condition selected from hearing loss, acute
renal failure, glaucoma, acute respiratory distress syndrome, an
acute lung injury, organ transplantation rejection,
ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal
cord injury, pressure sores, osteoarthritis, dry eye syndrome and
chronic obstructive pulmonary disease (COPD), in a subject in need
thereof, comprising administering to the subject an oligonucleotide
which inhibits expression of a gene whose mRNA sequence is set
forth in any one of SEQ ID NOS:1-41 or 46-48, in an amount
effective to treat the disease or condition.
[0080] In one embodiment, the present invention provides methods of
treating acute renal failure in a subject in need thereof,
comprising administering to the subject an oligonucleotide which
inhibits expression of any one of TP53BP (whose mRNA sequence is
set forth in SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set
forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in
SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID
NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID
NO:15), or RAC1 (whose mRNA sequence is set forth in SEQ ID
NO:24-26) in an amount effective to treat the acute renal
failure.
[0081] In one embodiment, the present invention provides methods of
treating spinal-cord injury in a subject in need thereof,
comprising administering to the subject an oligonucleotide which
inhibits expression of any one of RHOA (whose mRNA sequence is set
forth in SEQ ID NO:46); TP53BP (whose mRNA sequence is set forth in
SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID
NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6),
CASP2 (whose mRNA sequence is set forth in SEQ ID NO: 10-11), BNIP3
(whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose
mRNA sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA
sequence is set forth in SEQ ID NO:32) or BMP2 (whose mRNA sequence
is set forth in SEQ ID NO:34) in an amount effective to treat the
spinal cord injury.
[0082] In one embodiment, the present invention provides methods of
treating hearing loss in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of any one of TP53BP (whose mRNA sequence is set forth
in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ
ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6),
CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), NOX3
(whose mRNA sequence is set forth in SEQ ID NO:12), BNIP3 (whose
mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose mRNA
sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA
sequence is set forth in SEQ ID NO:32) or BMP2 (whose mRNA sequence
is set forth in SEQ ID NO:34) in an amount effective to treat the
hearing loss.
[0083] In one embodiment, the present invention provides methods of
treating a disease or condition selected from chronic obstructive
pulmonary disease, acute respiratory distress syndrome and acute
lung injury in a subject in need thereof, comprising administering
to the subject an oligonucleotide which inhibits expression of any
one of LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5);
CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2
(whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3 (whose
mRNA sequence is set forth in SEQ ID NO: 15), RAC1 (whose mRNA
sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA
sequence is set forth in SEQ ID NO:32), BMP2 (whose mRNA sequence
is set forth in SEQ ID NO:34), SPP1 (whose mRNA sequence is set
forth in SEQ ID NOS:39-41) or DUOX (whose mRNA sequence is set
forth in SEQ ID NOS:47-48) in an amount effective to treat the
disease or condition.
[0084] In one embodiment, the present invention provides methods of
treating a subject who is an organ transplant recipient or organ
transplant donor comprising administering to the subject an
oligonucleotide which inhibits expression of any one of TP53BP
(whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose
mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA
sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence
is set forth in SEQ ID NO: 10-11), BNIP3 (whose mRNA sequence is
set forth in SEQ ID NO:15), RAC1 (whose mRNA sequence is set forth
in SEQ ID NO:24-26), GSK3B (whose mRNA sequence is set forth in SEQ
ID NO:27), P2RX7 (whose mRNA sequence is set forth in SEQ ID
NO:28), TRPM2 (whose mRNA sequence is set forth in SEQ ID NO:30) or
PARG (whose mRNA sequence is set forth in SEQ ID NO:31), in an
amount effective to prevent rejection of the transplant.
[0085] In one embodiment, the present invention provides methods of
treating glaucoma in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of any one of TP53BP (whose mRNA sequence is set forth
in. SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set forth in SEQ
ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6),
CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3
(whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose
mRNA sequence is set forth in SEQ ID NO:24-26), SPP1 (whose mRNA
sequence is set forth in SEQ ID NOS:39-41), or RHOA (whose mRNA
sequence is set forth in SEQ ID NO:46) in an amount effective to
treat glaucoma.
[0086] In one embodiment, the present invention provides methods of
treating oral mucositis in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of any one of TP53BP (whose mRNA sequence is set forth
in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ
ID NOS:3-5); CASP2 (whose mRNA sequence is set forth in SEQ ID NOS:
10-11) or ATF3 (whose mRNA sequence is set forth in SEQ ID NOS:7-9)
in an amount effective to treat oral mucositis.
[0087] In one embodiment, the present invention provides methods of
treating osteoarthritis in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of SPP1 (whose mRNA sequence is set forth in SEQ ID
NOS:39-41), in an amount effective to treat osteoarthritis.
[0088] In one embodiment, the present invention provides methods of
treating dry eye syndrome in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of any one of TP53BP (whose mRNA sequence is set forth
in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ
ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6),
CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3
(whose mRNA sequence is set forth in SEQ ID NO:15), or RAC1 (whose
mRNA sequence is set forth in SEQ ID NO:24-26) in an amount
effective to treat the syndrome.
[0089] In one embodiment, the present invention provides methods of
treating a pressure sore in a subject in need thereof, comprising
administering to the subject an oligonucleotide which inhibits
expression of any one of CIQBP (whose mRNA sequence is set forth in
SEQ ID NO: 14), RAC1 (whose mRNA sequence is set forth in SEQ ID
NOS:24-26), GSK3B (whose mRNA sequence is set forth in SEQ ID
NO:27), P2RX7 (whose mRNA sequence is set forth in SEQ ID NO:28),
TRPM2 (whose mRNA sequence is set forth in SEQ ID NO:30), PARG
(whose mRNA sequence is set forth in SEQ ID NO:31), CD38 (whose
mRNA sequence is set forth in SEQ ID NO:32), STEAP4 (whose mRNA
sequence is set forth in SEQ ID NO:33), BMP2 (whose mRNA sequence
is set forth in SEQ ID NO:34), GJA1 (whose mRNA sequence is set
forth in SEQ ID NO:35), or TYROBP (whose mRNA sequence is set forth
in SEQ ID NOS:36-37) in an amount effective to treat the pressure
sore.
[0090] Lists of 19-mer, 21-mer and 23-mer sense and corresponding
antisense sequences useful in preparation of siRNA compounds are
set forth in SEQ ID NOS:277 to 50970 and 50993-68654, shown as
sequence pairs in Table B, Tables B1-B76.
[0091] In one embodiment, the present invention provides methods of
treating a disease or condition selected from hearing loss, acute
renal failure, glaucoma, acute respiratory distress syndrome, an
acute lung injury, organ transplantation rejection,
ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal
cord injury, pressure sores, osteoarthritis and chronic obstructive
pulmonary disease (COPD), in a subject in need thereof, comprising
administering to the subject an antibody which inhibits a
polypeptide whose sequence is set forth in any one of SEQ ID NOS:
90-93 in an amount effective to treat the disease or condition.
[0092] In one embodiment, the present invention provides a
pharmaceutical composition comprising an antibody which inhibits a
polypeptide whose sequence is set forth in any one of SEQ ID NOS:
90-93, in an amount effective to inhibit the polypeptide, and a
pharmaceutically acceptable carrier.
[0093] In one embodiment, the present invention relates to the use
of a therapeutically effective dose of an oligonucleotide for the
preparation of a composition for treating a subject suffering from
a disease or condition selected from hearing loss, acute renal
failure, glaucoma, acute respiratory distress syndrome, an acute
lung injury, organ transplantation rejection, ischemia-reperfusion
injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure
sores, osteoarthritis and chronic obstructive pulmonary disease
(COPD), wherein the oligonucleotide inhibits expression of a gene
whose mRNA sequence is set forth in any one of SEQ ID NOS: 1-41 or
46-48.
[0094] In one embodiment, the present invention relates to the use
of a therapeutically effective dose of an antibody for the
preparation of a composition for treating a subject suffering from
a disease or condition selected from hearing loss, acute renal
failure, glaucoma, acute respiratory distress syndrome, an acute
lung injury, organ transplantation rejection, ischemia-reperfusion
injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure
sores, osteoarthritis and chronic obstructive pulmonary disease
(COPD), wherein the antibody inhibits a polypeptide whose sequence
is set forth in any one of SEQ ID NOS: 90-93.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The present invention relates generally to compounds which
down-regulate expression of various genes including pro-apoptotic
genes, particularly to novel small interfering RNAs (siRNAs), and
to the use of these novel siRNAs in the treatment of various
diseases and medical conditions. Particular diseases and conditions
to be treated are hearing loss, acute renal failure (ARF),
glaucoma, acute respiratory distress syndrome (ARDS) and other
acute lung and respiratory injuries, ischemia-reperfusion injury
following lung transplantation, organ transplantation including
lung, liver, heart, bone marrow, pancreas, cornea and kidney
transplantation, spinal cord injury, pressure sores, age-related
macular degeneration (AMD), dry eye syndrome, oral mucositis and
chronic obstructive pulmonary disease (COPD). Other indications
include chemical-induced nephrotoxicity and chemical-induced
neurotoxicity, for example toxicity induced by cisplatin and
cisplatin-like compounds, by aminoglycosides, by loop diuretics,
and by hydroquinone and their analogs.
[0096] Lists of preferred siRNA to be used in the present invention
are provided in Table B, SEQ ID NOS:277 to 50970 and 50993-68654.
For each gene there is a separate list of 19-mer, 21-mer and 23-mer
sequences, which are prioritized based on their score in the
proprietary algorithm as the best sequences for targeting the human
gene expression. 21- or 23-mer siRNA sequences can also be
generated by 5' and/or 3' extension of the 19-mer sequences
disclosed herein. Such extension is preferably complementary to the
corresponding mRNA sequence. Certain 23-mer oligomers were devised
by this method where the order of the prioritization is the order
of the corresponding 19-mer.
[0097] Methods, molecules and compositions which inhibit the
pro-apoptotic genes of the invention are discussed herein at
length, and any of said molecules and/or compositions may be
beneficially employed in the treatment of a subject suffering from
any of said conditions. Where aspects or embodiments of the
invention are described in terms of Markush groups or other
grouping of alternatives, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the group.
[0098] Proapoptotic Genes
[0099] A "pro-apoptotic gene" is generally defined as a gene that
plays a positive role in apoptotic cell death. For the purposes of
this application, preferred pro-apoptotic genes and the preferred
uses of siRNA or other inhibitors of these pro-apoptotic genes are
listed in Table A below. It should be noted that whereas the
compounds of the present invention are useful in treating the
listed indications, certain compounds may be more effective in a
particular tissue than in another. Those preferred indications are
listed in Table A, hereinbelow.
TABLE-US-00001 TABLE A Preferred genes of the present invention
Preferred diseases/ No. Gene Full name and Human Gene ID conditions
1 TP53BP2 tumor protein p53 binding protein, 2 ARF, nephrotoxicity,
gi|112799848|ref|NM_001031685.2 (SEQ ID glaucoma, dry eye, kidney
NO: 1) transplantation gi|112799845|ref|NM_005426.2 (SEQ ID NO: 2):
hearing loss, acoustic trauma, oral mucositis 2 LRDD leucine-rich
repeats and death domain containing ARF, glaucoma, hearing
gi|61742781|ref|NM_018494.3 (SEQ ID NO: 3) loss, spinal-cord
injury, oral gi|61742783|ref|NM_145886.2 (SEQ ID NO: 4) mucositis;
kidney or lung gi|61742785|ref|NM_145887.2 (SEQ ID NO: 5)
transplantation, and ischemic-reperfusion lung injury, dry eye 3
CYBA cytochrome b-245, alpha polypeptide ARF, ARDS, hearing loss,
gi|68509913|ref|NM_000101.2|(SEQ ID NO: 6) spinal-cord injury,
glaucoma, kidney transplantation, lung transplantation and
ischemic-reperfusion lung injury 4 ATF3 activating transcription
factor 3 ARF, glaucoma, hearing gi|95102484|ref|NM_001030287.2|
(SEQ ID loss, spinal-cord injury, oral NO: 7) mucositis
gi|71902534|ref|NM_001674.2|(SEQ ID NO: 8)
gi|95102480|ref|NM_004024.4|(SEQ ID NO: 9) 5 CASP2 caspase 2,
apoptosis-related cysteine peptidase ARF, glaucoma, hearing
gi|39995058|ref|NM_032982.2 (SEQ ID NO: 10) loss, spinal-cord
injury, gi|39995060|ref|NM_032983.2 (SEQ ID NO: 11) kidney
transplantation, lung transplantation and ischemic-reperfusion lung
injury, oral mucositis, dry eye 6 NOX3 NADPH oxidase 3 Hearing
loss, acoustic gi|11136625|ref|NM_015718.1 gi| (SEQ ID trauma NO:
12) 7 HRK harakiri, BCL2 interacting protein (contains only BH3
domain) ARF, glaucoma, hearing gi|4504492|ref|NM_003806.1 (SEQ ID
NO: 13) loss, spinal-cord injury, ARDS 8 C1QBP complement component
1, q subcomponent ARF, COPD, hearing loss, binding protein
spinal-cord injury, pressure gi|28872801|ref|NM_001212.3 (SEQ ID
NO: 14) sores 9 BNIP3 BCL2/adenovirus E1B 19 kDa interacting ARF,
glaucoma, hearing protein 3 loss, acoustic trauma,
gi|7669480|ref|NM_004052.2 (SEQ ID NO: 15) spinal-cord injury,
ARDS, COPD, lung transplantation and ischemic-reperfusion lung
injury 10 MAPK8 mitogen-activated protein kinase 8 ARF, glaucoma,
hearing gi|20986493|ref|NM_002750.2(SEQ ID NO: 16) loss,
spinal-cord injury, gi|20986522|ref|NM_139049.1(SEQ ID NO: 17) ARDS
gi|20986518|ref|NM_139046.1(SEQ ID NO: 18)
gi|20986520|ref|NM_139047.1| (SEQ ID NO: 19) 11 MAPK14
mitogen-activated protein kinase 14 ARF, glaucoma, hearing
gi|20986511|ref|NM_139012.1(SEQ ID NO: 20) loss, spinal-cord
injury, gi|20986515|ref|NM_139014.1(SEQ ID NO: 21) ARDS
gi|4503068|ref|NM_001315.1(SEQ ID NO: 22)
gi|20986513|ref|NM_139013.1(SEQ ID NO: 23) 12 Rac1 ras-related C3
botulinum toxin substrate 1 (rho ARF, glaucoma, hearing family,
small GTP binding protein) loss, acoustic trauma,
gi|38505164|ref|NM_198829.1(SEQ ID NO: 24) spinal-cord injury,
ARDS, gi|156071511|ref|NM_018890.3(SEQ ID NO: 25) lung
transplantation and gi|156071503|ref|NM_006908.4(SEQ ID NO: 26)
ischemic-reperfusion lung injury, AMD, pressure sores 13 GSK3B
glycogen synthase kinase 3 beta ARF, hearing loss, spinal-
gi|21361339|ref|NM_002093.2(SEQ ID NO: 27) cord injury, COPD,
pressure sores, ARDS, transplantation 14 P2RX7 purinergic receptor
P2X, ligand-gated ion ARF, hearing loss, spinal- channel, 7 cord
injury, COPD, gi|34335273|ref|NM_002562.4 (SEQ ID NO: 28) pressure
sores, ARDS, transplantation 15 TRPM2 transient receptor potential
cation channel, ARF, hearing loss, spinal- subfamily M, member 2
cord injury, COPD, gi|6790681l|ref|NM_001001188.3 (SEQ ID pressure
sores, ARDS, NO: 29) transplantation gi|67906812|ref|NM_003307.3
(SEQ ID NO: 30) 16 PARG poly (ADP-ribose) glycohydrolase ARF,
hearing loss, spinal- gi|70610135|ref|NM_003631.2 (SEQ ID NO: 31)
cord injury, COPD, pressure sores, ARDS, transplantation 17 CD38
CD38 molecule ARF, hearing loss, spinal-
gi|38454325|ref|NM_001775.2 (SEQ ID NO: 32) cord injury, COPD,
pressure sores 18 STEAP4 STEAP family member 4 ARF, hearing loss,
spinal- gi|13375867|ref|NM_024636.1 (SEQ ID NO: 33) cord injury,
COPD, pressure sores 19 BMP2 bone morphogenetic protein 2 ARF,
hearing loss, spinal- gi|80861484|ref|NM_001200.2(SEQ ID NO: 34)
cord injury, COPD, pressure sores 20 GJA1 gap junction protein,
alpha 1, 43 kDa ARF, hearing loss, spinal-
gi|4755136|ref|NM_000165.2(SEQ ID NO: 35) cord injury, COPD,
pressure sores 21 TYROBP TYRO protein tyrosine kinase binding
protein ARF, hearing loss, spinal- gi|38157998|ref|NM_003332.2(SEQ
ID NO: 36) cord injury, COPD, gi|38158004|ref|NM_198125.1 (SEQ ID
NO: 37) pressure sores 22 CTGF connective tissue growth factor ARF,
hearing loss, spinal- gi|4503122|ref|NM_001901.1(SEQ ID NO: 38)
cord injury, COPD 23 SPP1 secreted phosphoprotein 1 ARF, glaucoma,
ARDS, gi|91206461|ref|NM_001040058.1 SEQ ID osteoarthritis NO: 39)
gi|38146097|ref|NM_000582.2 (SEQ ID NO: 40)
gi|91598938|ref|NM_001040060.1 (SEQ ID NO: 41) 24 RTN4R reticulon 4
receptor spinal-cord injury gi|47519383|ref|NM_023004.5 (SEQ ID NO:
42) 25 ANXA2 annexin A2 ARF, hearing loss, spinal-
gi|50845387|ref|NM_001002858.1| (SEQ ID cord injury, COPD NO: 43)
gi|50845389|ref|NM_004039.2| (SEQ ID NO: 44)
gi|4757756|ref|NP_004030.1 (SEQ ID NO: 45) 26 RHOA ras homolog gene
family member A spinal-cord injury, gi|50593005|ref|NM_001664.2(SEQ
ID NO: 46)| glaucoma 27 DUOX1 dual oxidase 1 Acute Respiratory
Distress gi|28872749|ref|NM_017434.3(SEQ ID NO: 47) Syndrome, COPD
gi|28872750|ref|NM_175940.1(SEQ ID NO: 48) ARDS: acute respiratory
distress syndrome; AMD: age-related macular degeneration; COPD:
Chronic obstructive pulmonary disease; ARF: acute renal failure
[0100] Table A comprises the polynucleotide SEQ ID NOS of the mRNA
of the genes targeted by the compounds of the present invention
(set forth as SEQ ID NOS: 1-48). The corresponding polypeptides are
set forth in SEQ ID NOS:49-96. The genes listed in Table A, supra,
are described in more detail as follows:
[0101] (1) Tumor Protein p53 Binding Protein, 2 (TP53BP2):
[0102] Gene aliases: BBP; 53BP2; ASPP2; p53BP2; PPP1R13A, A1746547,
X98550
[0103] This gene encodes a member of the ASPP
(apoptosis-stimulating protein of p53) family of p53 interacting
proteins. The corresponding protein contains four ankyrin repeats
and an SH3 domain involved in protein-protein interactions. It is
localized to the perinuclear region of the cytoplasm, and regulates
apoptosis and cell growth through interactions with other
regulatory molecules including members of the p53 family. Multiple
transcript variants encoding different isoforms have been found for
this gene. The polynucleotide sequences of human TP53BP2 mRNA
transcriptional variants 1 and 2 are SEQ ID NOS:1 and 2,
respectively, and the corresponding polypeptide sequence are set
forth in SEQ ID NOS:49-50, respectively.
[0104] (2) Leucine-Rich Repeats and Death Domain Containing
(LRDD)
[0105] Gene aliases: PIDD; MGC16925; DKFZp434D229, 1200011D09Rik,
AU042446
[0106] The protein encoded by this gene contains a leucine-rich
repeat and a death domain. This protein has been shown to interact
with other death domain proteins, such as Fas (TNFRSF6)-associated
via death domain (FADD) and MAP-kinase activating death
domain-containing protein (MADD), and thus may function as an
adaptor protein in cell death-related signaling processes. The
expression of the mouse counterpart of this gene has been found to
be positively regulated by the tumor suppressor p53 and to induce
cell apoptosis in response to DNA damage, which suggests a role for
this gene as an effector of p53-dependent apoptosis. Three
alternatively spliced transcript variants encoding distinct
isoforms have been reported. The polynucleotide sequence of human
LRDD transcriptional variants 2, 1 and 3 are set forth in SEQ ID
NOS: 3-5, respectively, and the corresponding polypeptide sequence
are set forth in SEQ ID NOS:51-53, respectively.
[0107] International Patent Publication WO 01/18037 discloses the
LRDD polynucleotide and polypeptide sequences. International Patent
Publication WO 03/087368 teaches compositions and methods for
inhibiting genes.
[0108] (3) Cytochrome b-245, Alpha Polypeptide (CYBA)
[0109] Gene aliases: cytochrome b light chain; cytochrome b(558)
alpha-subunit; cytochrome b, alpha polypeptide; flavocytochrome
b-558 alpha polypeptide; p22-phox.
[0110] Cytochrome b is comprised of a light chain (alpha) and a
heavy chain (beta). This gene encodes the light, alpha subunit
which has been proposed as a primary component of the microbicidal
oxidase system of phagocytes. Mutations in this gene are associated
with autosomal recessive chronic granulomatous disease (CGD), that
is characterized by the failure of activated phagocytes to generate
superoxide, which is important for the microbicidal activity of
these cells. The polynucleotide sequence of human CYBA mRNA is
depicted as SEQ ID NO:6, and the corresponding polypeptide sequence
is set forth in SEQ ID NO:54.
[0111] International Patent Publication WO 2005/103297 teaches
modulation of p22phox activity.
[0112] (4) Activating Transcription Factor 3 (ATF3)
[0113] Gene aliases: ATF3deltaZip2; ATF3deltaZip2c; ATF3deltaZip3,
LRG-21, LRF-1
[0114] ATF3 is a member of the mammalian activation transcription
factor/cAMP responsive element-binding (CREB) protein family of
transcription factors. Multiple transcript variants encoding two
different isoforms have been found for this gene. The longer
isoform represses rather than activates transcription from
promoters with ATF binding elements. The shorter isoform
(deltaZip2) lacks the leucine zipper protein-dimerization motif and
does not bind to DNA, and it stimulates transcription presumably by
sequestering inhibitory co-factors away from the promoter. It is
possible that alternative splicing of the ATF3 gene may be
physiologically important in the regulation of target genes. The
polynucleotide sequences of human ATF3 transcriptional variants 3,
1 and 2 are set forth in SEQ ID NOS: 7-9, respectively, and the
corresponding polypeptide sequences are set forth in SEQ ID
NOS:55-57, respectively.
[0115] US Patent Publication 2003/0125277 teaches antisense to
ATF3. International Patent Publication WO 2005/103297 relates to
the methods of treating neuronal disease.
[0116] (5) Caspase 2, Apoptosis-Related Cysteine Peptidase (Neural
Precursor Cell Expressed, Developmentally Down-Regulated 2
(CASP2)
[0117] Gene aliases: CASP-2, ICH-1L, ICH-1L/IS, ICH1, NEDD2; ICH-1
protease; NEDD2 apoptosis regulatory gene; caspase 2,
apoptosis-related cysteine protease.
[0118] This gene encodes a protein, which is a member of the
cysteine-aspartic acid protease (caspase) family. Sequential
activation of caspases plays a central role in the execution-phase
of cell apoptosis. Caspases exist as inactive proenzymes, which
undergo proteolytic processing at conserved aspartic residues to
produce two subunits, large and small, that dimerize to form the
active enzyme. The proteolytic cleavage of this protein is induced
by a variety of apoptotic stimuli. Alternative splicing of this
gene results in multiple transcript variants that encode different
isoforms. The polynucleotide sequences of human CASP2
transcriptional variants 1 and 3 are set forth in SEQ ID NOS:10-11,
respectively, and the corresponding polypeptide sequences are set
forth in SEQ ID NOS:58-59, respectively.
[0119] U.S. Pat. No. 6,083,735 relates to the alternative splicing
products of Casp2. U.S. Pat. Nos. 5,929,042 and 7,223,856 disclose
specific Casp2 antisense compounds for the treatment of
neurodegenerative disorders. International Patent Publication WO
02/024720 teaches Casp2 antisense. International Patent Publication
WO 02/034201 discloses methods of treating diabetic retinopathy; WO
03/05821 relates to the inhibition of apoptosis related genes; WO
2004/009797 teaches Casp2 antisense; and WO 2004/103389 relates to
methods for preventing cell death.
[0120] (6) NADPH Oxidase 3 (NOX3)
[0121] Gene aliases: GP91-3, MGC124289, het, nmf250; NADPH oxidase
catalytic subunit-like 3, NADPH oxidase 1; head-tilt
[0122] NADPH oxidases, such as NOX3, are plasma membrane-associated
enzymes found in many cell types. They catalyze the production of
superoxide by a 1-electron reduction of oxygen, using NADPH as the
electron donor. The polynucleotide sequence of human NOX3 mRNA is
set forth in SEQ ID NO: 12, and the corresponding polypeptide
sequence is set forth in SEQ ID NO:60.
[0123] International Patent Publication WO 2005/119251 relates to a
method of treating hearing loss.
[0124] (7) Harakiri, BCL2 Interacting Protein (Contains Only BH3
Domain) (HRK)
[0125] Gene aliases: DP5, Bid3; BCL2-interacting protein; activator
of apoptosis Hrk; BH3 interacting (with BCL2 family) domain,
apoptosis agonist.
[0126] As an activator of apoptosis, Hrk regulates apoptosis
through interaction with death-repressor proteins Bcl-2 and
Bcl-X(L). The HRK protein lacks significant homology to other BCL2
family members except for an 8-amino acid region that was similar
to the BCL2 homology domain-3 (BH3) motif of BIK. HRK interacts
with BCL2 and BCLXL via the BH3 domain, but not with the
death-promoting BCL2-related proteins BAX, BAK, or BCLXS. HRK
localizes to membranes of intracellular organelles in a pattern
similar to that previously reported for BCL2 and BCLXL. The
polynucleotide sequence of human HRK mRNA is set forth in SEQ ID
NO: 13 and the corresponding polypeptide sequence is set forth in
SEQ ID NO.61.
[0127] (8) Complement Component 1, q Subcomponent Binding Protein
(C1QBP)
[0128] Gene aliases: GC1QBP, HABP1, SF2p32, gC1Q-R, gC1qR, p32,
RP23-83113.1, AA407365, AA986492, D11Wsu182e, MGC91723; C1q
globular domain-binding protein; hyaluronan-binding protein 1;
splicing factor SF2-associated protein.
[0129] The human complement subcomponent C1q associates with C1r
and C1s in order to yield the first component of the serum
complement system. The protein encoded by this gene is known to
bind to the globular heads of C1q molecules and inhibit C1
activation. This protein has also been identified as the p32
subunit of pre-mRNA splicing factor SF2, as well as a hyaluronic
acid-binding protein. The polynucleotide sequence of human C1QBP
mRNA is set forth in SEQ ID NO: 14 and the corresponding
polypeptide sequence is set forth in SEQ ID NO:62.
[0130] (9) BCL2/Adenovirus E1B 19 kDa Interacting Protein 3
(BNIP3)
[0131] Gene aliases: NIP3, MGC93043; BCL2/adenovirus E1B 19
kD-interacting protein 3, BCL2/adenovirus E1B 19 kDa-interacting
protein 3 nuclear gene for mitochondrial product.
[0132] This gene is a member of the BCL2/adenovirus E1B 19
kd-interacting protein (BNIP) family. It interacts with the E1B 19
kDa protein, which is responsible for the protection of
virally-induced cell death, as well as E1B 19 kDa-like sequences of
BCL2, also an apoptotic protector. This gene contains a BH3 domain
and a transmembrane domain, which have been associated with
pro-apoptotic function. The dimeric mitochondrial protein encoded
by this gene is known to induce apoptosis, even in the presence of
BCL2. The polynucleotide sequence of human BNIP3 mRNA is set forth
in SEQ ID NO: 15 and the corresponding polypeptide sequence is set
forth in SEQ ID NO:63.
[0133] U.S. Pat. No. 5,858,678 relates to the BNIP3 polynucleotide
and polypeptide sequences. International Patent Publication WO
2004/009780 discloses methods of preventing ischemia induced cell
damage.
[0134] (10) Mitogen-Activated Protein Kinase 8 (MAPK8)
[0135] Gene aliases: JNK; JNK1; PRKM8; SAPK1; JNK1A2; JNK21B1/2;
JNK1 alpha protein kinase; JNK1 beta protein kinase; c-Jun
N-terminal kinase 1; mitogen-activated protein kinase 8 transcript
variant 2; protein kinase JNK1; stress-activated protein kinase
JNK1.
[0136] The protein encoded by this gene is a member of the MAP
kinase family. MAP kinases act as an integration point for multiple
biochemical signals, and are involved in a wide variety of cellular
processes such as proliferation, differentiation, transcription
regulation and development. This kinase is activated by various
cell stimuli, and targets specific transcription factors, and thus
mediates immediate-early gene expression in response to cell
stimuli. The activation of this kinase by tumor-necrosis factor
alpha (TNF-.alpha.) is found to be required for TNF-.alpha. induced
apoptosis. This kinase is also involved in UV radiation induced
apoptosis, which is thought to be related to cytochrome c-mediated
cell death pathway. Studies of the mouse counterpart of this gene
suggested its role in T cell proliferation, apoptosis and
differentiation. Four alternatively spliced transcript variants
encoding distinct isoforms have been reported. The polynucleotide
sequence of MAPK8 transcriptional variants 2, 1, 3 and 4 are set
forth in SEQ ID NOS:16-19 respectively and the corresponding
polypeptide sequences are set forth in SEQ ID NO:64-67.
[0137] International Patent Publication WO 99/09214 and U.S. Pat.
No. 5,877,309 disclose antisense to JNK family members.
[0138] (11) Mitogen-Activated Protein Kinase 14 (MAPK14)
[0139] Gene aliases: CSBP1; CSBP2; CSPB1; EXIP; Mxi2; PRKM14;
PRKM15; RK; SAPK2A; p38; p38ALPHA; MGC102436; p38MAPK; CSBP; Exip;
Hog; MGC105413; p38Hog; Csaids binding protein; MAP kinase Mxi2;
MAX-interacting protein 2; cytokine suppressive anti-inflammatory
drug binding protein; p38 MAP kinase; p38 mitogen activated protein
kinase; p38alpha Exip; stress-activated protein kinase 2A, tRNA
synthetase cofactor p38.
[0140] The protein encoded by this gene is a member of the MAP
kinase family, which act as an integration point for multiple
biochemical signals, and are involved in a wide variety of cellular
processes such as proliferation, differentiation, transcription
regulation and development. This kinase is activated by various
environmental stresses and proinflammatory cytokines. The
activation requires its phosphorylation by MAP kinase kinases
(MKKs), or its autophosphorylation triggered by its interaction
with MAP3K7IP1/TAB1 protein. The substrates of this kinase include
transcription regulator ATF2, MEF2C, and MAX, cell cycle regulator
CDC25B, and tumor suppressor p53, which suggest its role in stress
related transcription and cell cycle regulation, as well as in
genotoxic stress response. Four alternatively spliced transcript
variants of this gene encoding distinct isoforms have been
reported. The polynucleotide sequence of human MAPK14
transcriptional variants 2, 4, 1 and 3 are set forth in SEQ ID
NOS:20-23, respectively and the corresponding polypeptide sequences
are set forth in SEQ ID NO:68-71.
[0141] International Patent Publications WO 2000/59919 and WO
2005/016947 and U.S. Pat. Nos. 6,140,124 and 6,448,079 teach
antisense inhibition of p38.
[0142] (12) Ras-Related C3 Botulinum Toxin Substrate 1 (Rho Family,
Small GTP Binding Protein Rac1; RAC1)
[0143] Gene aliases: MGC111543, MIG5, TC-25, p21-Rac1;
migration-inducing gene 5; migration-inducing protein 5;
ras-related C3 botulinum toxin substrate 1; rho family, small GTP
binding protein Rac1, ras-related C3 botulinum toxin substrate 1
(rho family small GTP binding protein Rac1)
[0144] The protein encoded by this gene is a GTPase, which belongs
to the RAS superfamily of small GTP-binding proteins. Members of
this superfamily regulate a diverse array of cellular events,
including the control of cell growth, cytoskeletal reorganization,
and the activation of protein kinases. Several alternatively
spliced transcript variants of this gene have been described, but
the full-length nature of some of these variants has not been
determined. The polynucleotide sequences of human RAC1
transcriptional variants 1c, 1b and 1 are set forth in SEQ ID
NOS:24-26, respectively and the corresponding polypeptide sequences
are set forth in SEQ ID NO:72-74.
[0145] U.S. Pat. No. 6,180,597 relates to rho GTPase inhibitors
that increase endothelial cell nitric oxide synthase levels.
International Patent Publication WO 01/15739 teaches antisense
modulation of Rho family members. International Patent Publication
WO 2004/042052 teaches methods of suppressing TNF-.alpha.
secretion.
[0146] (13) Glycogen Synthase Kinase 3 Beta (GSK3B)
[0147] Gene aliases: 7330414F15Rik, 8430431H08Rik, C86142, GSK-3,
GSK-3beta, GSK3
[0148] Glycogen synthase kinase-3 (GSK3) is a proline-directed
serine-threonine kinase that was initially identified as a
phosphorylating and inactivating glycogen synthase. Two isoforms,
alpha (GSK3A; MIM 606784) and beta, show a high degree of amino
acid homology (Stambolic and Woodgett, Biochem J. 1994 303(Pt
3):701-4). GSK3B is involved in energy metabolism, neuronal cell
development, and body pattern formation (Plyte et al., Biochim
Biophys Acta. 1992. 1114(2-3):147-62). The polynucleotide sequence
of human GSK3B mRNA is set forth in SEQ ID NO:27, and the
corresponding polypeptide sequence is set forth in SEQ ID
NO:75.
[0149] U.S. Pat. No. 6,323,029 relates to antisense inhibition of
GSK3B.
[0150] (14) Purinergic Receptor P2X, Ligand-Gated Ion Channel, 7
(P2RX7)
[0151] Gene aliases: MGC20089, P2X7, A1467586; ATP receptor; P2X
purinoceptor 7; P2X7 receptor; P2Z receptor; purinergic receptor
P2X7.
[0152] The product of this gene belongs to the family of
purinoceptors for ATP. This receptor functions as a ligand-gated
ion channel and is responsible for ATP-dependent lysis of
macrophages through the formation of membrane pores permeable to
large molecules. Activation of this nuclear receptor by ATP in the
cytoplasm may be a mechanism by which cellular activity can be
coupled to changes in gene expression. Multiple alternatively
spliced variants which would encode different isoforms have been
identified although some fit nonsense-mediated decay (NMD)
criteria. The polynucleotide sequence of human P2RX7 mRNA is set
forth in SEQ ID NO:28 and the corresponding polypeptide sequence is
set forth in SEQ ID NO:76.
[0153] (15) Transient Receptor Potential Cation Channel, Subfamily
M, Member 2 (TRPM2)
[0154] Gene aliases: EREG1, KNP3, LTRPC2, MGC133383, NUDT9H,
NUDT9L1, TRPC7, 9830168K16Rik, C79133, Trp7, Trrp7; estrogen
responsive element associated gene 1; long transient receptor
potential channel 2; transient receptor potential channel 7,
transient receptor potential cation channel, subfamily M, member 2
(Trpm2); transient receptor potential channel 7; transient receptor
protein 7.
[0155] The protein encoded by this gene is a calcium-permeable
cation channel that is regulated by free intracellular ADP-ribose.
The encoded protein is activated by oxidative stress and confers
susceptibility to cell death. The polynucleotide sequences of the
human TRPM2 is set forth in SEQ ID NO:29 and the corresponding
polypeptide sequences is set forth in SEQ ID NO:77. (Two transcript
variants encoding different isoforms S and L had been found for
this gene. The S variant was removed by NCBI since it contains a
sequencing error and does not exist).
[0156] (16) Poly (ADP-Ribose) Glycohydrolase (PARG)
[0157] Gene aliases: PARG99; poly(ADP-ribose) glycohydrolase
[0158] Poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme
responsible for the catabolism of poly(ADP-ribose), a reversible
covalent-modifier of chromosomal proteins. The protein is found in
many tissues and may be subject to proteolysis generating smaller,
active products. The polynucleotide sequence of human PARG mRNA is
set forth in SEQ ID NO:31, and the corresponding polypeptide
sequence is set forth in SEQ ID NO:79.
[0159] (17) CD38 Molecule (CD38)
[0160] Gene aliases: T10, Cd38-rs1; ADP-ribosyl cyclase/cyclic
ADP-ribose hydrolase; CD38 antigen; CD38 antigen (p45); cyclic
ADP-ribose hydrolase.
[0161] CD38 is a novel multifunctional ectoenzyme widely expressed
in cells and tissues; especially in leukocytes. CD38 also functions
in cell adhesion, signal transduction and calcium signaling. The
polynucleotide sequence of human CD38 mRNA is set forth in SEQ ID
NO: 32, and the corresponding polypeptide sequences are set forth
in SEQ ID NO:80.
[0162] (18) STEAP Family Member 4 (STEAP4)
[0163] Gene aliases: DKFZp666D049, FLJ23153, STAMP2, TIARP,
TNFAIP9, 1110021017Rik, A1481214, dudulin 4; six transmembrane
prostate protein 2; tumor necrosis factor, alpha-induced protein 9;
tumor necrosis-alpha-induced adipose-related protein, m
TNFa-induced adipose-related protein; tumor necrosis factor,
alpha-induced protein 9.
[0164] A membrane protein induced by TNF-.alpha. and IL-6 in
adipose tissues. Both IL-6 and TNF-.alpha. were shown to be
unregulated in a spinal cord injury model (Ahn, et al., BBRC 2006
348(2):560-70) and are thought to promote apoptotic events. The
polynucleotide sequence of human STEAP4 mRNA is set forth in SEQ ID
NO:33, and the corresponding polypeptide sequence is set forth in
SEQ ID NO:81.
[0165] (19) Bone Morphogenetic Protein 2 (BMP2)
[0166] Gene aliases: BMP2A, AI467020, BC069214CR618407, M22489
[0167] The protein encoded by this gene belongs to the transforming
growth factor-beta (TGFB) superfamily. The encoded protein acts as
a disulfide-linked homodimer and induces bone and cartilage
formation. The polynucleotide sequence of human BMP2 mRNA is set
forth in SEQ ID NO:34, and the corresponding polypeptide sequence
is set forth in SEQ ID NO:82.
[0168] International Patent publication WO 2005/041857, coassigned
to the assignee of the present application, relates to BMP
inhibition for the treatment of ischemia and neurological
disease.
[0169] (20) Gap Junction Protein, Alpha 1, 43 kDa (Connexin 43,
GJA1)
[0170] Gene aliases: CX43, DFNB38, GJAL, ODD, ODDD, ODOD, SDTY3,
MGC93610, AU042049, AW546267, Cnx43, Cx43alpha1, Gja-1, Npm1, gap
junction protein, alpha-like; oculodentodigital dysplasia
(syndactyl)-type III), gap junction protein alpha 1 43 kD; gap
junction protein, alpha 1, 43 kD, alpha 1 connexin.
[0171] Gap junction protein, alpha 1 is a member of the connexin
gene family of proteins and is a component of gap junctions in the
heart, and is believed to have a crucial role in the synchronized
contraction of the heart and in embryonic development. Connexin 43
is targeted by several protein kinases that regulate myocardial
cell-cell coupling. A related intron-less connexin 43 pseudogene,
GJA1P, has been mapped to chromosome 5. The polynucleotide sequence
of human GJA1 mRNA is set forth in SEQ ID NO:35, and the
corresponding polypeptide sequence is set forth in SEQ ID NO:83.
U.S. Pat. No. 7,098,190 teaches antisense compounds for treating,
inter alia, wounds and spinal cord injury.
[0172] (21) TYRO Protein Tyrosine Kinase Binding Protein
(TYROBP)
[0173] Gene aliases: DAP12, KARAP, PLOSL, Ly83; DNAX-activation
protein 12; killer activating receptor associated protein.
[0174] This gene encodes a transmembrane signaling polypeptide that
contains an immunoreceptor tyrosine-based activation motif (ITAM)
in its cytoplasmic domain. The protein may associate with the
killer-cell inhibitory receptor (KIR) family of membrane
glycoproteins and may act as an activating signal transduction
element. This protein may bind zeta-chain (TCR) associated protein
kinase 70 kDa (ZAP-70) and spleen tyrosine kinase (SYK) and play a
role in signal transduction, bone modeling, brain myelination, and
inflammation. Mutations within the gene have been associated with
polycystic lipomembranous osteodysplasia with sclerosing
leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease. Its
putative receptor, triggering receptor expressed on myeloid cells 2
(TREM2), also causes PLOSL. Two alternative transcript variants
encoding distinct isoforms have been identified for this gene.
Other alternative splice variants have been described, but their
full-length nature has not been determined. The polynucleotide
sequences of human TYROBP transcriptional variants 1 and 2 are set
forth in SEQ ID NO:36-37, respectively, and the corresponding
polypeptide sequences are set forth in SEQ ID NO:84-85.
[0175] (22) Connective Tissue Growth Factor (CTGF)
[0176] Gene aliases: CCN2, HCS24, IGFBP8, MGC102839, NOV2, CTGRP,
Fisp12, Hcs24, fisp-12; hypertrophic chondrocyte-specific protein
24; insulin-like growth factor-binding protein 8, FISP-12 protein;
fibroblast inducible secreted protein; fibroblast inducible
secreted protein; hypertrophic chondrocyte-specific gene product
24.
[0177] A major connective tissue mitoattractant secreted by
vascular endothelial cells. Promotes proliferation and
differentiation of chondrocytes. The polynucleotide sequence of
human CTGF mRNA is set forth in SEQ ID NO:38 and the corresponding
polypeptide sequence is set forth in SEQ ID NO:86.
[0178] (23) Secreted Phosphoprotein 1 (SPP1)
[0179] Gene aliases: AA960535, A1790405, Apl-1, BNSP, BSPI, Bsp,
ETA-1, Eta, OP, Opn, Opnl, Ric, Spp-1, minopontin, OSP; 44 kDa bone
phosphoprotein; 44 kDa bone phosphoprotein; bone sialoprotein I;
osteopontin, early T-lymphocyte activation 1.
[0180] SSP1 is a secreted protein which acts as a cytokine involved
in enhancing production of interferon-gamma and interleukin-12 and
reducing production of interleukin-10 and which is essential in the
pathway that leads to type I immunity. The polynucleotide sequences
of human SPP1 transcriptional variants 1, 2 and 3 are set forth in
SEQ ID NOS:39-41, and the corresponding polypeptide sequences are
set forth in SEQ ID NOS:87-89.
[0181] U.S. Pat. No. 6,458,590 and US Patent Publications
2004/0142865 and 2006/0252684 relate to inhibition of
osteopontin.
[0182] (24) Reticulon 4 Receptor (RTN4R)
[0183] Gene aliases: NGR, NOGOR, NgR1; Nogo-66 receptor;
UNQ330/PRO526; nogo receptor; reticulon 4 receptor precursor.
[0184] This gene encodes the receptor for reticulon 4,
oligodendrocyte myelin glycoprotein and myelin-associated
glycoprotein. This receptor mediates axonal growth inhibition and
may play a role in regulating axonal regeneration and plasticity in
the adult central nervous system. The polynucleotide sequence of
human RTN4R mRNA is set forth in SEQ ID NO:42 and the corresponding
polypeptide sequence is set forth in SEQ ID NO:90.
[0185] (25) Annexin A2 (ANXA2)
[0186] Gene aliases: ANX2, ANX2L4, CAL1H, LIP2, LPC2, LPC2D, P36,
PAP-IV; annexin II; calpactin I heavy polypeptide; chromobindin 8;
lipocortin II; placental anticoagulant protein IV.
[0187] This gene encodes a member of the annexin family. Members of
this calcium-dependent phospholipid-binding protein family play a
role in the regulation of cellular growth and in signal
transduction pathways. This protein functions as an autocrine
factor, which heightens osteoclast formation and bone resorption.
This gene has three pseudogenes located on chromosomes 4, 9 and 10,
respectively. Multiple alternatively spliced transcript variants
encoding different isoforms have been found for this gene. The
polynucleotide sequences of human ANXA2 transcriptional variants 1,
3 and 2 are set forth in SEQ ID NOS:43-45 and the corresponding
polypeptide sequences are set forth in SEQ ID NOS:91-93.
[0188] (26) Ras Homolog Gene Family, Member A (RHOA)
[0189] Gene Aliases: ARH12, ARHA, RHO12, RHOH12, Aplysia
ras-related homolog 12; oncogene RHO H12; small GTP binding protein
RhoA.
[0190] RHOA is a small GTPase protein known to regulate the actin
cytoskeleton in the formation of stress fibers. It acts upon the
effector proteins: Rho kinase (ROCK) culminating in the inhibition
of axonal regeneration. In vitro studies using the Rho-A antagonist
C3 transferase enhances axonal growth on myelin substrates while in
vivo studies have not been effective. The polynucleotide sequence
of human RHOA mRNA is set forth in SEQ ID NO:46 and the
corresponding polypeptide sequence is set forth in SEQ ID
NO:94.
[0191] (27) Dual Oxidase 1 (DUOX1)
[0192] Gene Aliases: LNOX1, MGC138840, MGC138841, NOXEF1, THOX1,
NADPH thyroid oxidase 1; flavoprotein NADPH oxidase; nicotinamide
adenine dinucleotide phosphate oxidase.
[0193] The protein encoded by this gene is a glycoprotein and a
member of the NADPH oxidase family. The synthesis of thyroid
hormone is catalyzed by a protein complex located at the apical
membrane of thyroid follicular cells. This complex contains an
iodide transporter, thyroperoxidase, and a peroxide generating
system that includes this encoded protein and DUOX2. This protein
has both a peroxidase homology domain and a gp91phox domain. Two
alternatively spliced transcript variants encoding the same protein
have been described for this gene. The polynucleotide sequence of
human DUOX1 transcriptional variants 1 and 2 are set forth in SEQ
ID NOS:47-48 and the corresponding polypeptide sequences are set
forth in SEQ ID NOS:95-96.
[0194] A "pro-apoptotic polypeptide" refers to a polypeptide
encoded by any of the above listed genes, including splice
variants, isoforms, orthologs, or paralogs and the like.
[0195] An "inhibitor" is a compound which is capable of inhibiting
the expression of a gene or the activity of the product of such
gene to an extent sufficient to achieve a desired biological or
physiological effect. The term "inhibitor" as used herein refers to
one or more of an oligonucleotide inhibitor, including siRNA,
shRNA, aptamers, antisense molecules, miRNA and ribozymes, as well
as antibodies The inhibitor may cause complete or partial
inhibition.
[0196] The term "inhibit" as used herein refers to reducing the
expression of a gene or the activity of the product of such gene to
an extent sufficient to achieve a desired biological or
physiological effect. Inhibition may be complete or partial.
[0197] As used herein, the terms "polynucleotide" and "nucleic
acid" may be used interchangeably and refer to nucleotide sequences
comprising deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).
The terms should also be understood to include, as equivalents,
analogs of either RNA or DNA made from nucleotide analogs.
Throughout this application, mRNA sequences are set forth as
representing the corresponding genes.
[0198] "Oligonucleotide" refers to a compound comprising
deoxyribonucleotides and/or ribonucleotides from about 2 to about
50 nucleotides. Each DNA or RNA nucleotide may be independently
natural or synthetic, and or modified or unmodified. Modifications
include changes to the sugar moiety, the base moiety and or the
linkages between nucleotides in the oligonucleotide.
[0199] The present invention provides methods and compositions for
inhibiting expression of a target pro-apoptotic gene in vivo. In
general, the method includes administering oligoribonucleotides, in
particular small interfering RNAs (i.e., siRNAs) or a nucleic acid
material that can produce siRNA in a cell, that target an mRNA
transcribed from a pro-apoptotic gene in an amount sufficient to
down-regulate expression of a target gene by an RNA interference
mechanism. In particular, the subject method can be used to inhibit
expression of the pro-apoptotic gene for treatment of a
disease.
[0200] In accordance with the present invention, the siRNA
molecules or inhibitors of the pro-apoptotic gene are used as drugs
to treat various pathologies.
[0201] siRNA Oligoribonucleotides
[0202] Table B (B1-B76) comprises nucleic acid sequences of sense
and corresponding antisense oligomers, useful in preparing
corresponding siRNA compounds. Tables C1, C2 and C3 comprise
certain currently preferred nucleic acid sequences of sense and
corresponding antisense oligomers, useful in preparing the
corresponding siRNA compounds.
[0203] The selection and synthesis of siRNA corresponding to known
genes has been widely reported; see for example Ui-Tei et al., J
Biomed Biotechnol. 2006; 65052; Chalk et al., BBRC. 2004,
319(1):264-74; Sioud and Leirdal, Met. Mol Biol.; 2004, 252:457-69;
Levenkova et al., Bioinform. 2004, 20(3):430-2; Ui-Tei et al., NAR
2004, 32(3):936-48. For examples of the use of, and production of,
modified siRNA see Braasch et al., Biochem., 2003, 42(26):7967-75;
Chiu et al., RNA, 2003, 9(9): 1034-48; PCT publications WO
2004/015107 (atugen); WO 02/44321 (Tuschl et al), and U.S. Pat.
Nos. 5,898,031 and 6,107,094.
[0204] Several groups have described the development of DNA-based
vectors capable of generating siRNA within cells. The method
generally involves transcription of short hairpin RNAs that are
efficiently processed to form siRNAs within cells (Paddison et al.
PNAS USA 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002,
16:948-958; Sui et al. PNAS USA 2002, 8:5515-5520; and Brummelkamp
et al. Science 2002, 296:550-553). These reports describe methods
of generating siRNAs capable of specifically targeting numerous
endogenously and exogenously expressed genes.
[0205] The present invention provides double-stranded
oligoribonucleotides (eg. siRNAs), which down-regulate the
expression of the pro-apoptotic gene according to the present
invention. An siRNA of the invention is a duplex
oligoribonucleotide in which the sense strand is derived from the
mRNA sequence of the pro-apoptotic gene, and the antisense strand
is complementary to the sense strand. In general, some deviation
from the target mRNA sequence is tolerated without compromising the
siRNA activity (see e.g. Czauderna et al., 2003, NAR 31(11),
2705-2716). An siRNA of the invention inhibits gene expression on a
post-transcriptional level with or without destroying the mRNA.
Without being bound by theory, siRNA may target the mRNA for
specific cleavage and degradation and/or may inhibit translation
from the targeted message.
[0206] As used herein, the term "ribonucleotide" encompasses
natural and synthetic, unmodified and modified ribonucleotides.
Modifications include changes to the sugar moiety, to the base
moiety and/or to the linkages between ribonucleotides in the
oligonucleotide.
[0207] In some embodiments the oligoribonucleotide according to the
present invention comprises modified siRNA. In various embodiments
the siRNA comprises an RNA duplex comprising a first strand and a
second strand, whereby the first strand comprises a ribonucleotide
sequence at least partially complementary to about 18 to about 40
consecutive nucleotides of a target nucleic acid, and the second
strand comprises ribonucleotide sequence at least partially
complementary to the first strand and wherein said first strand
and/or said second strand comprises a plurality of groups of
modified ribonucleotides having a modification at the 2'-position
of the sugar moiety whereby within each strand each group of
modified ribonucleotides is flanked on one or both sides by a group
of flanking ribonucleotides whereby each ribonucleotide forming the
group of flanking ribonucleotides is selected from an unmodified
ribonucleotide or a ribonucleotide having a modification different
from the modification of the groups of modified
ribonucleotides.
[0208] In one embodiment, the group of modified ribonucleotides
and/or the group of flanking ribonucleotides comprise a number of
ribonucleotides selected from the group consisting of an integer
from 1 to 12. Accordingly, the group thus comprises one nucleotide,
two nucleotides, three nucleotides, four nucleotides, five
nucleotides, six nucleotides, seven nucleotides, eight nucleotides,
nine nucleotides, ten nucleotides, eleven nucleotides or twelve
nucleotides.
[0209] The groups of modified nucleotides and flanking nucleotides
may be organized in a pattern on at least one of the strands. In
some embodiments the first and second strands comprise a pattern of
modified nucleotides. In another embodiment, only one strand
comprises a pattern of modified nucleotides. In various embodiments
the pattern of modified nucleotides of said first strand is
identical relative to the pattern of modified nucleotides of the
second strand.
[0210] In other embodiments the pattern of modified nucleotides of
said first strand is shifted by one or more nucleotides relative to
the pattern of modified nucleotides of the second strand. In some
preferred embodiments the middle ribonucleotide in the antisense
strand is an unmodified nucleotide. For example, in a 19-oligomer
antisense strand, ribonucleotide number 10 is unmodified; in a
21-oligomer antisense strand, ribonucleotide number 11 is
unmodified; and in a 23-oligomer antisense strand, ribonucleotide
number 12 is unmodified. The modifications or pattern of
modification, if any, of the siRNA must be planned to allow for
this.
[0211] The modifications on the 2' moiety of the sugar residue
include amino, fluoro, alkoxy e.g. methoxy, alkyl, amino, fluoro,
chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl,
OCF.sub.3, OCN, O-, S-, or N-alkyl; O-, S, or N-alkenyl;
SOCH.sub.3; SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2, N.sub.3;
heterocycloalkyl; heterozycloalkaryl; aminoalkylamino;
polyalkylamino or substituted silyl, as, among others, described in
European patents EP 0 586 520 B1 or EP 0 618 925 B1.
[0212] In some embodiments the siRNA is blunt ended, at one or both
ends. More specifically, the siRNA may be blunt ended on the end
defined by the 5'-terminus of the first strand and the 3'-terminus
of the second strand, or the end defined by the 3'-terminus of the
first strand and the 5'-terminus of the second strand. In other
embodiments at least one of the two strands may have an overhang of
at least one nucleotide at the 5'-terminus. At least one of the
strands may also optionally have an overhang of at least one
nucleotide at the 3'-terminus. The overhang may consist of from
about 1 to about 5 consecutive nucleotides. A nucleotide of the
overhang may be a modified or unmodified ribonucleotide or
deoxyribonucleotide.
[0213] The length of RNA duplex is from about 18 to about 40
ribonucleotides, preferably 19, 21 or 23 ribonucleotides. Further,
the length of each strand may independently have a length selected
from the group consisting of about 15 to about 40 bases, preferably
18 to 23 bases and more preferably 19, 21 or 23
ribonucleotides.
[0214] Additionally, the complementarity between said first strand
and the target nucleic acid may be perfect. In some embodiments,
the strands are substantially complementary, i.e. having one, two
or up to three mismatches between said first strand and the target
nucleic acid. Substantially complementary refers to complementarity
of greater than about 84%, to another sequence. For example in a
duplex region consisting of 19 base pairs one mismatch results in
94.7% complementarity, two mismatches results in about 89.5%
complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0215] In certain embodiments the first strand and the second
strand each comprise at least one group of modified ribonucleotides
and at least one group of flanking ribonucleotides, whereby each
group of modified ribonucleotides comprises at least one
ribonucleotide and whereby each group of flanking ribonucleotides
comprises at least one ribonucleotide, wherein each group of
modified ribonucleotides of the first strand is aligned with a
group of flanking ribonucleotides on the second strand, and wherein
the 5' most terminal ribonucleotide is selected from a group of
modified ribonucleotides, and the 3' most terminal ribonucleotide
of the second strand is a selected from the group of flanking
ribonucleotide. In some embodiments each group of modified
ribonucleotides consists of a single ribonucleotide and each group
of flanking ribonucleotides consists of a single nucleotide.
[0216] In yet other embodiments the ribonucleotide forming the
group of flanking ribonucleotides on the first strand is an
unmodified ribonucleotide arranged in a 3' direction relative to
the ribonucleotide forming the group of modified ribonucleotides,
and the ribonucleotide forming the group of modified
ribonucleotides on the second strand is a modified ribonucleotide
which is arranged in 5' direction relative to the ribonucleotide
forming the group of flanking ribonucleotides. In some embodiments
the first strand of the siRNA comprises five to about twenty, eight
to twelve, preferably nine to twelve, groups of modified
ribonucleotides, and the second strand comprises seven to eleven,
preferably eight to eleven, groups of modified ribonucleotides.
[0217] The first strand and the second strand may be linked by a
loop structure, which may be comprised of a non-nucleic acid
polymer such as, inter alia, polyethylene glycol. Alternatively,
the loop structure may be comprised of a nucleic acid, including
modified and non-modified ribonucleotides and modified and
non-modified deoxyribonucleotides.
[0218] Further, the 5'-terminus of the first strand of the siRNA
may be linked to the 3'-terminus of the second strand, or the
3'-terminus of the first strand may be linked to the 5'-terminus of
the second strand, said linkage being via a nucleic acid linker or
a non-nucleic acid linker. In certain embodiments a nucleic acid
linker has a length of between about 2-100 nucleic acids,
preferably about 2 to about 30 nucleic acids.
[0219] In various embodiments, the present invention provides a
compound having the structure: [0220] 5' (N).sub.x-Z 3' (antisense
strand) [0221] 3' Z'-(N').sub.y 5' (sense strand) wherein each N
and N' is a ribonucleotide which may be modified or unmodified in
its sugar residue; and each of (N).sub.x and (N').sub.y is an
oligomer in which each consecutive N or N' is joined to the next N
or N' by a covalent bond; wherein each of x and y is an integer
between 18 and 40; wherein each of Z and Z' may be present or
absent, but if present is 1-5 consecutive nucleotides covalently
attached at the 3' terminus of the strand in which it is present;
and wherein the sequence of (N').sub.y comprises a sense sequence
having substantial identity to about 18 to about 40 consecutive
ribonucleotides in an mRNA set forth is one of SEQ ID NOS:1-48. In
preferred embodiments the sense sequence is selected from a
sequence presented in any one of Table B (B1-B76; SEQ ID NOS:277 to
50970 and 50993-68654) or Tables C1, C2 and C3 (SEQ ID NOS: 97-276
and 50971-50992).
[0222] In some embodiments the compound comprises a phosphodiester
bond.
[0223] In various embodiments the compound comprises
ribonucleotides wherein x=y and wherein x is an integer selected
from the group consisting of 18, 19, 20, 21, 22 and 23. In certain
embodiments x=y=19 or x=y=23.
[0224] In some embodiments the compound is blunt ended, for example
wherein Z and Z' are both absent. In an alternative embodiment, the
compound comprises at least one 3' overhang, wherein at least one
of Z or Z' is present. Z and Z' can be independently comprise one
or more covalently linked modified or non-modified nucleotides, as
described infra, for example inverted dT or dA; dT, LNA (locked
nucleic acids), mirror nucleotide and the like. In some embodiments
each of Z and Z' are independently selected from dT and dTdT.
[0225] In some embodiments the compound comprises one or more
ribonucleotides unmodified in their sugar residues. In other
embodiments the compound comprises at least one ribonucleotide
modified in the sugar residue. In some embodiments the compound
comprises a modification at the 2' position of the sugar residue.
Modifications in the 2' position of the sugar residue include
amino, fluoro, alkoxy and alkyl moieties. In certain preferred
embodiments the alkoxy modification is a methoxy moiety at the 2'
position of the sugar residue (2'-O-methyl; 2'-O-Me;
2'-O--CH.sub.3).
[0226] In some embodiments the compound comprises modified
alternating ribonucleotides in one or both of the antisense and the
sense strands. In certain embodiments the compound comprises
modified alternating ribonucleotides in the antisense and the sense
strands. In some preferred embodiments the middle ribonucleotide of
the antisense strand is not modified; e.g. ribonucleotide in
position 10 in a 19-mer strand.
[0227] In various embodiments the compound comprises an antisense
sequence present in Table B SEQ ID NOS:277 to 50970 and
50993-68654). In other embodiments the present invention provides a
mammalian expression vector comprising an antisense sequence
present in Table B (SEQ ID NOS:277 to 50970 and 50993-68654).
Certain presently preferred compounds are listed in Tables C1, C2
and C3, and their sequences are set forth in SEQ ID NOS: 97-276 and
SEQ ID NOS 50971-50992.
[0228] In certain embodiments the present invention provides a
compound having the structure [0229] 5' (N).sub.x 3' antisense
strand [0230] 3' (N').sub.y 5' sense strand wherein each of x and
y=19 and (N).sub.x and (N').sub.y are fully complementary; wherein
alternating ribonucleotides in (N).sub.x and (N').sub.y are
modified to result in a 2'-O-methyl modification in the sugar
residue of the ribonucleotides; wherein each N at the 5' and 3'
termini of (N).sub.x are modified; wherein each N' at the 5' and 3'
termini of (N').sub.y are unmodified; wherein each of (N).sub.x and
(N').sub.y is selected from the group of oligomers set forth in
Table B (B1-B25 and B76; SEQ ID NOS:277-15114 and 68647-68654).
[0231] Certain presently preferred compounds are listed in Tables
C1 and C3, and their sequences are set forth in SEQ ID NOS: 97-266
and SEQ ID NOS 50971-50992.
[0232] (N).sub.x and (N').sub.y may be phosphorylated or
non-phosphorylated at the 3' and 5' termini.
[0233] In certain embodiments of the invention, alternating
ribonucleotides are modified in the 2' position of the sugar
residue in both the antisense and the sense strands of the
compound. In particular the exemplified siRNA has been modified
such that a 2'-O-methyl (Me) group was present on the first, third,
fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth
and nineteenth nucleotide of the antisense strand, whereby the very
same modification, i.e. a 2'-O-Me group, was present at the second,
fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and
eighteenth nucleotide of the sense strand. Additionally, it is to
be noted that these particular siRNA compounds are also blunt
ended.
[0234] In certain embodiments the present invention provides a
compound having the structure [0235] 5' (N).sub.x 3' antisense
strand [0236] 3' (N').sub.y 5' sense strand wherein each of x and
y=23 and (N).sub.x and (N').sub.y are fully complementary wherein
alternating ribonucleotides in (N).sub.x and (N').sub.y are
modified to result in a 2'-O-methyl modification in the sugar
residue of the ribonucleotides; wherein each N at the 5' and 3'
termini of (N).sub.x are modified; wherein each N' at the 5' and 3'
termini of (N').sub.y are unmodified; wherein each of (N).sub.x and
(N').sub.y is selected from the group of oligomers set forth in
Table B (B51-B75; SEQ ID NOS:30939-68646).
[0237] (N).sub.x and (N').sub.y may be phosphorylated or
non-phosphorylated at the 3' and 5' termini. In certain embodiments
of the invention, alternating ribonucleotides are modified in both
the antisense and the sense strands of the compound. In particular
the exemplified siRNA has been modified such that a 2'-O-methyl
(2'-OMe) group was present on the first, third, fifth, seventh,
ninth, eleventh, thirteenth, fifteenth, seventeenth, nineteenth,
twenty-first and twenty-third nucleotide of the antisense strand
(N).sub.x, and whereby the very same modification, i.e. a 2'-OMe
group, was present at the second, fourth, sixth, eighth, tenth,
twelfth, fourteenth, sixteenth, eighteenth, twentieth and
twenty-second nucleotide of the sense strand (N').sub.y.
Additionally, it is to be noted that these particular siRNA
compounds are also blunt ended.
[0238] In certain embodiments of the compounds of the invention
having alternating ribonucleotides modified in one or both of the
antisense and the sense strands of the compound; for 19-mers and
23-mers the ribonucleotides at the 5' and 3' termini of the
antisense strand are modified in their sugar residues, and the
ribonucleotides at the 5' and 3' termini of the sense strand are
unmodified in their sugar residues. For 21-mers the ribonucleotides
at the 5' and 3' termini of the sense strand are modified in their
sugar residues, and the ribonucleotides at the 5' and 3' termini of
the antisense strand are unmodified in their sugar residues. As
mentioned above, it is preferred that the middle nucleotide of the
antisense strand is unmodified.
[0239] Additionally, the invention provides siRNA comprising a
nucleic acid sequence set forth in Table B (B1-B76; SEQ ID
NOS:277-50970 and 50993-68654) wherein 1, 2, or 3 of the
nucleotides in one strand or both strands are substituted thereby
providing at least one base pair mismatch. The substituted
nucleotides in each strand are preferably in the terminal region of
one strand or both strands.
[0240] In currently preferred embodiments the ribonucleic acid
sequences of the siRNA are SEQ ID NOS:97-276 and SEQ ID NOS:
50971-50992 of Tables C1, C2 and C3. In certain currently preferred
embodiments the ribonucleic acid sequences of the siRNA are set
forth in SEQ ID NOS:99-100; SEQ ID NOS:133-134; SEQ ID NOS:137-138;
SEQ ID NOS:211-212; SEQ ID NOS:213-214 as shown in Table C1.
[0241] According to one preferred embodiment of the invention, the
antisense and the sense strands of the siRNA are phosphorylated
only at the 3'-terminus and not at the 5'-terminus. According to
another preferred embodiment of the invention, the antisense and
the sense strands are non-phosphorylated. According to yet another
preferred embodiment of the invention, the 5' most ribonucleotide
in the sense strand is modified to abolish any possibility of in
vivo 5'-phosphorylation.
[0242] The invention further provides a vector capable of
expressing any of the aforementioned oligoribonucleotides in
unmodified form in a cell after which appropriate modification may
be made. In preferred embodiment the cell is a mammalian cell,
preferably a human cell.
[0243] Pharmaceutical Compositions
[0244] While it may be possible for the compounds of the present
invention to be administered as the raw chemical, it is preferable
to present them as a pharmaceutical composition. Accordingly the
present invention provides a pharmaceutical composition comprising
one or more of the compounds of the invention; and a
pharmaceutically acceptable carrier. This composition may comprise
a mixture of two or more different siRNAs. In one embodiment, this
composition may comprise a mixture of siRNA to RhoA and siRNA to
one or more of the other pro-apoptotic genes of the invention. In a
more particular embodiment, this composition may comprise a mixture
of siRNA to RhoA and siRNA to Casp2. Without being bound by theory,
RhoA is a small GTPase that when activated inhibits neurite
outgrowth. Its inhibition is relevant for spinal cord injury and it
can be combined for this indication with anti-apoptotic siRNAs of
the invention. The latter will protect, and siRNA to RhoA will
promote regeneration, and so a combined or even synergistic effect
is produced.
[0245] The invention further provides a pharmaceutical composition
comprising at least one compound of the invention covalently or
non-covalently bound to one or more compounds of the invention in
an amount effective to inhibit the pro-apoptotic genes of the
present invention; and a pharmaceutically acceptable carrier. The
compound may be processed intracellularly by endogenous cellular
complexes to produce one or more oligoribonucleotides of the
invention.
[0246] The invention further provides a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and one or more of
the compounds of the invention in an amount effective to inhibit
expression in a cell of a human pro-apoptotic gene of the present
invention, the compound comprising a sequence which is
substantially complementary to the sequence of (N).sub.x.
[0247] Substantially complementary refers to complementarity of
greater than about 84%, to another sequence. For example in a
duplex region consisting of 19 base pairs one mismatch results in
94.7% complementarity, two mismatches results in about 89.5%
complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0248] Additionally, the invention provides a method of inhibiting
the expression of the pro-apoptotic genes of the present invention
by at least 20%, preferably 30%, even more preferably 40% or even
50% as compared to a control comprising contacting an mRNA
transcript of the pro-apoptotic gene of the present invention with
one or more of the compounds of the invention.
[0249] In one embodiment the oligoribonucleotide is inhibiting one
or more of the pro-apoptotic genes of the present invention,
whereby the inhibition is selected from the group comprising
inhibition of gene function, inhibition of polypeptide and
inhibition of mRNA expression.
[0250] In one embodiment the compound is inhibiting a pro-apoptotic
polypeptide, whereby the inhibition is selected from the group
comprising inhibition of function (which may be examined by an
enzymatic assay or a binding assay with a known interactor of the
native gene/polypeptide, inter alia), inhibition of protein (which
may be examined by Western blotting, ELISA or immuno-precipitation,
inter alia) and inhibition of mRNA expression (which may be
examined by Northern blotting, quantitative RT-PCR, in-situ
hybridisation or microarray hybridisation, inter alia).
[0251] In additional embodiments the invention provides a method of
treating a subject suffering from a disease accompanied by an
elevated level of the pro-apoptotic genes of the present invention,
the method comprising administering to the subject a compound of
the invention in a therapeutically effective dose thereby treating
the subject.
[0252] More particularly, the invention provides an
oligoribonucleotide wherein one strand comprises consecutive
nucleotides having, from 5' to 3', the sequence set forth in any
one of SEQ ID NOS:277-50970 and 50993-68654, shown also in Table B,
or a homolog thereof wherein in up to two of the ribonucleotides in
each terminal region is altered.
[0253] Additionally, further nucleic acids according to the present
invention comprise at least 14 contiguous nucleotides of any one of
the polynucleotides in Table B (SEQ ID NOS:277-50970 and
50993-68654) and more preferably 14 contiguous nucleotide base
pairs at any end of the double-stranded structure comprised of the
first strand and second strand as described above.
[0254] It will be understood by one skilled in the art that given
the potential length of the nucleic acid according to the present
invention and particularly of the individual strands forming such
nucleic acid according to the present invention, some shifts
relative to the coding sequence of the pro-apoptotic genes of the
present invention to each side is possible, whereby such shifts can
be up to 1, 2, 3, 4, 5 and 6 nucleotides in both directions, and
whereby the thus generated double-stranded nucleic acid molecules
shall also be within the present invention.
[0255] Delivery
[0256] The siRNA molecules of the present invention may be
delivered to the target tissue by direct application of the naked
molecules prepared with a carrier or a diluent.
[0257] The term "naked siRNA" refers to siRNA molecules that are
free from any delivery vehicle that acts to assist, promote or
facilitate entry into the cell, including viral sequences, viral
particles, liposome formulations, lipofectin or precipitating
agents and the like. For example, siRNA in PBS is "naked
siRNA".
[0258] However, in some embodiments the siRNA molecules of the
invention are delivered in liposome formulations and lipofectin
formulations and the like and can be prepared by methods well known
to those skilled in the art. Such methods are described, for
example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859,
which are herein incorporated by reference.
[0259] Delivery systems aimed specifically at the enhanced and
improved delivery of siRNA into mammalian cells have been
developed, (see, for example, Shen et al., FEBS Let. 2003,
539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et
al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol.
2003. 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and
Simeoni et al., NAR 2003, 31, 11: 2717-2724). siRNA has recently
been successfully used for inhibition of gene expression in
primates (see for example, Tolentino et al., Retina 24(4):660).
[0260] The pharmaceutically acceptable carriers, solvents,
diluents, excipients, adjuvants and vehicles as well as implant
carriers generally refer to inert, non-toxic solid or liquid
fillers, diluents or encapsulating material not reacting with the
active ingredients of the invention and they include liposomes and
microspheres. Examples of delivery systems useful in the present
invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;
4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;
4,439,196; and 4,475,196. Many other such implants, delivery
systems, and modules are well known to those skilled in the art. In
one specific embodiment of this invention topical and transdermal
formulations may be selected. The siRNAs or pharmaceutical
compositions of the present invention are administered and dosed in
accordance with good medical practice, taking into account the
clinical condition of the individual subject, the disease to be
treated, the site and method of administration, scheduling of
administration, patient age, sex, body weight and other factors
known to medical practitioners.
[0261] The "therapeutically effective dose" for purposes herein is
thus determined by such considerations as are known in the art. The
dose must be effective to achieve improvement including but not
limited to improved survival rate or more rapid recovery, or
improvement or elimination of symptoms and other indicators as are
selected as appropriate measures by those skilled in the art.
[0262] In general, the active dose of compound for humans is in the
range of from 1 ng/kg to about 20-100 mg/kg body weight per day,
preferably about 0.01 mg to about 2-10 mg/kg body weight per day,
in a regimen of one dose per day or twice or three or more times
per day for a period of 1-4 weeks or longer. The compounds of the
present invention can be administered by any of the conventional
routes of administration. It should be noted that the compound can
be administered as the compound or as pharmaceutically acceptable
salt and can be administered alone or as an active ingredient in
combination with pharmaceutically acceptable carriers, solvents,
diluents, excipients, adjuvants and vehicles. The compounds can be
administered orally, subcutaneously or parenterally including
intravenous, intraarterial, intramuscular, intraperitoneally, and
intranasal administration as well as intrathecal and infusion
techniques. Implants of the compounds are also useful. Liquid forms
may be prepared for injection, the term including subcutaneous,
transdermal, intravenous, intramuscular, intrathecal, and other
parental routes of administration. The liquid compositions include
aqueous solutions, with and without organic co-solvents, aqueous or
oil suspensions, emulsions with edible oils, as well as similar
pharmaceutical vehicles. In a particular embodiment, the
administration comprises intravenous administration. In another
embodiment the administration comprises topical or local
administration. In addition, in certain embodiments the
compositions for use in the novel treatments of the present
invention may be formed as aerosols, for example for intranasal
administration. In certain embodiments, oral compositions (such as
tablets, suspensions, solutions) may be effective for local
delivery to the oral cavity such as oral composition suitable for
mouthwash for the treatment of oral mucositis.
[0263] Methods of Treatment
[0264] In another aspect, the present invention relates to a method
for the treatment of a subject in need of treatment for a disease
or disorder associated with the abnormal expression of the
proapoptotic genes of Table A, comprising administering to the
subject an amount of an inhibitor which reduces or inhibits
expression of these genes.
[0265] In preferred embodiments the subject being treated is a
warm-blooded animal and, in particular, mammals including
human.
[0266] The methods of the invention comprise administering to the
subject one or more inhibitory compounds which down-regulate
expression of the proapoptotic genes of Table A; and in particular
siRNA in a therapeutically effective dose so as to thereby treat
the subject.
[0267] In various embodiments the inhibitor is selected from the
group consisting of siRNA, shRNA, an aptamer, an antisense
molecule, miRNA, a ribozyme, and an antibody. In the presently
preferred embodiments the inhibitor is siRNA.
[0268] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) pro-apoptotic-related disorder as
listed above. Those in need of treatment include those already
experiencing the disease or condition, those prone to having the
disease or condition, and those in which the disease or condition
is to be prevented. The compounds of the invention may be
administered before, during or subsequent to the onset of the
disease or condition or symptoms associated therewith. In cases
where treatment is for the purpose of prevention, then the present
invention relates to a method for delaying the onset of or averting
the development of the disease or disorder.
[0269] The present invention relates to the use of compounds which
down-regulate the expression of the pro-apoptotic genes of the
invention particularly to novel small interfering RNAs (siRNAs), in
the treatment of the following diseases or conditions in which
inhibition of the expression of the pro-apoptotic genes is
beneficial: hearing loss, acute renal failure (ARF), glaucoma,
acute respiratory distress syndrome (ARDS) and other acute lung and
respiratory injuries, ischemia-reperfusion injury following lung
transplantation, organ transplantation including lung, liver,
heart, bone marrow, pancreas, cornea and kidney transplantation,
spinal cord injury, pressure sores, age-related macular
degeneration (AMD), dry eye syndrome, oral mucositis and chronic
obstructive pulmonary disease (COPD). Other indications include
chemical-induced nephrotoxicity and chemical-induced neurotoxicity,
for example toxicity induced by cisplatin and cisplatin-like
compounds, by aminoglycosides, by loop diuretics, and by
hydroquinone and their analogs.
[0270] Methods, molecules and compositions which inhibit the
pro-apoptotic genes of the invention are discussed herein at
length, and any of said molecules and/or compositions may be
beneficially employed in the treatment of a subject suffering from
any of said conditions. Preferred oligomer sequences useful in the
preparation of siRNA directed to selected pro-apoptotic genes are
set forth in SEQ ID NOS:277-50970 and 50993-68654, listed in Table
B.
[0271] The method of the invention includes administering a
therapeutically effective amount of one or more compounds which
down-regulate expression of the pro-apoptotic genes, particularly
the novel siRNAs of the present invention, small molecule
inhibitors of the pro-apoptotic genes as described herein or
antibodies to the pro-apoptotic proteins.
[0272] In some preferred embodiments, the methods of the invention
are applied to various conditions of hearing loss. Without being
bound by theory, the hearing loss may be due to apoptotic inner ear
hair cell damage or loss, wherein the damage or loss is caused by
infection, mechanical injury, loud sound, aging (presbycusis), or
chemical-induced ototoxicity. Ototoxins include therapeutic drugs
including antineoplastic agents, salicylates, quinines, and
aminoglycoside antibiotics, contaminants in foods or medicinals,
and environmental or industrial pollutants. Typically, treatment is
performed to prevent or reduce ototoxicity, especially resulting
from or expected to result from administration of therapeutic
drugs. Preferably a therapeutically effective composition is given
immediately after the exposure to prevent or reduce the ototoxic
effect. More preferably, treatment is provided prophylactically,
either by administration of the composition prior to or
concomitantly with the ototoxic pharmaceutical or the exposure to
the ototoxin.
[0273] By "ototoxin" in the context of the present invention is
meant a substance that through its chemical action injures, impairs
or inhibits the activity of the sound receptors component of the
nervous system related to hearing, which in turn impairs hearing
(and/or balance). In the context of the present invention,
ototoxicity includes a deleterious effect on the inner ear hair
cells. Ototoxic agents that cause hearing impairments include, but
are not limited to, antineoplastic agents such as vincristine,
vinblastine, cisplatin and cisplatin-like compounds, taxol and
taxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine;
alcohol; metals; industrial toxins involved in occupational or
environmental exposure; contaminants of food or medicinals; and
over-doses of vitamins or therapeutic drugs, e.g., antibiotics such
as penicillin or chloramphenicol, and megadoses of vitamins A, D,
or B6, salicylates, quinines, loop diuretics, and phosphodiesterase
type 5 (PDE5) inhibitors such as sildenafil citrate
(Viagra.RTM.).
[0274] By "exposure to an ototoxic agent" is meant that the
ototoxic agent is made available to, or comes into contact with, a
mammal. Exposure to an ototoxic agent can occur by direct
administration, e.g., by ingestion or administration of a food,
medicinal, or therapeutic agent, e.g., a chemotherapeutic agent, by
accidental contamination, or by environmental exposure, e.g.,
aerial or aqueous exposure.
[0275] Hearing may be due to end-organ lesions involving inner ear
hair cells, e.g., acoustic trauma, viral endolymphatic
labyrinthitis, Meniere's disease. Hearing impairments include
tinnitus, which is a perception of sound in the absence of an
acoustic stimulus, and may be intermittent or continuous, wherein
there is diagnosed a sensorineural loss. Hearing loss may be due to
bacterial or viral infection, such as in herpes zoster oticus,
purulent labyrinthitis arising from acute otitis media, purulent
meningitis, chronic otitis media, sudden deafness including that of
viral origin, e.g., viral endolymphatic labyrinthitis caused by
viruses including mumps, measles, influenza, chicken pox,
mononucleosis and adenoviruses. The hearing loss can be congenital,
such as that caused by rubella, anoxia during birth, bleeding into
the inner ear due to trauma during delivery, ototoxic drugs
administered to the mother, erythroblastosis fetalis, and
hereditary conditions including Waardenburg's syndrome and Hurler's
syndrome.
[0276] The hearing loss can be noise-induced, generally due to a
noise greater than 85 decibels (db) that damages the inner ear. In
a particular aspect of the invention, the hearing loss is caused by
an ototoxic drug that effects the auditory portion of the inner
ear, particularly inner ear hair cells. Incorporated herein by
reference are chapters 196, 197, 198 and 199 of The Merck Manual of
Diagnosis and Therapy, 14th Edition, (1982), Merck Sharp & Dome
Research Laboratories, N.J. and corresponding chapters in the most
recent 16th edition, including Chapters 207 and 210) relating to
description and diagnosis of hearing and balance impairments.
[0277] It is the object of the present invention to provide a
method and compositions for treating a mammal, to prevent, reduce,
or treat a hearing impairment, disorder or imbalance, preferably an
ototoxin-induced hearing condition, by administering to a mammal in
need of such treatment a composition of the invention. One
embodiment of the invention is a method for treating a hearing
disorder or impairment wherein the ototoxicity results from
administration of a therapeutically effective amount of an ototoxic
pharmaceutical drug. Typical ototoxic drugs are chemotherapeutic
agents, e.g. antineoplastic agents, and antibiotics. Other possible
candidates include loop-diuretics, quinines or a quinine-like
compound, and salicylate or salicylate-like compounds.
[0278] The methods and compositions of the present invention are
especially effective when the ototoxic compound is an antibiotic,
preferably an aminoglycoside antibiotic. Ototoxic aminoglycoside
antibiotics include but are not limited to neomycin, paromomycin,
ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,
viomycin, gentamicin, sisomicin, netilmicin, streptomycin,
dibekacin, fortimicin, and dihydrostreptomycin, or combinations
thereof. Particular antibiotics include neomycin B, kanamycin A,
kanamycin B, gentamicin C1, gentamicin C1a, and gentamicin C2.
[0279] The methods and compositions of the present invention are
also effective when the ototoxic compound is a antineoplastic agent
such as vincristine, vinblastine, cisplatin and cisplatin-like
compounds and taxol and taxol-like compounds.
[0280] The methods and compositions of the present invention are
also effective in the treatment of acoustic trauma or mechanical
trauma, preferably acoustic or mechanical trauma that leads to
inner ear hair cell loss. Acoustic trauma to be treated in the
present invention may be caused by a single exposure to an
extremely loud sound, or following long-term exposure to everyday
loud sounds above 85 decibels. Mechanical inner ear trauma to be
treated in the present invention is for example the inner ear
trauma following an operation of electronic device insertion in the
inner ear. The compositions of the present invention prevent or
minimize the damage to inner ear hair cells associated with the
operation.
[0281] In some embodiments the composition of the invention is
co-administered with an ototoxin. For example, an improved method
is provided for treatment of infection of a mammal by
administration of an aminoglycoside antibiotic, the improvement
comprising administering a therapeutically effective amount of one
or more compounds (particularly novel siRNAs) which down-regulate
expression of the pro-apoptotic genes, to the subject in need of
such treatment to reduce or prevent ototoxin-induced hearing
impairment associated with the antibiotic. The compounds which
down-regulate expression of the pro-apoptotic genes particularly
novel siRNAs are preferably administered locally within the inner
ear.
[0282] In yet another embodiment an improved method for treatment
of cancer in a mammal by administration of a chemotherapeutic
compound is provided, wherein the improvement comprises
administering a therapeutically effective amount of a composition
of the invention to the subject in need of such treatment to reduce
or prevent ototoxin-induced hearing impairment associated with the
chemotherapeutic drug. The compounds which reduce or prevent the
ototoxin-induced hearing impairment, eg. the novel siRNAs inter
alia are preferably administered locally within the inner ear.
[0283] In another embodiment the methods of treatment are applied
to treatment of hearing loss resulting from the administration of a
chemotherapeutic agent in order to treat its ototoxic side-effect.
Ototoxic chemotherapeutic agents amenable to the methods of the
invention include, but are not limited to an antineoplastic agent,
including cisplatin or cisplatin-like compounds, taxol or
taxol-like compounds, and other chemotherapeutic agents believed to
cause ototoxin-induced hearing impairments, e.g., vincristine, an
antineoplastic drug used to treat hematological malignancies and
sarcomas. Cisplatin-like compounds include carboplatin
(Paraplatin.RTM.), tetraplatin, oxaliplatin, aroplatin and
transplatin inter alia.
[0284] In another embodiment the methods of the invention are
applied to hearing impairments resulting from the administration of
quinine and its synthetic substitutes, typically used in the
treatment of malaria, to treat its ototoxic side-effect.
[0285] In another embodiment the methods of the invention are
applied to hearing impairments resulting from administration of a
diuretic to treat its ototoxic side-effect. Diuretics, particularly
"loop" diuretics, i.e. those that act primarily in the Loop of
Henle, are candidate ototoxins. Illustrative examples, not limiting
to the invention method, include furosemide, ethacrylic acid, and
mercurials. Diuretics are typically used to prevent or eliminate
edema. Diuretics are also used in nonedematous states for example
hypertension, hypercalcemia, idiopathic hypercalciuria, and
nephrogenic diabetes insipidus.
[0286] In another preferred embodiment, the compounds of the
invention are used for treating acute renal failure, in particular
acute renal failure due to ischemia in post surgical patients, and
acute renal failure due to chemotherapy treatment such as cisplatin
administration or sepsis-associated acute renal failure. A
preferred use of the compounds of the invention is for the
prevention of acute renal failure in high-risk patients undergoing
major cardiac surgery or vascular surgery. The patients at
high-risk of developing acute renal failure can be identified using
various scoring methods such as the Cleveland Clinic algorithm or
that developed by US Academic Hospitals (QMMI) and by Veterans'
Administration (CICSS). Other preferred uses of the compounds of
the invention are for the prevention of ischemic acute renal
failure in kidney transplant patients or for the prevention of
toxic acute renal failure in patients receiving chemotherapy.
[0287] In another preferred embodiment, the compounds of the
invention are used for treating glaucoma. Main types of glaucoma
are primary open angle glaucoma (POAG), angle closure glaucoma,
normal tension glaucoma and pediatric glaucoma. These are marked by
an increase of intraocular pressure (IOP), or pressure inside the
eye. When optic nerve damage has occurred despite a normal IOP,
this is called normal tension glaucoma. Secondary glaucoma refers
to any case in which another disease causes or contributes to
increased eye pressure, resulting in optic nerve damage and vision
loss.
[0288] In another preferred embodiment, the compounds of the
invention are used for treating or preventing the damage caused by
nephrotoxins such as diuretics, .beta.-blockers, vasodilator
agents, ACE inhibitors, cyclosporin, aminoglycoside antibiotics
(e.g. gentamicin), amphotericin B, cisplatin, radiocontrast media,
immunoglobulins, mannitol, NSAIDs (eg aspirin, ibuprofen,
diclofenac), cyclophosphamide, methotrexate, amciclovir,
polyethylene glycol, .beta.-lactam antibiotics, vancomycin,
rifampicin, sulphonamides, ciprofloxacin, ranitidine, cimetidine,
furosemide, thiazides, phenyloin, penicillamine, lithium salts,
fluoride, demeclocycline, foscamet, aristolochic acid.
[0289] In another preferred embodiment, the compounds of the
invention are used for treating or preventing the damage caused by
spinal-cord injury especially spinal cord trauma caused by motor
vehicle accidents, falls, sports injuries, industrial accidents,
gunshot wounds, spinal cord trauma caused by spine weakening (such
as from rheumatoid arthritis or osteoporosis) or if the spinal
canal protecting the spinal cord has become too narrow (spinal
stenosis) due to the normal aging process, direct damage that occur
when the spinal cord is pulled, pressed sideways, or compressed,
damage to the spinal-cord following bleeding, fluid accumulation,
and swelling inside the spinal cord or outside the spinal cord (but
within the spinal canal). The compounds of the invention are also
used for treating or preventing the damage caused by spinal-cord
injury due to disease such as polio or spina bifida.
[0290] In other embodiments the compounds and methods of the
invention are useful for treating or preventing the incidence or
severity of acute lung injury, in particular conditions which
result from ischemic/reperfusion injury or oxidative stress. For
example, acute respiratory distress syndrome (ARDS) due to
coronavirus infection or endotoxins, severe acute respiratory
syndrome (SARS), ischemia reperfusion injury associated with lung
transplantation and other acute lung injuries.
[0291] In other embodiments the compounds and methods of the
invention are useful for treating or preventing damage following
organ transplantation including lung, liver, heart, bone pancreas,
intestine, skin, blood vessels, heart valve, bone and kidney
transplantation.
[0292] The term "organ transplant" is meant to encompass transplant
of any one or more of the following organs including, inter alia,
lung, kidney, heart, skin, vein, bone, cartilage, liver
transplantation. Although a xenotransplant can be contemplated in
certain situations, an allotransplant is usually preferable. An
autograft can be considered for bone marrow, skin, bone, cartilage
and or blood vessel transplantation.
[0293] The siRNA compounds of the present invention are
particularly useful in treating a subject experiencing the adverse
effects of organ transplant, including ameliorating, treating or
preventing perfusion injury.
[0294] For organ transplantation, either the donor or the recipient
or both may be treated with a compound or composition of the
present invention. Accordingly, the present invention relates to a
method of treating an organ donor or an organ recipient comprising
the step of administering to the organ donor or organ recipient a
therapeutically effective amount of a compound according to the
present invention.
[0295] The invention further relates to a method for preserving an
organ comprising contacting the organ with an effective amount of
compound of the present invention. Also provided is a method for
reducing or preventing injury (in particular reperfusion injury) of
an organ during surgery and/or following removal of the organ from
a subject comprising placing the organ in an organ preserving
solution wherein the solution comprises a compound according to the
present invention.
[0296] In other embodiments the compounds and methods of the
invention are useful for treating or preventing the incidence or
severity of other diseases and conditions in a patient. These
diseases and conditions include stroke and stroke-like situations
(e.g. cerebral, renal, cardiac failure), neuronal cell death, brain
injuries with or without reperfusion issues, chronic degenerative
diseases e.g. neurodegenerative disease including Alzheimer's
disease, Huntington's disease, Parkinson's disease, multiple
sclerosis, amyotrophic lateral sclerosis, spinobulbar atrophy,
prion disease, and apoptosis resulting from traumatic brain injury
(TBI).
[0297] The compounds and methods of the invention are directed to
providing neuroprotection, or to provide cerebroprotection, or to
prevent and/or treat cytotoxic T cell and natural killer
cell-mediated apoptosis associated with autoimmune disease and
transplant rejection, to prevent cell death of cardiac cells
including heart failure, cardiomyopathy, viral infection or
bacterial infection of heart, myocardial ischemia, myocardial
infarct, and myocardial ischemia, coronary artery by-pass graft, to
prevent and/or treat mitochondrial drug toxicity e.g. as a result
of chemotherapy or HIV therapy, to prevent cell death during viral
infection or bacterial infection, or to prevent and/or treat
inflammation or inflammatory diseases, inflammatory bowel disease,
sepsis and septic shock, or to prevent cell death from follicle to
ovocyte stages, from ovocyte to mature egg stages and sperm (for
example, methods of freezing and transplanting ovarian tissue,
artificial fertilization), or to preserve fertility in mammals
after chemotherapy, in particular human mammals, or to prevent
and/or treat, macular degeneration, or to prevent and/or treat
acute hepatitis, chronic active hepatitis, hepatitis-B, and
hepatitis-C, or to prevent hair loss, (e.g. hair loss due-to
male-pattern baldness, or hair loss due to radiation, chemotherapy
or emotional stress), or to treat or ameliorate skin damage whereby
the skin damage may be due to exposure to high levels of radiation,
heat, chemicals, sun, or to burns and autoimmune diseases), or to
prevent cell death of bone marrow cells in myelodysplastic
syndromes (MDS), to treat pancreatitis, to treat rheumatoid
arthritis, psoriasis, glomerulonephritis, atherosclerosis, and
graft versus host disease (GVHD), or to treat retinal pericyte
apoptosis, retinal damages resulting from ischemia, diabetic
retinopathy, or to treat any disease states associated with an
increase of apoptotic cell death.
[0298] The methods comprising administering to the subject a
composition comprising one or more inhibitors (such as siRNA) which
inhibit at least one gene of the present invention in a
therapeutically effective dose, thereby treating the subject. In
one preferred embodiment, siRNA compounds directed to two
pro-apoptotic genes are combined in order to obtain a synergistic
therapeutic effect. In one specific example, siRNA compounds
directed to RhoA (whose mRNA sequence is set forth as SEQ ID NO:46)
are combined with siRNA compounds directed to any other
pro-apoptotic gene of Table A, preferably with siRNA compounds
directed to Casp2 (whose mRNA sequence is set forth as SEQ ID NOS:
10-11).
[0299] The present invention also provides for a process of
preparing a pharmaceutical composition, which comprises: [0300]
providing one or more double stranded compound of the invention;
and [0301] admixing said compound with a pharmaceutically
acceptable carrier.
[0302] The present invention also provides for a process of
preparing a pharmaceutical composition, which comprises admixing
one or more compounds of the present invention with a
pharmaceutically acceptable carrier.
[0303] In a preferred embodiment, the compound used in the
preparation of a pharmaceutical composition is admixed with a
carrier in a pharmaceutically effective dose. In a particular
embodiment the compound of the present invention is conjugated to a
steroid or to a lipid or to another suitable molecule e.g. to
cholesterol.
[0304] Modifications
[0305] Modifications or analogs of nucleotides can be introduced to
improve the therapeutic properties of the nucleotides. Improved
properties include increased nuclease resistance and/or increased
ability to permeate cell membranes.
[0306] Accordingly, the present invention also includes all analogs
of, or modifications to, a oligonucleotide of the invention that
does not substantially affect the function of the polynucleotide or
oligonucleotide. In a preferred embodiment such modification is
related to the base moiety of the nucleotide, to the sugar moiety
of the nucleotide and/or to the phosphate moiety of the
nucleotide.
[0307] In one embodiment the modification is a modification of the
phosphate moiety, whereby the modified phosphate moiety is selected
from the group comprising phosphothioate.
[0308] The compounds of the present invention can be synthesized by
any of the methods that are well-known in the art for synthesis of
ribonucleic (or deoxyribonucleic) oligonucleotides. Such synthesis
is, among others, described in Beaucage and Iyer, Tetrahedron 1992;
48:2223-2311; Beaucage and Iyer, Tetrahedron 1993; 49: 6123-6194
and Caruthers, et. al., Methods Enzymol. 1987; 154: 287-313; the
synthesis of thiolates is, among others, described in Eckstein,
Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA
molecules is described in Sproat, in Humana Press 2005 edited by
Herdewijn P.; Kap. 2: 17-31 and respective downstream processes
are, among others, described in Pingoud et. al., in IRL Press 1989
edited by Oliver R. W. A.; Kap. 7: 183-208.
[0309] Other synthetic procedures are known in the art e.g. the
procedures as described in Usman et al., 1987, J. Am. Chem. Soc.,
109, 7845; Scaringe et al., 1990, NAR., 18, 5433; Wincott et al.,
1995, NAR. 23, 2677-2684; and Wincott et al., 1997, Methods Mol.
Bio., 74, 59, and these procedures may make use of common nucleic
acid protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end. The modified (e.g.
2'-O-methylated) nucleotides and unmodified nucleotides are
incorporated as desired.
[0310] The oligonucleotides of the present invention can be
synthesized separately and joined together post-synthetically, for
example, by ligation (Moore et al., 1992, Science 256, 9923; Draper
et al., International Patent Publication No. WO 93/23569; Shabarova
et al., 1991, NAR 19, 4247; Bellon et al., 1997, Nucleosides &
Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8,
204), or by hybridization following synthesis and/or
deprotection.
[0311] It is noted that a commercially available machine
(available, inter alia, from Applied Biosystems) can be used; the
oligonucleotides are prepared according to the sequences disclosed
herein. Overlapping pairs of chemically synthesized fragments can
be ligated using methods well known in the art (e.g., see U.S. Pat.
No. 6,121,426). The strands are synthesized separately and then are
annealed to each other in the tube. Then, the double-stranded
siRNAs are separated from the single-stranded oligonucleotides that
were not annealed (e.g. because of the excess of one of them) by
HPLC. In relation to the siRNAs or siRNA fragments of the present
invention, two or more such sequences can be synthesized and linked
together for use in the present invention.
[0312] The compounds of the invention can also be synthesized via
tandem synthesis methodology, as described for example in US Patent
Publication No. US 2004/0019001 (McSwiggen), wherein both siRNA
strands are synthesized as a single contiguous oligonucleotide
fragment or strand separated by a cleavable linker which is
subsequently cleaved to provide separate siRNA fragments or strands
that hybridize and permit purification of the siRNA duplex. The
linker can be a polynucleotide linker or a non-nucleotide
linker.
[0313] The present invention further provides for a pharmaceutical
composition comprising two or more siRNA molecules for the
treatment of any of the diseases and conditions mentioned herein,
whereby said two molecules may be physically mixed together in the
pharmaceutical composition in amounts which generate equal or
otherwise beneficial activity, or may be covalently or
non-covalently bound, or joined together by a nucleic acid linker
of a length ranging from 2-100, preferably 2-50 or 2-30
nucleotides. In one embodiment, the siRNA molecules are comprised
of a double-stranded nucleic acid structure as described herein,
wherein the two siRNA sequences are selected from the nucleic acids
set forth in Table B.
[0314] Thus, the siRNA molecules may be covalently or
non-covalently bound or joined by a linker to form a tandem siRNA
compound. Such tandem siRNA compounds comprising two siRNA
sequences are typically of 38-150 nucleotides in length, more
preferably 38 or 40-60 nucleotides in length, and longer
accordingly if more than two siRNA sequences are included in the
tandem molecule. A longer tandem compound comprised of two or more
longer sequences which encode siRNA produced via internal cellular
processing, e.g., long dsRNAs, is also envisaged, as is a tandem
molecule encoding two or more shRNAs. Such tandem molecules are
also considered to be a part of the present invention. A tandem
compound comprising two or more siRNAs sequences of the invention
is envisaged. In a particular embodiment, the tandem comprises RhoA
siRNA covalently linked to one or more of the other siRNAs of the
invention. In a more particular embodiment, the tandem compound may
comprise a sequence comprising siRNA to RhoA and a sequence
comprising siRNA to Casp2. Without being bound by theory RhoA is a
small GTPase that when activated inhibits neurite outgrowth and its
inhibition is relevant for spinal cord injury. Thus a tandem
compound for this indication can comprise RhoA siRNA sequence and
one or more siRNA sequences to anti-apoptotic siRNAs of the
invention. The latter will protect, and siRNA to RhoA will promote
regeneration, and so a combined or even synergistic effect is
produced.
[0315] siRNA molecules that target the pro-apoptotic genes of the
invention may be the main active component in a pharmaceutical
composition, or may be one active component of a pharmaceutical
composition containing two or more siRNAs (or molecules which
encode or endogenously produce two or more siRNAs, be it a mixture
of molecules or one or more tandem molecules which encode two or
more siRNAs), said pharmaceutical composition further being
comprised of one or more additional siRNA molecule which targets
one or more additional gene. Simultaneous inhibition of said
additional gene(s) will likely have an additive or synergistic
effect for treatment of the diseases disclosed herein.
[0316] Additionally, the pro-apoptotic siRNA disclosed herein or
any nucleic acid molecule comprising or encoding such siRNA can be
linked or bound (covalently or non-covalently) to antibodies
(including aptamer molecules) against cell surface internalizable
molecules expressed on the target cells, in order to achieve
enhanced targeting for treatment of the diseases disclosed herein.
For example, anti-Fas antibody (preferably a neutralizing antibody)
may be combined (covalently or non-covalently) with any
pro-apoptotic siRNA. In another example, an aptamer which can act
like a ligand/antibody may be combined (covalently or
non-covalently) with any pro-apoptotic siRNA.
[0317] The compounds of the present invention can be delivered
either directly or with viral or non-viral vectors. When delivered
directly the sequences are generally rendered nuclease resistant.
Alternatively the sequences can be incorporated into expression
cassettes or constructs such that the sequence is expressed in the
cell as discussed herein below. Generally the construct contains
the proper regulatory sequence or promoter to allow the sequence to
be expressed in the targeted cell. Vectors optionally used for
delivery of the compounds of the present invention are commercially
available, and may be modified for the purpose of delivery of the
compounds of the present invention by methods known to one of skill
in the art.
[0318] It is also envisaged that a long oligonucleotide (typically
25-500 nucleotides in length) comprising one or more stem and loop
structures, where stem regions comprise the sequences of the
oligonucleotides of the invention, may be delivered in a carrier,
preferably a pharmaceutically acceptable carrier, and may be
processed intracellularly by endogenous cellular complexes (e.g. by
DROSHA and DICER as described above) to produce one or more smaller
double stranded oligonucleotides (siRNAs) which are
oligonucleotides of the invention. This oligonucleotide can be
termed a tandem shRNA construct. It is envisaged that this long
oligonucleotide is a single stranded oligonucleotide comprising one
or more stem and loop structures, wherein each stem region
comprises a sense and corresponding antisense siRNA sequence of the
pro-apoptotic genes of the invention. In particular, it is
envisaged that this oligonucleotide comprises sense and antisense
siRNA sequences as depicted in Table B, set forth in SEQ ID
NOS:277-50970 and 50993-68654.
[0319] All analogues of, or modifications to, a
nucleotide/oligonucleotide may be employed with the present
invention, provided that said analogue or modification does not
substantially affect the function of the
nucleotide/oligonucleotide. The nucleotides can be selected from
naturally occurring or synthetic modified bases. Naturally
occurring bases include adenine, guanine, cytosine, thymine and
uracil. Modified bases of nucleotides include inosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl
adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza
thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,
8-aminoadenine, 8-thiol adenine, 8-thioalkyl adenines, 8-hydroxyl
adenine and other 8-substituted adenines, 8-halo guanines, 8-amino
guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine
and other substituted guanines, other aza and deaza adenines, other
aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro
cytosine. In some embodiments one or more nucleotides in an
oligomer is substituted with inosine.
[0320] In addition, analogues of polynucleotides can be prepared
wherein the structure of one or more nucleotide is fundamentally
altered and better suited as therapeutic or experimental reagents.
An example of a nucleotide analogue is a peptide nucleic acid (PNA)
wherein the deoxyribose (or ribose) phosphate backbone in DNA (or
RNA is replaced with a polyamide backbone which is similar to that
found in peptides. PNA analogues have been shown to be resistant to
enzymatic degradation and to have extended stability in vivo and in
vitro. Other modifications that can be made to oligonucleotides
include polymer backbones, cyclic backbones, acyclic backbones,
thiophosphate-D-ribose backbones, triester backbones, thiolate
backbones, 2'-5' bridged backbone, artificial nucleic acids,
morpholino nucleic acids, locked nucleic acid (LNA), glycol nucleic
acid (GNA), threose nucleic acid (TNA), arabinoside, and mirror
nucleoside (for example, beta-L-deoxynucleoside instead of
beta-D-deoxynucleoside). Examples of siRNA compounds comprising LNA
nucleotides are disclosed in Elmen et al., (NAR 2005,
33(1):439-447).
[0321] The compounds of the present invention can be synthesized
using one or more inverted nucleotides, for example inverted
thymidine or inverted adenine (see, for example, Takei, et al.,
2002, JBC 277(26):23800-06).
[0322] A "mirror" nucleotide is a nucleotide with reversed
chirality to the naturally occurring or commonly employed
nucleotide, i.e., a mirror image (L-nucleotide) of the naturally
occurring (D-nucleotide). The nucleotide can be a ribonucleotide or
a deoxyribonucleotide and my further comprise at least one sugar,
base and or backbone modification. U.S. Pat. No. 6,602,858
discloses nucleic acid catalysts comprising at least one
L-nucleotide substitution.
[0323] Although the inhibitors of the present invention are
preferably siRNA molecules, other inhibitors contemplated to be
used in the methods of the invention to inhibit a pro-apoptotic
gene and to treat the diseases and conditions described herein are
inter alia antibodies, preferably neutralizing antibodies or
fragments thereof, including single chain antibodies, antisense
oligonucleotides, antisense DNA or RNA molecules, ribozymes,
proteins, polypeptides and peptides including peptidomimetics and
dominant negatives, and also expression vectors expressing all the
above. Additional inhibitors may be small chemical molecules, which
generally have a molecular weight of less than 2000 daltons, more
preferably less than 1000 daltons, even more preferably less than
500 daltons. These inhibitors may act as follows: small molecules
may affect expression and/or activity; antibodies may affect
activity; all kinds of antisense may affect the pro-apoptotic gene
expression; and dominant negative polypeptides and peptidomimetics
may affect activity; expression vectors may be used inter alia for
delivery of antisense or dominant-negative polypeptides or
antibodies.
[0324] Antibodies
[0325] The term "antibody" refers to IgG, IgM, IgD, IgA, and IgE
antibody, inter alia. The definition includes polyclonal antibodies
or monoclonal antibodies. This term refers to whole antibodies or
fragments of antibodies comprising an antigen-binding domain, e.g.
antibodies without the Fc portion, single chain antibodies,
miniantibodies, fragments consisting of essentially only the
variable, antigen-binding domain of the antibody, etc. The term
"antibody" may also refer to antibodies against polynucleotide
sequences obtained by cDNA vaccination. The term also encompasses
antibody fragments which retain the ability to selectively bind
with their antigen or receptor and are exemplified as follows,
inter alia: [0326] (1) Fab, the fragment which contains a
monovalent antigen-binding fragment of an antibody molecule which
can be produced by digestion of whole antibody with the enzyme
papain to yield a light chain and a portion of the heavy chain;
[0327] (2) (Fab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab'.sub.2) is a dimer of two Fab fragments
held together by two disulfide bonds; [0328] (3) Fv, defined as a
genetically engineered fragment containing the variable region of
the light chain and the variable region of the heavy chain
expressed as two chains; and [0329] (4) Single chain antibody
(SCA), defined as a genetically engineered molecule containing the
variable region of the light chain and the variable region of the
heavy chain linked by a suitable polypeptide linker as a
genetically fused single chain molecule.
[0330] The genes of the present invention that are preferably
inhibited using specific antibodies for the treatment of a desired
disease are reticulon 4 receptor (RTN4R) and annexin A2
(ANXA2).
[0331] Antisense Molecules
[0332] By the term "antisense" (AS) or "antisense fragment" is
meant a polynucleotide fragment (comprising either
deoxyribonucleotides, ribonucleotides or a mixture of both) having
inhibitory antisense activity, said activity causing a decrease in
the expression of the endogenous genomic copy of the corresponding
gene. An AS polynucleotide is a polynucleotide which comprises
consecutive nucleotides having a sequence of sufficient length and
homology to a sequence present within the sequence of the target
gene to permit hybridization of the AS to the gene. Many reviews
have covered the main aspects of antisense (AS) technology and its
enormous therapeutic potential (Aboul-Fadl, Curr Med Chem. 2005,
12(19):2193-214; Crooke, Curr Mol Med. 2004. 4(5):465-87; Crooke,
Annu Rev Med. 2004; 55:61-95; Vacek et al., Cell Mol Life Sci.
2003. 60(5):825-33; Cho-Chung, Arch Pharm Res. 2003. 26(3):183-91.
There are further reviews on the chemical (Crooke, 1995; Uhlmann et
al, 1990), cellular (Wagner, 1994. Nature. 24; 372(6504):333-5) and
therapeutic (Hanania, et al, 1995; Scanlon, et al, 1995; Gewirtz,
1993) aspects of this technology. Antisense intervention in the
expression of specific genes can be achieved by the use of
synthetic AS oligonucleotide sequences (see for example, Zhang et
al., Curr Cancer Drug Targets. 2005 5(1):43-9.)
[0333] AS oligonucleotide sequences may be short sequences of DNA,
typically 15-30 mer but may be as small as 7 mer (Wagner et al,
1996 Nat Biotechnol. 14(7):840-4), designed to complement a target
mRNA of interest and form an RNA:AS duplex. This duplex formation
can prevent processing, splicing, transport or translation of the
relevant mRNA. Moreover, certain AS nucleotide sequences can elicit
cellular RNase H activity when hybridized with their target mRNA,
resulting in mRNA degradation (Calabretta et al, 1996 Semin Oncol.
23(1):78-87). In that case, RNase H will cleave the RNA component
of the duplex and can potentially release the AS to further
hybridize with additional molecules of the target RNA. An
additional mode of action results from the interaction of AS with
genomic DNA to form a triple helix which can be transcriptionally
inactive.
[0334] The sequence target segment for the antisense
oligonucleotide is selected such that the sequence exhibits
suitable energy related characteristics important for
oligonucleotide duplex formation with their complementary
templates, and shows a low potential for self-dimerization or
self-complementation (Anazodo et al., 1996 BBRC. 229(1):305-9). For
example, the computer program OLIGO (Primer Analysis Software,
Version 3.4), can be used to determine antisense sequence melting
temperature, free energy properties, and to estimate potential
self-dimer formation and self-complimentary properties. The program
allows the determination of a qualitative estimation of these two
parameters (potential self-dimer formation and self-complimentary)
and provides an indication of "no potential" or "some potential" or
"essentially complete potential". Using this program target
segments are generally selected that have estimates of no potential
in these parameters. However, segments can be used that have "some
potential" in one of the categories. A balance of the parameters is
used in the selection as is known in the art. Further, the
oligonucleotides are also selected as needed so that analogue
substitution do not substantially affect function.
[0335] Phosphorothioate antisense oligonucleotides do not normally
show significant toxicity at concentrations that are effective and
exhibit sufficient pharmacodynamic half-lives in animals (Agarwal
et al., 1996) and are nuclease resistant. Antisense induced
loss-of-function phenotypes related with cellular development were
shown for a variety of different genes including integrin (Galileo
et al., Neuron. 1992 9(6):1117-31.) and for the N-myc protein
(Rosolen et al., 1990 Prog Clin Biol Res. 366:29-36). Antisense
oligonucleotide inhibition of basic fibroblast growth factor
(bFGF), having mitogenic and angiogenic properties, suppressed 80%
of growth in glioma cells (Morrison, J B C 1991 266(2):728-34) in a
saturable and specific manner. Being hydrophobic, antisense
oligonucleotides interact well with phospholipid membranes (Akhter
et al., NAR. 1991, 19:5551-5559). Following their interaction with
the cellular plasma membrane, they are actively (or passively)
transported into living cells (Loke et al., PNAS 1989,
86(10):3474-8), in a saturable mechanism predicted to involve
specific receptors (Yakubov et al., PNAS, 1989 86(17):6454-58).
[0336] Ribozymes
[0337] A "ribozyme" is an RNA molecule that possesses RNA catalytic
ability (see Cech Biochem Soc Trans. 2002 November; 30(Pt
6):1162-6, for review) and cleaves a specific site in a target RNA.
In accordance with the present invention, ribozymes which cleave
mRNA may be utilized as inhibitors. This may be necessary in cases
where antisense therapy is limited by stoichiometric considerations
(Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325).
Ribozymes can then be used that will target the a gene associated
with a bone marrow disease. The number of RNA molecules that are
cleaved by a ribozyme is greater than the number predicted by
stochiochemistry (Hampel and Tritz, Biochem. 1989, 28(12):4929-33;
Uhlenbeck, Nature. 1987 328(6131):596-600).
[0338] Ribozymes catalyze the phosphodiester bond cleavage of RNA.
Several ribozyme structural families have been identified including
Group I introns, RNase P, the hepatitis delta virus ribozyme,
hammerhead ribozymes and the hairpin ribozyme originally derived
from the negative strand of the tobacco ringspot virus satellite
RNA (sTRSV) (Sullivan, 1994; U.S. Pat. No. 5,225,347). The latter
two families are derived from viroids and virusoids, in which the
ribozyme is believed to separate monomers from oligomers created
during rolling circle replication (Symons, 1989 and 1992).
Hammerhead and hairpin ribozyme motifs are most commonly adapted
for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994). In
general the ribozyme has a length of from about 30-100 nucleotides.
Delivery of ribozymes is similar to that of AS fragments and/or
siRNA molecules.
[0339] Screening of Inactivation Compounds for Pro-Apoptotic
Genes:
[0340] Some of the compounds and compositions of the present
invention may be used in a screening assay for identifying and
isolating compounds that modulate the activity of a pro-apoptotic
gene, in particular compounds that modulate a disorder accompanied
by an elevated level of the pro-apoptotic genes of the invention.
The compounds to be screened comprise inter alia substances such as
small chemical molecules and antisense oligonucleotides.
[0341] The inhibitory activity of the compounds of the present
invention on pro-apoptotic genes or binding of the compounds of the
present invention to pro-apoptotic genes may be used to determine
the interaction of an additional compound with the pro-apoptotic
polypeptide, e.g., if the additional compound competes with the
oligonucleotides of the present invention for inhibition of a
pro-apoptotic gene, or if the additional compound rescues said
inhibition. The inhibition or activation can be tested by various
means, such as, inter alia, assaying for the product of the
activity of the pro-apoptotic polypeptide or displacement of
binding compound from the pro-apoptotic polypeptide in radioactive
or fluorescent competition assays.
[0342] The present invention is illustrated in detail below with
reference to examples, but is not to be construed as being limited
thereto.
[0343] Citation of any document herein is not intended as an
admission that such document is pertinent prior art, or considered
material to the patentability of any claim of the present
application. Any statement as to content or a date of any document
is based on the information available to applicant at the time of
filing and does not constitute an admission as to the correctness
of such a statement.
EXAMPLES
General Methods in Molecular Biology
[0344] Standard molecular biology techniques known in the art and
not specifically described were generally followed as in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York (1989), and as in Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1989) and as in Perbal, A Practical Guide to
Molecular Cloning, John Wiley & Sons, New York (1988), and as
in Watson et al., Recombinant DNA, Scientific American Books, New
York and in Birren et al (eds) Genome Analysis: A Laboratory Manual
Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York
(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;
4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated
herein by reference. Polymerase chain reaction (PCR) was carried
out generally as in PCR Protocols: A Guide To Methods And
Applications, Academic Press, San Diego, Calif. (1990). In situ (In
cell) PCR in combination with Flow Cytometry can be used for
detection of cells containing specific DNA and mRNA sequences
(Testoni et al., 1996, Blood 87:3822.) Methods of performing RT-PCR
are also well known in the art.
Example 1
In Vitro Testing of the siRNA Compounds for Pro-Apoptotic Genes
[0345] 1. General
[0346] About 1.5-2.times.10.sup.5 test cells (HeLa cells or 293T
cells for siRNA targeting the human gene and NRK52 cells or NMUMG
cells for siRNA targeting the rat/mouse gene) were seeded per well
in 6 wells plate (70-80% confluent).
[0347] After 24 h cells were transfected with siRNA oligomers using
Lipofectamine.TM. 2000 reagent (Invitrogene) at final concentration
of 500 .mu.M, 5 nM, 20 nM or 40 nM. The cells were incubated at
37.degree. C. in a CO2 incubator for 72 h.
[0348] As positive control for cells transfection PTEN-Cy3 labeled
siRNA oligos were used. As negative control for siRNA activity GFP
siRNA oligos were used.
[0349] About 72 h after transfection cells were harvested and RNA
was extracted from cells. Transfection efficiency was tested by
fluorescent microscopy.
[0350] The siRNAs used in the in vitro experiments described in
Example 1 were 19-mers or 23-mers, having alternating
ribonucleotides modified in both the antisense and the sense
strands of the compound. For 19-mers, the modification was such
that a 2'-O-methyl (Me) group was present on the first, third,
fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth
and nineteenth nucleotide of the antisense strand, whereby the very
same modification, i.e. a 2'-O-Me group, was present at the second,
fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and
eighteenth nucleotide of the sense strand. These particular siRNA
compounds were also blunt ended and were non-phosphorylated at the
termini; however, comparative experiments have shown that siRNAs
phosphorylated at the 3'-termini have similar activity.
[0351] Results:
[0352] The percent of inhibition of gene expression using specific
siRNAs was determined using qPCR analysis of target gene in cells
expressing the endogenous gene. The data in Tables C1, C2 and C3
below demonstrate the percent of residual expression of the target
gene in cells following treatment with specific siRNA molecules
(Tables C1 and C3: 19-mer siRNA compounds; Table C2: 23-mer siRNA
compounds). In general, the siRNAs having specific sequences that
were selected for in vitro testing were specific for both human and
the rat/rabbit genes. Similar results of reduced expression of
specific genes are obtained with other siRNAs, the sequences of
which are listed in Table B.
TABLE-US-00002 TABLE C1 Percent of knockdown of the expression of
the target human gene in cells using 19-mer siRNA molecules. Target
% of gene Cell line* siRNA tested Sequence control** TP53BP2 293T
TP53BP2_1 Sense: 118,75 SEQ ID NOS: 97-98 GAGGGUGAAAUUCAACCCC
Antisense: GGGGUUGAAUUUCACCCUC TP53BP2 293T TP53BP2_2 Sense: 44, 26
SEQ ID NOS: 99-100 CACCCAGAGAACAUUUAUU Antisense:
AAUAAAUGUUCUCUGGGUG TP53BP2 293T TP53BP2_3 Sense: 134, 87 SEQ ID
NOS: 101-102 GGGUGAAAUUCAACCCCCU Antisense: AGGGGGUUGAAUUUCACCC
TP53BP2 293T TP53BP2_4 Sense: 123, 115 SEQ ID No. 103-104
AGGGUGAAAUUCAACCCCC Antisense: GGGGGUUGAAUUUCACCCU TP53BP2 293T
TP53BP2_5 Sense: 89, 30 SEQ ID NOS: 105-106 AGGGAGUGUUUGAAUAAGC
Antisense: GCUUAUUCAAACACUCCCU TP53BP2 293T TP53BP2_6 Sense: 93 SEQ
ID NOS: 107-108 ACCCAGAGAACAUUUAUUC Antisense: GAAUAAAUGUUCUCUGGGU
TP53BP2 293T TP53BP2_8 Sense: 67 SEQ ID NOS: 109-110
CGCUGAGGGAGAAAGAGAA Antisense: UUCUCUUUCUCCCUCAGCG LRDD PC-3 LRDD_1
Sense: 25 SEQ ID NOS: 111-112 CGCACCUGAAGAAUGUGAA Antisense:
UUCACAUUCUUCAGGUGCG LRDD PC-3 LRDD_2 Sense: 38 SEQ ID NOS: 113-114
GUCUUCUACUCGCACCUGA Antisense: UCAGGUGCGAGUAGAAGAC LRDD PC-3 LRDD_3
Sense: 12, 38, 18 SEQ ID Nos. 115-116 GACUGUUCCUGACCUCAGA
Antisense: UCUGAGGUCAGGAACAGUC LRDD PC-3 LRDD_5 Sense: 34, 47, 21
SEQ ID NOS: 117-118 ACCUCAGAUUUGGACAGCU Antisense:
AGCUGUCCAAAUCUGAGGU CYBA MDA- CYBA_15 Sense: 5,3 MB-4 SEQ ID NOS:
119-120 UGGGGACAGAAGUACAUGA Antisense: UCAUGUACUUCUGUCCCCA CYBA
MDA- CYBA_16 Sense: 28,20 MB-4 SEQ ID NOS: 121-122
GGGCCCUUUACCAGGAAUU Antisense: AAUUCCUGGUAAAGGGCCC CYBA MDA-
CYBA_17 Sense: 5,2 MB-4 SEQ ID NOS: 123-124 CCCUUUACCAGGAAUUACU
Antisense: AGUAAUUCCUGGUAAAGGG ATF3 293T ATF3_2 Sense: 107, 72 SEQ
ID NOS: 125-126 GAAGGAACAUUGCAGAGCU Antisense: AGCUCUGCAAUGUUCCUUC
ATF3 293T ATF3_3 Sense: 109, 80 SEQ ID NOS: 127-128
ACAGAUAAAAGAAGGAACA Antisense: UGUUCCUUCUUUUAUCUGU ATF3 293T ATF3_4
Sense: 79, 60 SEQ ID NOS: 129-130 AUCCUAGUAUUCCUAACCU Antisense:
AGGUUAGGAAUACUAGGAU ATF3 293T ATF3_5 Sense: 93, 90 SEQ ID NOS:
131-132 AUCCCAGUAUUCCUAGCCU Antisense: AGGCUAGGAAUACUGGGAU CASP2
HeLa- CASP2_1 Sense: 12, 8 SEQ ID NOS: 133-134 GCACUCCUGAAUUUUAUCA
Antisense: UGAUAAAAUUCAGGAGUGC CASP2 HeLa- CASP2_2 Sense: 25, 38
SEQ ID NOS: 135-136 GCACAGGAAAUGCAAGAGA Antisense:
UCUCUUGCAUUUCCUGUGC CASP2 HeLa- CASP2_3 Sense: 22, 39 SEQ ID NOS:
137-138 GGGCUUGUGAUAUGCACGU Antisense: ACGUGCAUAUCACAAGCCC CASP2
HeLa- CASP2_4 Sense: 11, 18 SEQ ID NOS: 139-140 GCCAGAAUGUGGAACUCCU
Antisense: AGGAGUUCCACAUUCUGGC NOX3 293 NOX_4 Sense: 21, 32 SEQ ID
NOS: 141-142 UCCUGGAACUUCACAUGAA Antisense: UUCAUGUGAAGUUCCAGGA
NOX3 293 NOX_5 Sense: 28 SEQ ID NOS: 143-144 GGUGUUCAUUUCUAUUACA
Antisense: UGUAAUAGAAAUGAACACC NOX3 293 NOX_6 Sense: 41 SEQ ID NOS:
145-146 ACACACACCAUGUUUUCAU Antisense: AUGAAAACAUGGUGUGUGU NOX3 293
NOX_7 Sense: 26, 36 SEQ ID NOS: 147-148 GGUACACACACCAUGUUUU
Antisense: AAAACAUGGUGUGUGUACC NOX3 293 NOX_8 Sense: 31 SEQ ID NOS:
149-150 CACUUUCUGAGUUAUCAUA Antisense: UAUGAUAACUCAGAAAGUG NOX3 293
NOX_9 Sense: 34 SEQ ID NOS: 151-152 CUGAAAUCUAUAUGGUACA Antisense:
UGUACCAUAUAGAUUUCAG NOX3 293 NOX_10 Sense: 49 SEQ ID NOS: 153-154
CUGGCGAUUUCAACAAGAA Antisense: UUCUUGUUGAAAUCGCCAG NOX3 293 NOX_11
Sense: 39 SEQ ID NOS: 155-156 UCUGGCGAUUUCAACAAGA Antisense:
UCUUGUUGAAAUCGCCAGA HRK MDA- HRK_1 Sense: 15, 20 MB-468 SEQ ID NOS:
157-158 CCCCAAUGCUAUUUACAUA Antisense: UAUGUAAAUAGCAUUGGGG HRK MDA-
HRK_2 Sense: 22, 68 MB-468 SEQ ID NOS: 159-160 AUGCUAUUUACAUACAGCU
Antisense: AGCUGUAUGUAAAUAGCAU CIQBP HeLa- CIQBP_1 Sense: 60 SEQ ID
NOS: 161-162 CCCCAAUGCUAUUUACAUA Antisense: UAUGUAAAUAGCAUUGGGG
CIQBP HeLa- CIQBP2 Sense: 70 SEQ ID NOS: 163-164
AUGCUAUUUACAUACAGCU Antisense: AGCUGUAUGUAAAUAGCAU CIQBP HeLa-
CIQBP3 Sense: 6, 4 SEQ ID NOS: 165-166 GAGCCUGAACUGACAUCAA
Antisense: UUGAUGUCAGUUCAGGCUC BNIP3 293T BNIP3_1 Sense: 58 SEQ ID
NOS: 167-168 GAGACAUGGAAAAAAUACU Antisense: AGUAUUUUUUCCAUGUCUC
BNIP3 293T BNIP3_2 Sense: 96, 73 SEQ ID NOS: 169-170
GACAUGGAAAAAAUACUGC Antisense: GCAGUAUUUUUUCCAUGUC BNIP3 293T
BNIP3_3 Sense: 116 SEQ ID NOS: 171-172 ACCCUCAGCAUGAGGAACA
Antisense: UGUUCCUCAUGCUGAGGGU BNIP3 293T BNIP3_4 Sense: 89, 90 SEQ
ID NOS: 173-174 GAAAAACUCAGAUUGGAUA Antisense: UAUCCAAUCUGAGUUUUUC
BNIP3 293T BNIP3_11 Sense: 69 SEQ ID NOS: CUGCAUUGGUGAAUUUAAU
Antisense: AUUAAAUUCACCAAUGCAG BNIP3 293T BNIP3_12 Sense: 56 SEQ ID
NOS: CAGGUUGUCUACUAAAGAA Antisense: UUCUUUAGUAGACAACCUG BNIP3 293T
BNIP3_13 Sense: 76 SEQ ID NOS: GCCUUAUAUAUCACACUAU Antisense:
AUAGUGUGAUAUAUAAGGC BNIP3 293T BNIP3_15 Sense: 56 SEQ ID NOS:
GGAAUUAAGUCUCCGAUUA Antisense: UAAUCGGAGACUUAAUUCC BNIP3 293T
BNIP3_22 Sense: 78 SEQ ID NOS: AGGUUGUCUACUAAAGAAA Antisense:
UUUCUUUAGUAGACAACCU BNIP3 293T BNIP3_23 Sense: 92 SEQ ID NOS:
GAGAAAAACAGCUCACAGU Antisense ACUGUGAGCUGUUUUUCUC BNIP3 293T
BNIP3_24 Sense: 59 SEQ ID NOS: CCAAGAUAGAGCUACAAAC Antisense
GUUUGUAGCUCUAUCUUGG BNIP3 293T BNIP3_25 Sense: 67 SEQ ID NOS:
189-190 CACUCUGCAUUGGUGAAUU Antisense AAUUCACCAAUGCAGAGUG BNIP3
293T BNIP3_26 Sense: 80 SEQ ID NOS: 191-192 CCUUAAUUCAGCUGAAGUA
Antisense UACUUCAGCUGAAUUAAGG BNIP3 293T BNIP3_27 Sense: 82 SEQ ID
NOS: 193-194 GUUCAACUUUUGUGUGCUU Antisense
AAGCACACAAAAGUUGAAC BNIP3 293T BNIP3_28 Sense: 42 SEQ ID NOS.
195-196 UCCUUUGUGUUCAACUUUU Antisense AAAAGUUGAACACAAAGGA MAPK8
293T MAPK8_1 Sense: 40, 51 SEQ ID NOS: 197-198 ACCACAGAAAUCCCUAGAA
Antisense: UUCUAGGGAUUUCUGUGGU MAPK8 293T MAPK8_2 Sense: 60 SEQ ID
NOS: 199-200 GCCGACCAUUUCAGAAUCA Antisense: UGAUUCUGAAAUGGUCGGC
MAPK8 293T MAPK8_3 Sense: 70 SEQ ID NOS: 201-202
GGACUUACGUUGAAAACAG Antisense: CUGUUUUCAACGUAAGUCC MAPK8 293T
MAPK8_4 Sense: 100 SEQ ID NOS: 203-204 UGGAUGCAAAUCUUUGCCA
Antisense: UGGCAAAGAUUUGCAUCCA MAPK14 A431 MAPK14_1 Sense: + in SEQ
ID NOS: 205-2-6 GACCAUUUCAGUCCAUCAU Western Antisense: blot
AUGAUGGACUGAAAUGGUC MAPK14 A431 MAPK14_2 Sense: + + in SEQ ID NOS:
207-208 GAGGUCUAAAGUAUAUACA Western Antisense: blot
UGUAUAUACUUUAGACCUC MAPK14 A431 MAPK14_3 Sense: - in SEQ ID NOS:
209-210 GUGCUGCUUUUGACACAAA Western Antisense: blot
UUUGUGUCAAAAGCAGCAC RAC1 293T RAC1_1 Sense: 27, 21, 41 SEQ ID NOS:
211-212 UUGGUGCUGUAAAAUACCU Antisense: AGGUAUUUUACAGCACCAA RAC1
293T RAC1_2 Sense: 32, 17, 27 SEQ ID NOS: 213-214
GAGUCCUGCAUCAUUUGAA Antisense: UUCAAAUGAUGCAGGACUC RAC1 293T RAC1_3
Sense: 23, 19, 29 SEQ ID NOS: 215-216 GAUGUGUUCUUAAUUUGCU
Antisense: AGCAAAUUAAGAACACAUC BMP2 Hela BMP2_5 Sense: 58 (in SEQ
ID NOS: 217-218 GUCAAGCCAAACACAAACA 10 nM) Antisense: 15 (in
UGUUUGUGUUUGGCUUGAC 10 nM) SPP1 HEPG2 SPPL_1 Sense: 87 SEQ ID NOS:
219-220 GUCCAGCAAUUAAUAAAAC Antisense: GUUUUAUUAAUUGCUGGAC SPP1
HEPG2 SPP1_2 Sense: 19, 28 SEQ ID NOS: 221-222 GUGCCAUACCAGUUAAACA
Antisense: UGUUUAACUGGUAUGGCAC SPP1 HEPG2 SPP1_3 Sense: 51,39 SEQ
ID NOS: 223-224 GCAAAAUGAAAGAGAACAU Antisense: AUGUUCUCUUUCAUUUUGC
SPP1 HEPG2 SPP1_5 Sense: 26 SEQ ID NOS: 225-226 GCAUUUCUCAUGAAUUAGA
Antisense: UCUAAUUCAUGAGAAAUGC SPP1 HEPG2 SPP1_6 Sense: 26 SEQ ID
NOS: 227-228 CCGCAUUUCUCAUGAAUUA Antisense: UAAUUCAUGAGAAAUGCGG
RHOA 293T- RHOA_1 Sense: 47, 20 human SEQ ID NOS: 229-230
GUACCAGUUAAUUUUUCCA Antisense: UGGAAAAAUUAACUGGUAC RHOA 293T-
RHOA_2 Sense: 34, 14 human SEQ ID NOS: 231-232 UAGAAAACAUCCCAGAAAA
Antisense: UUUUCUGGGAUGUUUUCUA RHOA 293T- RHOA_3 Sense: 49, 26
human SEQ ID NOS: 233-234 ACCAGUUAAUUUUUCCAAC Antisense:
GUUGGAAAAAUUAACUGGU RHOA 293T- RHOA_4 Sense: 29, 20 human SEQ ID
NOS: 235-236 GCCACUUAAUGUAUGUUAC Antisense: GUAACAUACAUUAAGUGGC
RHOA 293T- RHOA_5 Sense: 78 human SEQ ID NOS: 237-238
GGGCAGUUUUUUGAAAAUG Antisense: CAUUUUCAAAAAACUGCCC RHOA 293T-
RHOA_6 Sense: 34 human SEQ ID NOS: 239-240 GGCUAAGUAAAUAGGAAUU
Antisense: AAUUCCUAUUUACUUAGCC RHOA 293T- RHOA_7 Sense: 25 human
SEQ ID NOS: 241-242 CCUGUGGAAAGACAUGCUU Antisense:
AAGCAUGUCUUUCCACAGG RHOA 293T- RHOA_8 Sense: 35 human SEQ ID NOS:
243-244 GUGCUCUUUUCUCCUCACU Antisense: AGUGAGGAGAAAAGAGCAC RHOA
293T- RHOA_9 Sense: 23 human SEQ ID NOS: 245-246
GGGCUAAGUAAAUAGGAAU Antisense: AUUCCUAUUUACUUAGCCC RHOA 293T-
RHOA_10 Sense: 61 human SEQ ID NOS: 247-248 GUGGGCAGUUUUUUGAAAA
Antisense: UUUUCAAAAAACUGCCCAC RHOA 293T- RHOA_11 Sense: 33 human
SEQ ID NOS: 249-250 GGUGCCUUGUCUUGUGAAA Antisense:
UUUCACAAGACAAGGCACC RHOA 293T- RHOA_12 Sense: 79 human SEQ ID NOS:
251-252 CCCAAGUUCAUGCAGCUGU Antisense: ACAGCUGCAUGAACUUGGG RHOA
293T- RHOA_13 Sense: 36 human SEQ ID NOS: 253-254
GGCACUCAGUCUCUCUUCU Antisense: AGAAGAGAGACUGAGUGCC RHOA 293T-
RHOA_14 Sense: 41 human SEQ ID NOS: 255-256 CACUUUGGAAGAUGGCAUA
Antisense: UAUGCCAUCUUCCAAAGUG Duox1 exogenous Duox1_1 Sense: 60
expression SEQ ID NOS: 257-258 GAGAGAAGUUCCAACGCAG Antisense:
CUGCGUUGGAACUUCUCUC Duox1 exogenous Duox1_2 Sense: 72 expression
SEQ ID NOS: 259-260 CGAGAGAAGUUCCAACGCA Antisense:
UGCGUUGGAACUUCUCUCG Duox1 exogenous Duox1_3 Sense: 85 expression
SEQ ID NOS: 261-262 ACCGAGAGAAGUUCCAACG Antisense:
CGUUGGAACUUCUCUCGGU Duox1 exogenous Duox1_4 Sense: 84 expression
SEQ ID NOS: 263-264 AGAUCCCCAAGGAGUAUGA Antisense:
UCAUACUCCUUGGGGAUCU Duox1 exogenous Duox1_6 Sense: 80 expression
SEQ ID NOS: 265-266 UUGCCUCCAUCCUCAAAGA Antisense:
UCUUUGAGGAUGGAGGCAA *cell line used in assay, **% of control in
separate tests using 20 nM concentration of siRNA molecules. All
sequences are presented in a 5'-3' orientation.
TABLE-US-00003 TABLE C2 Percent of knockdown of the expression of
the target human gene in cells using 23 mer siRNA molecules. Cell
line % of Target used for control gene analysis siRNA tested
Sequence ** CASP2 HeLa- CASP2_1-1 Sense: 16 SEQ ID No.
CCUUGCACUCCUGAAUUUUAUCA 267-268 Antisense: UGAUAAAAUUCAGGAGUGCAAGG
CASP2 HeLa- CASP2_1-2 Sense: 23 SEQ ID No. CUUGCACUCCUGAAUUUUAUCAA
269-270 Antisense: UUGAUAAAAUUCAGGAGUGCAAG CASP2 HeLa- CASP2_1-3
Sense: 7 SEQ ID No. UUGCACUCCUGAAUUUUAUCAAA 271-272 Antisense:
UUUGAUAAAAUUCAGGAGUGCAA CASP2 HeLa- CASP2_1-4 Sense: 30 SEQ ID No.
UGCACUCCUGAAUUUUAUCAAAC 273-274 Antisense: GUUUGAUAAAAUUCAGGAGUGCA
CASP2 HeLa- CASP2_1-5 Sense: 10 SEQ ID No. GCACUCCUGAAUUUUAUCAAACA
275-276 Antisense: UGUUUGAUAAAAUUCAGGAGUGC *cell line used in
assay, **% of control in separate tests using 20 nM concentration
of siRNA molecules. All sequences are presented in a 5'-3'
orientation.
TABLE-US-00004 TABLE C3 Percent of knockdown of the expression of
the target human gene in cells using 19-mer siRNAs molecules.
Target Cell % of gene line* siRNA tested Sequence control** P2RX7
Nalm6 P2RX7_6 Sense: 78 SEQ ID NOS: 50971-50972 CCGAGAAACAGGCGAUAAU
Antisense: AUUAUCGCCUGUUUCUCGG P2RX7 Nalm6 P2RX7_7 Sense: 73 SEQ ID
NOS: 50973-50974 CCAGACGCCAIJUUAAAAGU Antisense:
ACUUUUAAAUGGCGUCUGG P2RX7 Nalm6 P2RX7_8 Sense: 75 SEQ ID NOS:
50975-50976 GUGGCUCUGAUUGCUUUAU Antisense: AUAAAGCAAUCAGAGCCAC
P2RX7 Nalm6 P2RX7_9 Sense: 60 (in SEQ ID NOS: 50977-50978
CCAAAGGGAAAUAUGCUUU 5 nM) Antisense: AAAGCAUAUUUCCCUUUGG P2RX7
Nalm6 P2RX7_10 Sense: 87 SEQ ID NOS: 50979-50980
CACAACUACACCACGAGAA Antisense: UUCUCGUGGUGUAGUUGUG TRPM2 Nalm6
TRPM2_5 Sense: 85 SEQ ID NOS:50981-50982 GACAAUGCCUGGAUCGAGA
Antisense: UCUCGAUCCAGGCAUUGUC TRPM2 Nalm6 TRPM2_6 Sense: 46 SEQ ID
NOS:50983-50984 CCAAGAACUUCAACAUGAA Antisense: UUCAUGUUGAAGUUCUUGG
PARG Nalm6 PARG_2 Sense: 59 SEQ ID NOS:50985-50986
GACAGAGUCUUGAAGAUUU Antisense: AAAUCUUCAAGACUCUGUC PARG Nalm6
PARG_3 Sense: 60 SEQ ID NOS:50987-50988 GUGGCAUAUUCUAAGAAAU
Antisense: AUUUCUUAGAAUAUGCCAC PARG Nalm6 PARG_5 Sense: 68 SEQ ID
NOS:50989-50990 CCCAGACAUUAACUUCAAU Antisense: AUUGAAGUUAAUGUCUGGG
CD38 Nalm6 CD38_5 Sense: 0 (in SEQ ID NOS:50991-50992
GGGUGCAUUUAUUUCAAAA 20 nM) Antisense: 23 (in UUUUGAAAUAAAUGCACCC 5
nM) *cell line used in assay, **% of control in separate tests
using 20 nM concentration of siRNA molecules (except if indicated
otherwise). All sequences are presented in a 5'-3' orientation.
Example 2
Model Systems of Acute Renal Failure (ARF)
[0353] ARF is a clinical syndrome characterized by rapid
deterioration of renal function that occurs within days. Without
being bound by theory the acute kidney injury may be the result of
renal ischemia-reperfusion injury such as renal
ischemia-reperfusion injury in patients undergoing major surgery
such as major cardiac surgery. The principal feature of ARF is an
abrupt decline in glomerular filtration rate (GFR), resulting in
the retention of nitrogenous wastes (urea, creatinine). Recent
studies, support that apoptosis in renal tissues is prominent in
most human cases of ARF. The principal site of apoptotic cell death
is the distal nephron. During the initial phase of ischemic injury,
loss of integrity of the actin cytoskeleton leads to flattening of
the epithelium, with loss of the brush border, loss of focal cell
contacts, and subsequent disengagement of the cell from the
underlying substratum.
[0354] Testing an active siRNA compound was performed using an
animal model for ischemia-reperfusion-induced ARF.
[0355] Protection Against Ischemia-Reperfusion Injury Induced ARF
Using Specific Rac1, TP53BP2 and Casp2 siRNA Compounds
[0356] Ischemia-reperfusion injury was induced in rats following 45
minutes bilateral kidney arterial clamp and subsequent release of
the clamp to allow 24 hours of reperfusion. A dose of 12 mg/kg of
the following siRNA compounds was injected into the jugular vein of
individual experimental animals 30 minutes prior to and 4 hours
following the clamp. ARF progression was monitored by measurement
of serum creatinine levels before (baseline) and 24 hrs post
surgery.
TABLE-US-00005 SEQ ID NO:213 Rac1_2: Sense sequence:
GAGUCCUGCAUCAUUUGAA,; SEQ ID NO:214 Antisense sequence:
UUCAAAUGAUGCAGGACUC,. SEQ ID NO:99 TP53BP2_2: Sense sequence:
CACCCAGAGAACAUUUAUU,; SEQ ID NO:100 Antisense sequence:
AAUAAAUGUUCUCUGGGUG,. SEQ ID NO:139 Casp2_4: Sense sequence:
GCCAGAAUGUGGAACUCCU,; SEQ ID NO:140 Antisense sequence:
AGGAGUUCCACAUUCUGGC,.
[0357] At the end of the experiment, the rats were perfused via an
indwelling femoral line with warm PBS followed by 4%
paraformaldehyde. The left kidneys were surgically removed and
stored in 4% paraformaldehyde for subsequent histological analysis.
Acute renal failure is frequently defined as an acute increase of
the serum creatinine level from baseline. An increase of at least
0.5 mg per dL or 44.2 .mu.mol per L of serum creatinine is
considered as an indication for acute renal failure. Serum
creatinine was measured at time zero before the surgery and at 24
hours post ARF surgery. Tables D1-D3 below demonstrate the results
obtained in the ARF model in rats. As revealed from the results,
RAC1, TP53BP2 and Casp2 siRNA compounds reduced creatinine levels
following ischemia-reperfusion induced ARF in an experimental rat
model.
[0358] Table D1: Treatment with Rac1.sub.--2 siRNA (SEQ ID
NOS:213-214)
[0359] Values represent creatinine levels [in mg/dL] prior to
(Baseline) and 24 hours following ischemia-reperfusion induced ARF
in placebo group (PBS), and in RAC1.sub.--2 siRNA treated rats 30
min prior to the ischemic injury (-30') and in RAC1.sub.--2 siRNA
treated rats 4 hours post ischemic injury (+4 h).
TABLE-US-00006 TABLE D1 creatinine creatinine creatinine creatinine
levels with levels levels levels PBS 24 h post with RAC1_2 with
RAC1_2 Animal Baseline ischemic injury siRNA (-30') siRNA (+4 h) 1
0.2 2.4 1.4 1.3 2 0.3 2.7 1.2 1.2 3 0.3 2.7 1.7 1.1 4 0.3 2.2 1.8
1.5 5 0.3 2.4 1.8 1.5 6 0.3 2.5 2.0 1.6 Mean 0.3 2.5 1.7 1.4
[0360] Table D2: Treatment with TP53BP2.sub.--2 siRNA (SEQ ID
NOS:99-100). Values represent creatinine levels 24 hours following
ischemia-reperfusion induced ARF in placebo group (PBS), in
non-relevant GFP siRNA treated rats 4 hours post ischemic injury
(GFP siRNA (+4 h)), in TP53BP2.sub.--2 siRNA treated rats 30 min
prior to the ischemic injury (TP53BP2.sub.--2 siRNA (-30')) and in
TP53BP2.sub.--2 siRNA treated rats 4 hours post ischemic injury
(TP53BP2.sub.--2 siRNA (+4 h)).
TABLE-US-00007 TABLE D2 creatinine creatinine creatinine creatinine
levels with levels with levels with levels with PBS 24 h post GFP
siRNA TP53BP2_2 TP53BP2_2 Animal ischemic injury (+4 h) siRNA
(-30') siRNA (+4 h) 1 3.5 3.10 2.20 1.80 2 4.10 2.80 2.20 1.90 3
4.20 3.20 2.50 1.1 4 3.30 2.30 1.40 5 2.40 6 2.20 Mean 3.78 3.03
2.30 1.55 STD 0.44 0.21 0.13 0.37
[0361] Table D3: Treatment with Casp2.sub.--4 siRNA (SEQ ID
NO:139-140). Values represent creatinine levels prior to (baseline)
and 24 hours following ischemia-reperfusion induced ARF in placebo
group (PBS), in Casp2.sub.--4 siRNA treated rats 30 min prior to
the ischemic injury (-30') and in Casp2.sub.--4 siRNA treated rats
4 hours post ischemic injury (+4 h).
TABLE-US-00008 TABLE D3 creatinine creatinine creatinine creatinine
levels with levels with levels with levels PBS 24 h post Casp2_4
Casp2_4 Animal baseline ischemic injury siRNA (-30') siRNA (+4 h) 1
0.30 2.40 1.10 1.30 2 0.30 3.80 1.00 0.90 3 0.10 2.70 1.70 1.40 4
0.20 2.40 1.90 1.00 5 0.10 3.40 0.90 1.30 6 0.20 2.40 1.20 1.10
Mean 0.20 2.85 1.30 1.17 STD 0.09 0.61 0.40 0.20
[0362] Similar results are obtained following administration of
other siRNA compounds from Table B, in particular siRNAs directed
to particular genes TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, CIQBP,
BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38,
STEAP4, BMP2, CX43, TYROBP, CTGF, and SPP1.
Example 3
Model Systems of Pressure Sores or Pressure Ulcers
[0363] Pressure sores or pressure ulcers including diabetic ulcers,
are areas of damaged skin and tissue that develop when sustained
pressure (usually from a bed or wheelchair) cuts off circulation to
vulnerable parts of the body, especially the skin on the buttocks,
hips and heels. The lack of adequate blood flow leads to ischemic
necrosis and ulceration of the affected tissue. Pressure sores
occur most often in patients with diminished or absent sensation or
who are debilitated, emaciated, paralyzed, or long bedridden.
Tissues over the sacrum, ischia, greater trochanters, external
malleoli, and heels are especially susceptible; other sites may be
involved depending on the patient's situation.
[0364] Testing the active inhibitors of the invention (such as
siRNA compounds) for treating pressure sore, ulcers and similar
wounds is performed in the mouse model described in Reid et al., (J
Surgical Research. 116:172-180, 2004).
[0365] An additional rabbit model (described by Mustoe et al, (JCI,
1991. 87(2):694-703; Ahn and Mustoe, Ann Pl Surg, 1991.
24(1):17-23) is used for testing the siRNA compounds of the
invention. siRNA according to Table B and specifically compounds
directed to genes CIQBP, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38,
STEAP4, BMP2, CX43, or TYROBP are tested in animal models where it
is shown that these siRNA compounds treat and prevent pressure
sores and ulcers.
Example 4
Model Systems of Chronic Obstructive Pulmonary Disease (COPD)
[0366] Chronic obstructive pulmonary disease (COPD) is
characterized mainly by emphysema, a permanent destruction of
peripheral air spaces distal to terminal bronchioles. Emphysema is
also characterized by accumulation of inflammatory cells such as
macrophages and neutrophils in bronchioles and alveolar structures.
Emphysema and chronic bronchitis may occur as part of COPD or
independently.
[0367] Testing the active inhibitors of the invention (such as
siRNA) for treating COPD/emphysema/chronic bronchitis is performed
in animal models such as those disclosed as follows:
[0368] Starcher and Williams, 1989. Lab. Animals, 23:234-240; Peng,
et al., 2004.; Am J Respir Crit Care Med, 169:1245-1251; Jeyaseelan
et al., 2004. Infect. Immunol, 72: 7247-56. Additional models are
described in PCT Patent Publication WO 2006/023544 assigned to the
assignee of the present application, which is hereby incorporated
by reference into this application.
[0369] siRNA according to Table B, and in particular to siRNA to
genes CIQBP, BNIP3, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2,
CX43, TYROBP, CTGF, and DUOX1 are tested in these animal models,
which show that these siRNA compounds may treat and/or prevent
emphysema, chronic bronchitis and COPD.
Example 5
Model Systems of Spinal Cord Injury
[0370] Spinal cord injury, or myelopathy, is a disturbance of the
spinal cord that results in loss of sensation and/or mobility. The
two common types of spinal cord injury are due to trauma and
disease. Traumatic injury can be due to automobile accidents,
falls, gunshot, diving accidents inter alia, and diseases which can
affect the spinal cord include polio, spina bifida, tumors and
Friedreich's ataxia.
[0371] Uptake of siRNA Molecules into Neurons Following Injection
into Injured Spinal-Cord:
[0372] The uptake of Cy3 labeled siRNA (delivered by injection into
the injured cord) in different types of cells was examined
following spinal cord contusion in 18 rats and in uninjured rats (9
rats). Sagittal cryosections were produced and immunostaining using
four different groups of antibodies was performed in order to
determine whether uptake has occurred in neurons, astroglia,
oligodendroglia and/or macrophages/microglia. Markers for neurons
were NeuN, or GAP43; markers for astroglia and potential neural
stem cells were GFAP, nestin or vimentin; markers for
oligodendroglia were NG2 or APC; markers for macrophages/microglia
were ED1 or Iba-1 (Hasegawa et al., 2005. Exp Neurol 193
394-410).
[0373] Six rats were injected with two different doses of Cy3
labeled siRNA (1 .mu.g/l, 10 .mu.g/.mu.l) and were left for 1 and 3
days before sacrifice. Histological analyses indicate that many
long filamentous profiles have taken up the labeled siRNA as well
as other processes and cell bodies. Immunostaining with antibodies
to MAP2 has identified uptake of label into dendrites and into cell
bodies of neurons including motorneurons. Staining with other
antibodies specific to astrocytes or macrophages revealed lower
uptake of Cy3 labeled siRNA as compared to neurons. These results
indicate that siRNA molecules injected to the injured spinal-cord
reach the cell body and dendrites of neurons including
motorneurons.
[0374] Protection Against Spinal-Cord Injury Using Specific RhoA
siRNA Compounds:
[0375] The Spinal-Cord Injury Animal Model:
[0376] Six adult female Sprague-Dawley rats were anesthetized with
40 mg/kg of pentobarbital and the spinal thoracic T9-10 was exposed
by laminectomy. Contusive injury was produced by dropping a 10 gm
rod onto the exposed spinal cord from a height of 12.5 mm using
MASCIS (Multicenter Animal Spinal Cord Injury Study) impactor (as
described In Basso et al., Journal of Neurotrauma Vol 12 (1), p
1-21 1995 and in Basso et al., Journal of Neurotrauma Vol 13 (7), p
343-59 1996). Prior to injury, three point injections of
RhoA.sub.--4 siRNA (Sense sequence: GCCACUUAAUGUAUGUUAC, SEQ ID
NO:235; Antisense sequence: GUAACAUACAUUAAGUGGC, SEQ ID NO:236) at
the concentration of 1 .mu.g/.mu.l were performed at the injury
epicenter 2 mm rostral and caudal to the epicenter (total dose of
30 .mu.g). GFP siRNA was injected in additional five rats as a
control. Each injection was conducted slowly during a period of 10
min into dorsal column (.about.1 mm depth) of T10 using a Hamilton
syringe. Following injections, muscles and skin were closed
separately. Cefazolin (25 mg/kg) was administered for 7 days after
surgery. The behavioral assessment of the recovery following the
spinal cord contusion was preformed using an open field locomotor
test as described by Basso et al (the BBB locomotor rating
scale).
[0377] Table D4 below demonstrates the results obtained in the open
field locomotor test following spinal-cord injury in rats. As
summarized in Table D4 below, RhoA siRNA compounds protect against
spinal-cord injury in an experimental rat model as revealed by
significantly higher BBB locomotor score up to 6 weeks post injury
in the RhoA siRNA treated rats.
[0378] Table D4: Treatment with RhoA.sub.--4 siRNA (SEQ ID
NOS:235-236). Values represent mean BBB locomotor score following
spinal-cord injury in placebo group (GFP siRNA) and in RhoA.sub.--4
siRNA treated group.
TABLE-US-00009 TABLE D4 BBB score BBB score BBB score BBB score BBB
score BBB score BBB score 6 in day 2 1 week 2 weeks 3 weeks 4 weeks
5 weeks weeks post post spinal post spinal post spinal post spinal
post spinal post spinal spinal cord Animal cord injury cord injury
cord injury cord injury cord injury cord injury injury RhoA 1 9 11
RhoA 2 9 13 RhoA 0.5 8 10 10.5 10.5 12 13 RhoA 4 9 12 18 12 16 18
RhoA 4 11 13 14 18 18 14 RhoA 4 8.5 12 13 12 12 16 Mean 2.583 9.083
11.833 13.875 13.125 14.5 15.25 (p = 0.042) (p = 0.0006) (p =
0.0301 (p = 0.0302) (p = 0.0140) STD 1.625 1.021 1.169 3.119 3.326
3 2.217 GFP 2 7 9 9 9.5 10 11 GFP 0 7 9 9.5 10 11 12 GFP 3 7 9.5 11
11 11 12 GFP 0.5 7 9 11 12 12 13 GFP 0.5 8 9 9 9 9 10.5 Mean 1.2
7.2 9.1 9.9 10.3 10.6 11.7 STD 1.255 0.447 0.224 1.025 1.204 1.14
0.975
[0379] siRNA compounds according to Table B and in particular siRNA
directed to genes LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3,
MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2,
CX43, TYROBP, CTGF, and RHOA are tested in this animal model, which
show that these siRNA compounds promote functional recovery
following spinal cord injury and thus may be used to treat spinal
cord injury.
Example 6
Model Systems of Glaucoma
[0380] Testing the active inhibitors of the invention (such as
siRNA) for treating or preventing glaucoma is done in the animal
model for example as described by Pease et al., J. Glaucoma, 2006,
15(6):512-9 (Manometric calibration and comparison of TonoLab and
TonoPen tonometers in rats with experimental glaucoma and in normal
mice).
[0381] siRNA according to Table B in particular to genes TP53BP2,
LRDD, CYBA, ATF3, CASP2, HRK, BNIP3, MAPK8, MAPK14, RAC1, and RHOA
are tested in this animal model which show that these siRNA
compounds treat and/or prevent glaucoma.
Example 7
Model Systems of Ischemia/Reperfusion Injury Following Lung
Transplantation in Rats
[0382] Testing the active inhibitors of the invention (such as
siRNA) for treating or preventing ischemia/reperfusion injury or
hypoxic injury following lung transplantation is done in one or
more of the experimental animal models, for example as described by
Mizobuchi et al., (2004. J. Heart Lung Transplant, 23:889-93);
Huang, et al., (1995. J. Heart Lung Transplant. 14: S49);
Matsumura, et al., (1995. Transplantation 59: 1509-1517); Wilkes,
et al., (1999. Transplantation 67:890-896); Naka, et al., (1996.
Circulation Research, 79: 773-783).
[0383] siRNA according to Table B and in particular to TP53BP2,
LRDD, CYBA, CASP2, BNIP3, RAC1, and DUOX1 are tested in these
animal models, which show that these siRNA compounds treat and/or
prevent ischemia-reperfusion injury following lung transplantation
and thus may be used in conjunction with transplant surgery.
Example 8
Model Systems of Acute Respiratory Distress Syndrome
[0384] Testing the active inhibitors of the invention (such as
siRNA) for treating acute respiratory distress syndrome is done in
the animal model as described by Chen, et al (J Biomed Sci. 2003;
10(6 Pt 1):588-92). siRNA compounds according to Table B in
particular to genes CYBA, HRK, BNIP3, MAPK8, MAPK14, RAC1, GSK3B,
P2RX7, TRPM2, PARG, SPP1, and DUOX1 are tested in this animal model
which shows that these siRNAs treat and/or prevent acute
respiratory distress syndrome and thus may be used to treat this
condition.
Example 9
Model Systems of Hearing Loss Conditions
[0385] (i) Distribution of Cy3-PTEN siRNA in the cochlea following
local application to the round window of the ear:
[0386] A solution of 1 .mu.g/100 .mu.l of Cy3-PTEN siRNA (total of
0.3-0.4 .mu.g) PBS was applied to the round window of chinchillas.
The Cy3-labelled cells within the treated cochlea were analyzed
24-48 hours post siRNA round window application after sacrifice of
the chinchillas. The pattern of labeling within the cochlea was
similar following 24 h and 48 h and includes labeling in the basal
turn of cochlea, in the middle turn of cochlea and in the apical
turn of cochlea. Application of Cy3-PTEN siRNA onto scala tympani
revealed labelling mainly in the basal turn of the cochlea and the
middle turn of the cochlea. The Cy3 signal was persistence to up to
15 days after the application of the Cy3-PTEN siRNA. These results
indicated for the first time that local application of siRNA
molecules within the round window led to significant penetration of
the siRNA molecules to the basal, middle and apical turns of the
cochlea. The siRNA compounds of the invention are tested in this
animal model which shows that there is significant penetration of
these siRNA compounds to the basal, middle and apical turns of the
cochlea, and that these compounds may be used in the treatment of
hearing loss.
[0387] (ii) Chinchilla model of carboplatin-induced or
cisplatin-induced cochlea hair cell death Chinchillas are
pre-treated by direct administration of specific siRNA in saline to
the left ear of each animal. Saline is given to the right ear of
each animal as placebo. Two days following the administration of
the specific siRNA compounds of the invention, the animals are
treated with carboplatin (75 mg/kg ip) or cisplatin
(intraperitoneal infusion of 13 mg/kg over 30 minutes). After
sacrifice of the chinchillas (two weeks post carboplatin treatment)
the % of dead cells of inner hair cells (IHC) and outer hair cells
(OHC) is calculated in the left ear (siRNA treated) and in the
right ear (saline treated). It is calculated that the % of dead
cells of inner hair cells (IHC) and outer hair cells (OHC) is lower
in the left ear (siRNA treated) than in the right ear (saline
treated).
[0388] (iii)) Chinchilla model of acoustic-induced cochlea hair
cell death:
[0389] The activity of specific siRNA in an acoustic trauma model
is studied in chinchilla. The animals are exposed to an octave band
of noise centered at 4 kHz for 2.5 h at 105 dB. The left ear of the
noise-exposed chinchillas is pre-treated (48 h before the acoustic
trauma) with 30 .mu.g of siRNA in .about.10 .mu.L of saline; the
right ear is pre-treated with vehicle (saline). The compound action
potential (CAP) is a convenient and reliable electrophysiological
method for measuring the neural activity transmitted from the
cochlea. The CAP is recorded by placing an electrode near the base
of the cochlea in order to detect the local field potential that is
generated when a sound stimulus, such as click or tone burst, is
abruptly turned on. The functional status of each ear is assessed
2.5 weeks after the acoustic trauma. Specifically, the mean
threshold of the compound action potential recorded from the round
window is determined 2.5 weeks after the acoustic trauma in order
to determine if the thresholds in the siRNA-treated ear are lower
(better) than the untreated (saline) ear. In addition, the amount
of inner and outer hair cell loss is determined in the
siRNA-treated and the control ear.
[0390] siRNA molecules according to Table B in particular to genes
TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8,
MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43,
TYROBP, and CTGF are tested in this animal model which shows that
the thresholds in the siRNA-treated ear are lower (better) than in
the untreated (saline) ear. In addition, the amount of inner and
outer hair cell loss is lower in the siRNA-treated ear than in the
control ear.
Example 10
Animal Models of Osteoarthritis (OA)
[0391] Collagen induced arthritis (CIA): CIA in mice is described
in Trentham et al. (1977. J. Exp. Med. 146: 857-868).
Adjuvant-induced arthritis (AA):AA is described in Kong et al.,
(1999. Nature, 402:304-308). A menisectomy model is described in
Han et al., (1999. Nagoya J Med Sci 62(3-4):115-26).
[0392] The effect of different siRNA inhibitors, such as siRNA to
SSP1, on different parameters related to OA such as chondrocyte
proliferation, terminal differentiation and development of
arthritis, is evaluated using one or more of the above models, in
addition to in vitro models known in the art. siRNA compounds
according to Table B, in particular to SSP1, are tested in these
animal models which show that these siRNAs treat and/or prevent OA
and thus may be used to treat this condition.
[0393] The siRNAs used in the in vivo experiments described herein
were all 19-mers, having alternating ribonucleotides modified in
both the antisense and the sense strands of the compound. The
modification was such that a 2'-O-methyl (Me) group was present on
the first, third, fifth, seventh, ninth, eleventh, thirteenth,
fifteenth, seventeenth and nineteenth nucleotide of the antisense
strand, whereby the very same modification, i.e. a 2'-O-Me group,
was present at the second, fourth, sixth, eighth, tenth, twelfth,
fourteenth, sixteenth and eighteenth nucleotide of the sense
strand. These particular siRNA compounds were also blunt ended and
were non-phosphorylated at the termini; however, comparative
experiments have shown that siRNAs phosphorylated at the 3'-termini
have similar activity.
Example 11
Generation of Sequences for Active siRNA Compounds to Pro-Apoptotic
Genes and Production of the siRNAs
[0394] Using proprietary algorithms and the known sequence of the
pro-apoptotic genes, the sequences of many potential siRNAs were
generated. In addition to the algorithm, some of the 23-mer
oligomer sequences were generated by 5' and/or 3' extension of the
19-mer sequences. The sequences that have been generated using this
method are fully complementary to the corresponding mRNA
sequence.
[0395] Table B (SEQ ID NOS:277-50970 and 50993-68654) shows siRNAs
for the following pro-apoptotic genes: TP53BP2, LRDD, CYBA, ATF3,
CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7,
TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RHOA,
and DUOX1 For each gene there is a separate list of 19-mer, 21-mer
and 23-mer siRNA sequences, which are prioritized based on their
score in the proprietary algorithm as the best sequences for
targeting the human gene expression.
[0396] The following abbreviations are used in the Table B herein:
"other spec or Sp." refers to cross species identity; chn:
chinchilla; chp: chimpanzee, chk: chicken; guinea-pig: GP; mnk:
monkey; ms: mouse; rt: rat; sp: sheep; rb: rabbit; ORF: open
reading frame. 19-mers, 21-mers and 23-mers refer to oligomers of
19, 21 and 23 ribonucleic acids in length.
TABLE-US-00010 Lengthy table referenced here
US20090162365A1-20090625-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090162365A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090162365A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090162365A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References