# Patent

## Title
Treatment or prevention of oto-pathologies by inhibition of pro-apoptotic genes

## Abstract

The invention relates to one or more inhibitors, in particular siRNAs, which down-regulate the expression of human pro-apoptotic genes. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating or preventing the incidence or severity of hearing impairment (or balance impairment), particularly hearing impairment associated with cell death of the inner ear hair cells or outer ear hair cells, comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient.

## Background
This application claims the benefit of U.S. Provisional patent application No. 60/760,795, filed Jan. 20, 2006, of U.S. Provisional patent application No. 60/781,037, filed Mar. 9, 2006 and of U.S. Provisional patent application No. 60/854,503, filed Oct. 25, 2006, all of which are hereby incorporated by reference in their entirety.


Throughout this application various patent and scientific publications are cited. The disclosures for these publications 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.
BACKGROUND OF THE INVENTION
siRNAs and RNA Interference
RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, 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; see Gil et al. 2000, Apoptosis, 5:107-114. Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defense mechanisms (see Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747). As a result, small interfering RNAs (siRNAs), which are short double-stranded RNAs, have become powerful tools in attempting to understand gene function.
Thus, RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) or microRNAs (miRNAs) (Ambros V. Nature 431:7006, 350-355 (2004); and Bartel D P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs: genomics, biogenesis, mechanism, and function). The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. An siRNA is a double-stranded RNA molecule which down-regulates or silences (prevents) the expression of a gene/mRNA of its endogenous (cellular) counterpart. RNA interference is based on the ability of dsRNA species to enter a specific protein complex, where it is then targeted to the complementary cellular RNA and specifically degrades it. 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 2001, Genes Dev., 15, 188). In more detail, longer dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein et al., Nature, 2001, v.409, p. 363-6; Lee et al., Nature, 2003, 425, p. 415-9). The RISC protein complex recognizes these fragments and complementary mRNA. The whole process is culminated by endonuclease cleavage of target mRNA (McManus & Sharp, Nature Rev Genet, 2002, v.3, p. 737-47; Paddison & Hannon, Curr Opin Mol Ther. 2003 June; 5(3): 217-24). For information on these terms and proposed mechanisms, see Bernstein E., Denli A M. Hannon G J: 2001 The rest is silence. RNA. 1; 7(11): 1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 116; 107(4): 415-8 and PCT publication WO 01/36646 (Glover et al).
The selection and synthesis of siRNA corresponding to known genes has been widely reported; see for example Chalk A M, Wahlestedt C, Sonnhammer E L. 2004 Improved and automated prediction of effective siRNA Biochem. Biophys. Res. Commun. June 18; 319(1): 264-74; Sioud M, Leirdal M., 2004, Potential design rules and enzymatic synthesis of siRNAs, Methods Mol Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004, Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. and Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K., Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res. 2004 I 9; 32(3):936-48. Se also Liu Y, Braasch D A, Nulf C J, Corey D R. Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 I 24; 43(7):1921-7. See also PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemical modification analysis, RNA 2003 September; 9(9):1034-48 and I patent Nos. 5898031 and 6107094 (Crooke) for production of modified/more stable siRNAs.
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 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. These reports describe methods to generate siRNAs capable of specifically targeting numerous endogenously and exogenously expressed genes.
Several studies have revealed that siRNA therapeutics are effective in vivo in both mammals and in humans. Bitko et al., have shown that specific siRNA molecules directed against the respiratory syncytial virus (RSV) nucleocapsid N gene are effective in treating mice when administered intranasally (Bitko et al., “Inhibition of respiratory viruses by nasally administered siRNA”, Nat. Med. 2005, 11(1):50-55). A review of the use of siRNA in medicine was recently published by Barik S. in J. Mol. Med (2005) 83: 764-773). Furthermore, a phase I clinical study with short siRNA molecule that targets the VEGFR1 receptor for the treatment of Age-Related Macular Degeneration (AMD) has been conducted in human patients. The siRNA drug administered by an intravitreal inter-ocular injection was found effective and safe in 14 patients tested after a maximum of 157 days of follow up (Boston Globe Jan. 21, 2005).
The p53 Gene and Polypeptide
The human p53 gene is a well-known and highly studied gene. The p53 polypeptide plays a key role in cellular stress response mechanisms by converting a variety of different stimuli, for example DNA damaging conditions, such as gamma-irradiation, deregulation of transcription or replication, and oncogene transformation, into cell growth arrest or apoptosis (Gottlieb et al, 1996, Biochem. Biophys. Acta, 1287, p. 77). The p53 polypeptide is essential for the induction of programmed cell death or “apoptosis” as a response to such stimuli.
Most anti-cancer therapies damage or kill also normal cells that contain native p53, causing severe side effects associated with the damage or death of healthy cells. Since such side effects are to a great extent determined by p53-mediated death of normal cells, the temporary suppression of p53 during the acute phase of anti-cancer therapy has been suggested as a therapeutic strategy to avoid these severe toxic events. This was described in U.S. Pat. No. 6,593,353 and in Komarov P G et al, 1999, A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy., Science, 285(5434):1651, 1653. p53 has been shown to be involved in chemotherapy and radiation-induced alopecia. (Botcharev et al, 2000, p53 is essential for Chemotherapy-induced Hair Loss, Cancer Research, 60, 5002-5006).
RTP801
Gene RTP801, was first reported by the assignee of the instant application. U.S. Pat. Nos. 6,455,674, 6,555,667, and 6740738, all assigned to the assignee of the instant application, disclose and claim per se the RTP801 polynucleotide and polypeptide, and antibodies directed toward the polypeptide. RTP801 represents a unique gene target for hypoxia-inducible factor-1 (HIF-1) that may regulate hypoxia-induced pathogenesis independent of growth factors such as VEGF.
Pro-Apoptotic Genes
Pro-apoptotic genes are genes that play a role in apoptotic cell death. A non-limiting list of pro-apoptotic genes includes p53 and RTP801 and also Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 12, Caspase 14, Apaf-1, Nod1, Nod2, Ipaf, DEFCAP, RAIDD, RICK, Bcl10, ASC, TUCAN, ARC, CLARP, FADD, DEDD, DEDD2, Cryopirin, PYC1, Pyrin, TRADD, UNC5a, UNC5b, UNC5c, ZUD, p84N5, LRDD, CDK1, CDK2, CDK4, CDK5, CDK9, PITSLRE A, CHK2, LATS1, Prk, MAP4K1, MAP4K2, STK4, SLK, GSK3alpha, GSK3beta, MEKK1, MAP3K5 (Ask1), MAP3K7, MAP3K8, MAP3K9, MAP3K10, MAP3K11, MAP3K12, DRP-1, MKK6, p38, JNK3, DAPK1, DRAK1, DRAK2, IRAK, RIP, RIP3, RIP5, PKR, IRE1, MSK1, PKCalpha, PKCbeta, PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta, PKCzeta, CAMK2A, HIPK2, LKB1, BTK, c-Src, FYN, Lck, ABL2, ZAP70, TrkA, TrkC, MYLK, FGFR2, EphA2, AATYK, c-Met, RET, PRKAA2, PLA2G2A, SMPD1, SMPD2, SPP1, FAN, PLCG2, IP6K2, PTEN, SHIP, AIF, AMID, Cytochrome c, Smac, HtrA2, TSAP6, DAP-1, FEM-1, DAP-3, Granzyme B, DIO-1, DAXX, CAD, CIDE-A, CIDE-B, Fsp27, Ape1, ERCC2, ERCC3, BAP31, Bit1, AES, Huntingtin, HIP1, hSir2, PHAP1, GADD45b, GADD34, RAD21, MSH6, ADAR, MBD4, WW45, ATM, mTOR, TIP49, diubiquitin/FAT10, FAF1, p193, Scythe/BAT3, Amida, IGFBP-3, TDAG51, MCG10, PACT, p52/RAP, ALG2, ALG3, presenelin-1, PSAP, AIP1/Alix, ES18, mda-7, p14ARF, ANT1, p33ING1, p33ING2, p53AIP1, p53DINP1, MGC35083, NRAGE, GRIM19, lipocalin 2, glycodelin A, NADE, Porimin, STAG1, DAB2, Galectin-7, Galectin-9, SPRC, FLJ21908, WWOX, XK, DKK-1, Fzd1, Fzd2, SARP2, axin 1, RGS3, DVL1, NFkB2, IkBalpha, NF-ATC1, NF-ATC2, NF-ATC4, zf3/ZNF319, Egr1, Egr2, Egr3, Sp1, TIEG, WT1, Zac1, Icaros, ZNF148, ZK1/ZNF443, ZNF274, WIG1, HIVEP1, HIVEP3, Fliz1, ZPR9, GATA3, TR3, PPARG, CSMF, RXRa, RARa, RARb, RAR9, T3Ra, ERbeta, VDR, GR/GCCR, p53, p73alpha, p63(human [ta alpha, ta beta, ta gamma, da alpha, da beta, da gamma], 53BP2, ASPP1, E2F1, E2F2, E2F3, HIF1 alpha, TCF4, c-Myc, Max, Mad, MITF, Id2, Id3, Id4, c-Jun, c-Fos, ATF3, NF-IL6, CHOP, NRF1, c-Maf, Bach2, Msx2, Csx, Hoxa5, Ets-1, PU1/Spi1, Ets-2, ELK1, TEL1, c-Myb, TBX5, IRF1, IRF3, IRF4, IRF9, AP-2 alpha, FKHR, FOXO1A, FKHRL1, FOXO3a, AFX1, MLLT7, Tip60, BTG1, AUF1, HNRPD, TIA1, NDG1, PCBP4, MCG10, FXR2, TNFR2, LTbR, CD40, CD27, CD30, 4-1BB, TNFRSF19, XEDAR, Fn14, OPG, DcR3, FAS, TNFR1, WSL-1, p75NTR, DR4, DR5, DR6, EDAR, TNF alpha, FAS ligand, TRAIL, Lymphotoxin alpha, Lymphotoxin beta, 4-1BBL, RANKL, TL1, TWEAK, LIGHT, APRIL, IL-1-alpha, IL-1-beta, IL-18, FGF8, IL-2, IL-21, IL-5, IL-4, IL-6, LIF, IL-12, IL-7, IL-10, IL-19, IL-24, IFN alpha, IFN beta, IFN gamma, M-CSF, prolactin, TLR2, TLR3, TLR4, MyD88, TRIF, RIG-1, CD14, TCR alpha, CD3 gamma, CD8, CD4, CD7, CD19, CD28, CTLA4, SEMA3A, SEMA3B, HLA-A, HLA-B, HLA-L, HLA-DMalpha, CD22, CD33, CALL, DCC, ICAM1, ICAM3, CD66a, PVR, CD47, CD2, Thy-1, SIRPa1, CD5, E-cadherin, ITGAM, ITGAV, CD18, ITGB3, CD9, IgE Fc R beta, CD82, CD81, PERP, CD24, CD69, KLRD1, galectin 1, B4GALT1, Clq alpha, C5R1, MIP1alpha, MIP1beta, RANTES, SDF1, XCL1, CCCKR5, OIAS/OAS1, INDO, MxA, IFI16, AIM2, iNOS, HB-EGF, HGF, MIF, TRAF3, TRAF4, TRAF6, PAR-4, IKKGamma, FIP2, TXBP151, FLASH, TRF1, IEX-1S, Dok1, BLNK, CIN85, Bif-1, HEF1, Vav1, RasGRP1, POSH, Rac1, RhoA, RhoB, RhoC, ALG4, SPP1, TRIP, SIVA, TRABID, TSC-22, BRCA1, BARD1, 53BP1, MDC1, Mdm4, Siah-1, Siah-2, RoRet, TRIM35, PML, RFWD1, DIP1, Socs1, PARC, USP7, CYLD, TP53BP2, CYBA, NOX3, HRK, C1QBP, BNIP3, MAPK8, MAPK14, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, GJA1, TYROBP, CTGF, RTN4R, ANXA2, DUOX1, SLC5A1, SLC2A2, AKR1B1, SORD, SLC2A1 and MME.
Chemical-induced Ototoxicity
The toxic effects of various ototoxic therapeutic drugs on auditory cells and spiral ganglion neurons are often the limiting factor for their therapeutic usefulness. Main ototoxic drugs include the widely used chemotherapeutic agent cisplatin and its analogs, commonly used aminoglycoside antibiotics, e.g. gentamicin, for the treatment of infections caused by gram-negative bacteria, quinine and its analogs, salicylate and its analogs, and loop-diuretics.
For example, antibacterial aminoglycosides such as gentamicins, streptomycins, kanamycins, tobramycins, and the like are known to have serious toxicity, particularly ototoxicity and nephrotoxicity, which reduces the usefulness of such antimicrobial agents (see Goodman and Gilman's The Pharmacological Basis of Therapeutics, 6th ed., A. Goodman Gilman et al., eds; Macmillan Publishing Co., Inc., New York, pp. 1169-71 (1980)). Clearly, ototoxicity is a dose-limiting side-effect of antibiotic administration. From 4 to 15% of patients receiving 1 gram per day for greater than 1 week develop measurable hearing loss, which slowly becomes worse and can lead to complete permanent deafness if treatment continues.
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®) damages auditory and vestibular systems. Salicylates, such as aspirin, are the most commonly used therapeutic drugs for their anti-inflammatory, analgesic, anti-pyretic and anti-thrombotic effects. Unfortunately, they too have ototoxic side effects. They often lead to tinnitus (“ringing in the ears”) and temporary hearing loss. Moreover, if the drug is used at high doses for a prolonged time, the hearing impairment can become persistent and irreversible.
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. Of particular interest are those conditions arising as an unwanted side-effect of ototoxic therapeutic drugs including cisplatin and its analogs, aminoglycoside antibiotics, salicylate and its analogs, or loop diuretics. In addition, there exits a need for methods which will allow higher and thus more effective dosing with these ototoxicity-inducing pharmaceutical drugs, while concomitantly preventing or reducing ototoxic effects caused by these drugs. What is needed is a method that provides 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.
Without being bound by theory, it is believed that cisplatin drugs and other drugs that induce ototoxicity (such as aminoglycoside antibiotics) may induce the ototoxic effects via programmed cell death or apoptosis in inner ear tissue, particularly inner ear hair cells (Zhang et al., Neuroscience 120 (2003) 191-205; Wang et al., J. Neuroscience 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, 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.
SUMMARY OF THE INVENTION
The invention provides novel double stranded oligoribonucleotides that inhibit the p53 gene or any other pro-apoptotic genes. The invention also provides a pharmaceutical composition comprising one or more such oligoribonucleotides, and a vector capable of expressing the oligoribonucleotide. The present invention also relates to methods and compositions for treating or preventing the incidence or severity of hearing impairment (or balance impairment), particularly hearing impairment associated with cell death of the inner ear hair cells or outer ear hair cells. The methods and compositions involve administering to a mammal in need of such treatment a prophylactically or therapeutically effective amount of one or more compounds which down-regulate expression of the p53 gene or other pro-apoptotic genes, particularly novel small interfering RNAs (siRNAs), small molecule inhibitors of p53 or other pro-apoptotic genes as described herein or antibodies to p53 polypeptide or other pro-apoptotic genes.
More specifically, the present invention provides methods and compositions for treating a patient suffering from hearing impairment, or other oto-pathologies associated with cell death of inner ear hair cells or outer ear hair cells. Such oto-pathologies may be the result of acoustic trauma, mechanical trauma, or ototoxin-induced hearing loss. The methods of the invention comprising administering to the patient one or more compounds which down-regulate expression of the p53 gene or other pro-apoptotic genes, particularly siRNAs that inhibit p53 or other pro-apoptotic genes, typically as a pharmaceutical composition, in a therapeutically effective dose so as to thereby treat the patient. Since long-term p53 inactivation may significantly increase the risk of cancer, it is preferred that the inhibition of p53 using the molecules of the present invention be temporary or local.
In one embodiment, the present invention provides for improved compositions and methods for treatments requiring administration of a pharmaceutical drug having an ototoxic, hearing-impairing side-effect, in combination with a therapeutically effective amount of one or more siRNA molecules that inhibit p53 or other pro-apoptotic genes, to treat or prevent the ototoxicity induced by the pharmaceutical drug. The compositions of the invention can be administered at a suitable interval(s) either prior to, subsequent to, or substantially concurrently with the administration of the ototoxic, hearing-impairing drug that induces inner ear apoptotic tissue damage.
Accordingly, it is an object of the invention to provide an improved composition containing a therapeutically effective amount of one or more siRNA molecules that inhibit p53 or other pro-apoptotic genes in combination with an ototoxic, hearing-impairing pharmaceutical drug for administration to a mammal. Preferably, the combination drugs are administered separately; the siRNA molecules that inhibit p53 or other pro-apoptotic genes are administered locally while the ototoxic, hearing-impairing pharmaceutical drug is administered systemically. The siRNA molecules may be administered prior to, simultaneously with or subsequent to the ototoxic drug. Such combination compositions can further contain a pharmaceutically acceptable carrier. The pharmaceutical composition will have lower ototoxicity than the ototoxic pharmaceutical alone, and, preferably, will have a higher dosage of the ototoxic pharmaceutical than typically used. Examples of such improved compositions include cisplatin or other ototoxic neoplastic agent or an aminoglycoside antibiotic(s) in combination with the therapeutically effective amount of one or more siRNA molecules that inhibit p53 or other pro-apoptotic genes.
Still further, the invention relates to the use of the compositions of the invention in cases where diuretics are needed. The present invention provides a solution to the art that has long sought a therapy and a medicament which can treat the ototoxic effects currently associated with certain diuretics, and particular with the more popular and commonly used loop-diuretics, without sacrificing their diuretic effectiveness.
Still further, the invention relates to the use of the compositions of the invention in cases where quinine or quinine-like compounds are needed. The present invention provides a solution to the art that has long sought a therapy and a medicament which can treat the ototoxic effects currently associated with certain quinines without sacrificing their effectiveness.
Still further, the invention relates to a method for treating or preventing the incidence or severity of hearing impairment in a patient comprising administering to the patient a composition comprising an effective amount of naked siRNA molecules. Preferably, the naked siRNA molecules are applied directly to the round window membrane of the cochlea or administered by transtympanic injection. Further, the naked siRNA molecules are preferably directed against at least one pro-apoptotic gene.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. This figure represents the nucleotide sequence of the human p53 gene-SEQ ID NO:1.
FIG. 2. This figure represents the amino acid sequence of the human p53 polypeptide—SEQ ID NO:2.
FIG. 3. This figure shows Western Blot results demonstrating the effect of various human p53 siRNAs on p53 expression.
FIG. 4. This figure shows Western Blot results demonstrating the effect of various mouse p53 siRNAs on p53 expression.
FIG. 5. This figure shows the effect of p53 siRNA treatment on acoustic-induced hair cell death in the cochlea of chinchilla.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates generally to compounds which down-regulate expression of the p53 gene and other 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 in particular various forms of hearing impairment as described above Preferred lists of such siRNA are in Tables A, B, anc C.
The inventors of the present invention have found that it is beneficial to induce temporary inhibition of p53 or a pro-apoptotic gene in order to treat any of the above diseases or disorders. Methods, molecules and compositions which inhibit p53 or a pro-apoptotic gene are discussed herein at length, and any of said molecules and/or compositions may be beneficially employed in the treatment of a patient suffering from any of said conditions.
A “pro-apoptotic gene” is defined as gene that plays a role in apoptotic cell death. A non-limiting list of pro-apoptotic genes includes p53 and RTP801 and also Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 12, Caspase 14, Apaf-1, Nod1, Nod2, Ipaf, DEFCAP, RAIDD, RICK, Bcl10, ASC, TUCAN, ARC, CLARP, FADD, DEDD, DEDD2, Cryopirin, PYC1, Pyrin, TRADD, UNC5a, UNC5b, UNC5c, ZUD, p84N5, LRDD, CDK1, CDK2, CDK4, CDK5, CDK9, PITSLRE A, CHK2, LATS1, Prk, MAP4K1, MAP4K2, STK4, SLK, GSK3alpha, GSK3beta, MEKK1, MAP3K5 (Ask1), MAP3K7, MAP3K8, MAP3K9, MAP3K10, MAP3K11, MAP3K12, DRP-1, MKK6, p38, JNK3, DAPK1, DRAK1, DRAK2, IRAK, RIP, RIP3, RIP5, PKR, IRE1, MSK1, PKCalpha, PKCbeta, PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta, PKCzeta, CAMK2A, HIPK2, LKB1, BTK, c-Src, FYN, Lck, ABL2, ZAP70, TrkA, TrkC, MYLK, FGFR2, EphA2, AATYK, c-Met, RET, PRKAA2, PLA2G2A, SMPD1, SMPD2, SPP1, FAN, PLCG2, IP6K2, PTEN, SHIP, AIF, AMID, Cytochrome c, Smac, HtrA2, TSAP6, DAP-1, FEM-1, DAP-3, Granzyme B, DIO-1, DAXX, CAD, CIDE-A, CIDE-B, Fsp27, Ape1, ERCC2, ERCC3, BAP31, Bit1, AES, Huntingtin, HIP1, hSir2, PHAP1, GADD45b, GADD34, RAD21, MSH6, ADAR, MBD4, WW45, ATM, mTOR, TIP49, diubiquitin/FAT10, FAF1, p193, Scythe/BAT3, Amida, IGFBP-3, TDAG51, MCG10, PACT, p52/RAP, ALG2, ALG3, presenelin-1, PSAP, AIP1/Alix, ES18, mda-7, p14ARF, ANT1, p33ING1, p33ING2, p53AIP1, p53DINP1, MGC35083, NRAGE, GRIM19, lipocalin 2, glycodelin A, NADE, Porimin, STAG1, DAB2, Galectin-7, Galectin-9, SPRC, FLJ21908, WWOX, XK, DKK-1, Fzd1, Fzd2, SARP2, axin 1, RGS3, DVL1, NFkB2, IkBalpha, NF-ATC1, NF-ATC2, NF-ATC4, zf3/ZNF319, Egr1, Egr2, Egr3, Sp1, TIEG, WT1, Zac1, Icaros, ZNF148, ZK1/ZNF443, ZNF274, WIG1, HIVEP1, HIVEP3, Fliz1, ZPR9, GATA3, TR3, PPARG, CSMF, RXRa, RARa, RARb, RARg, T3Ra, ERbeta, VDR, GR/GCCR, p53, p73alpha, p63(human [ta alpha, ta beta, ta gamma, da alpha, da beta, da gamma], 53BP2, ASPP1, E2F1, E2F2, E2F3, HIF1 alpha, TCF4, c-Myc, Max, Mad, MITF, Id2, Id3, Id4, c-Jun, c-Fos, ATF3, NF-IL6, CHOP, NRF1, c-Maf, Bach2, Msx2, Csx, Hoxa5, Ets-1, PU1/Spi1, Ets-2, ELK1, TEL1, c-Myb, TBX5, IRF1, IRF3, IRF4, IRF9, AP-2 alpha, FKHR, FOXO1A, FKHRL1, FOXO3a, AFX1, MLLT7, Tip60, BTG1, AUF1, HNRPD, TIA1, NDG1, PCBP4, MCG10, FXR2, TNFR2, LTbR, CD40, CD27, CD30, 4-1BB, TNFRSF19, XEDAR, Fn14, OPG, DcR3, FAS, TNFR1, WSL-1, p75NTR, DR4, DR5, DR6, EDAR, TNF alpha, FAS ligand, TRAIL, Lymphotoxin alpha, Lymphotoxin beta, 4-1BBL, RANKL, TL1, TWEAK, LIGHT, APRIL, IL-1-alpha, IL-1-beta, IL-18, FGF8, IL-2, IL-21, IL-5, IL-4, IL-6, LIF, IL-12, IL-7, IL-10, IL-19, IL-24, IFN alpha, IFN beta, IFN gamma, M-CSF, prolactin, TLR2, TLR3, TLR4, MyD88, TRIF, RIG-1, CD14, TCR alpha, CD3 gamma, CD8, CD4, CD7, CD19, CD28, CTLA4, SEMA3A, SEMA3B, HLA-A, HLA-B, HLA-L, HLA-DMalpha, CD22, CD33, CALL, DCC, ICAM1, ICAM3, CD66a, PVR, CD47, CD2, Thy-1, SIRPa1, CD5, E-cadherin, ITGAM, ITGAV, CD18, ITGB3, CD9, IgE Fc R beta, CD82, CD81, PERP, CD24, CD69, KLRD1, galectin 1, B4GALT1, Clq alpha, C5R1, MIP1alpha, MIP1beta, RANTES, SDF1, XCL1, CCCKR5, OIAS/OAS1, INDO, MxA, IFI16, AIM2, iNOS, HB-EGF, HGF, MIF, TRAF3, TRAF4, TRAF6, PAR-4, IKKGamma, FIP2, TXBP151, FLASH, TRF1, IEX-1S, Dok1, BLNK, CIN85, Bif-1, HEF1, Vav1, RasGRP1, POSH, Rac1, RhoA, RhoB, RhoC, ALG4, SPP1, TRIP, SIVA, TRABID, TSC-22, BRCA1, BARD1, 53BP1, MDC1, Mdm4, Siah-1, Siah-2, RoRet, TRIM35, PML, RFWD1, DIP1, Socs1, PARC, USP7, CYLD, TP53BP2, CYBA, NOX3, HRK, C1QBP, BNIP3, MAPK8, MAPK14, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, GJA1, TYROBP, CTGF, RTN4R, ANXA2, DUOX1, SLC5A1, SLC2A2, AKR1B1, SORD, SLC2A1 and MME.
A “pro-apoptotic polypeptide” is a polypeptide encoded by any of the above pro-apoptotic genes.
The present invention provides methods and compositions for inhibiting expression of a target p53 gene or a pro-apoptotic gene in vivo. In general, the method includes administering oligoribonucleotides, such as small interfering RNAs (i.e., siRNAs) that are targeted to a particular p53, RTP801 or any pro-apoptotic gene mRNA and hybridize to, or interact with, the mRNAs under biological conditions (within the cell), or a nucleic acid material that can produce siRNA in a cell, 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 p53 gene or any pro-apoptotic gene for treatment of a disease.
In accordance with the present invention, the siRNA molecules or inhibitors of the p53 gene or any pro-apoptotic gene may be used as drugs to treat various pathologies accompanied by an elevated level of p53 polypeptide. Since long-term p53 inactivation can significantly increase the risk of cancer, it is preferred that the inhibition of p53 using the molecules of the present invention be temporary/reversible.
The present invention provides double-stranded oligoribonucleotides (siRNAs), which down-regulate the expression of the p53 gene, RTP801 or any pro-apoptotic gene. An siRNA of the invention is a duplex oligoribonucleotide in which the sense strand is derived from the mRNA sequence of the p53 gene, RTP801 or any 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 Nucleic Acids Research 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.
There are at least four variant p53 polypeptides (see Bourdon et al. Genes Dev. 2005; 19: 2122-2137). The sequence given in FIG. 1 is the nucleotide sequence of gi-8400737. The corresponding polypeptide sequence has 393 amino acids; see FIG. 2. All variants and any other similar minor variants are included in the definition of p53 polypeptide and in the definition of the p53 genes encoding them.
As used herein, the term “p53 gene” is defined as any homolog of the p53 gene having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to the amino acid encoding region of SEQ ID NO:1 or nucleic acid sequences which bind to the p53 gene under conditions of highly stringent hybridization, which are well-known in the art (for example, see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998.
As used herein, the term “p53”, or “p53 polypeptide” is defined as any homolog of the p53 polypeptide having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to SEQ ID NO:2, as either full-length or a fragment or a domain thereof, as a mutant or the polypeptide encoded by a spliced variant nucleic acid sequence, as a chimera with other polypeptides, provided that any of the above has the same or substantially the same biological function as the p53 polypeptide.
Generally, the siRNAs used in the present invention comprise a ribonucleic acid comprising a double stranded structure, whereby the double-stranded structure comprises a first strand and a second strand, whereby the first strand comprises a first stretch of contiguous nucleotides and whereby said first stretch is at least partially complementary to a target nucleic acid, and the second strand comprises a second stretch of contiguous nucleotides and whereby said second stretch is at least partially identical to a target nucleic acid, whereby said first strand and/or said second strand comprises a plurality of groups of modified nucleotides having a modification at the 2′-position whereby within the strand each group of modified nucleotides is flanked on one or both sides by a flanking group of nucleotides whereby the flanking nucleotides forming the flanking group of nucleotides is either an unmodified nucleotide or a nucleotide having a modification different from the modification of the modified nucleotides. Further, said first strand and/or said second strand may comprise said plurality of modified nucleotides and may comprises said plurality of groups of modified nucleotides.
The group of modified nucleotides and/or the group of flanking nucleotides may comprise a number of nucleotides whereby the number is selected from the group comprising one nucleotide to 10 nucleotides. In connection with any ranges specified herein it is to be understood that each range discloses any individual integer between the respective figures used to define the range including said two figures defining said range. In the present case the group thus comprises one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides and ten nucleotides.
The pattern of modified nucleotides of said first strand may be shifted by one or more nucleotides relative to the pattern of modified nucleotides of the second strand.
The modifications discussed above may be selected from the group comprising amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O—, S—, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.
The double stranded structure of the siRNA may be blunt ended, on one or both sides. More specifically, the double stranded structure may be blunt ended on the double stranded structure's side which is defined by the 5′-end of the first strand and the 3′-end of the second strand, or the double stranded structure may be blunt ended on the double stranded structure's side which is defined by at the 3′-end of the first strand and the 5′-end of the second strand.
Additionally, at least one of the two strands may have an overhang of at least one nucleotide at the 5′-end; the overhang may consist of at least one deoxyribonucleotide. At least one of the strands may also optionally have an overhang of at least one nucleotide at the 3′-end.
The length of the double-stranded structure of the siRNA is typically from about 17 to 21 and more preferably 18 or 19 bases. Further, the length of said first strand and/or the length of said second strand may independently from each other be selected from the group comprising the ranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19 bases.
Additionally, the complementarily between said first strand and the target nucleic acid may be perfect, or the duplex formed between the first strand and the target nucleic acid may comprise at least 15 nucleotides wherein there is one mismatch or two mismatches between said first strand and the target nucleic acid forming said double-stranded structure.
In some cases both the first strand and the second strand each comprise at least one group of modified nucleotides and at least one flanking group of nucleotides, whereby each group of modified nucleotides comprises at least one nucleotide and whereby each flanking group of nucleotides comprising at least one nucleotide with each group of modified nucleotides of the first strand being aligned with a flanking group of nucleotides on the second strand, whereby the most terminal 5′ nucleotide of the first strand is a nucleotide of the group of modified nucleotides, and the most terminal 3′ nucleotide of the second strand is a nucleotide of the flanking group of nucleotides. Each group of modified nucleotides may consist of a single nucleotide and/or each flanking group of nucleotides may consist of a single nucleotide.
Additionally, it is possible that on the first strand the nucleotide forming the flanking group of nucleotides is an unmodified nucleotide which is arranged in a 3′ direction relative to the nucleotide forming the group of modified nucleotides, and on the second strand the nucleotide forming the group of modified nucleotides is a modified nucleotide which is arranged in 5′ direction relative to the nucleotide forming the flanking group of nucleotides.
Further the first strand of the siRNA may comprise eight to twelve, preferably nine to eleven, groups of modified nucleotides, and the second strand may comprise seven to eleven, preferably eight to ten, groups of modified nucleotides.
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.
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′-end of the first strand may be linked to the 5′-terminus of the second strand, said linkage being via a nucleic acid linker typically having a length between 10-2000 nucleobases.
In particular, the invention provides a compound having structure A:

5′(N)x—Z 3′ (antisense strand)

3′Z′—(N′)y5′ (sense strand)

    
    
        wherein each N and N′ is a ribonucleotide which may be modified or unmodified in its sugar residue and (N), and (N′)y is 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 dTdT and is covalently attached at the 3′ terminus of the strand in which it is present;
        and wherein the sequence of (N)x comprises an antisense sequence to mRNA of p53 or any pro-apoptotic gene in particular any of the antisense sequences present in any of Tables A, B, and C.
    
    


It will be readily understood by those skilled in the art that the compounds of the present invention consist of a plurality of nucleotides, which are linked through covalent linkages. Each such covalent linkage may be a phosphodiester linkage, a phosphothioate linkage, or a combination of both, along the length of the nucleotide sequence of the individual strand. Other possible backbone modifications are described inter alia in U.S. Pat. Nos. 5,587,361; 6,242,589; 6,277,967; 6,326,358; 5,399,676; 5,489,677; and 5,596,086.
In particular embodiments, x and y are preferably an integer between about 19 to about 27, most preferably from about 19 to about 23. In a particular embodiment of the compound of the invention, x may be equal to y (viz., x=y) and in preferred embodiments x=y=19 or x=y=21. In a particularly preferred embodiment x=y=19.
In one embodiment of the compound of the invention, Z and Z′ are both absent; in another embodiment one of Z or Z′ is present.
In one embodiment of the compound of the invention, all of the ribonucleotides of the compound are unmodified in their sugar residues.
In preferred embodiments of the compound of the invention, at least one ribonucleotide is modified in its sugar residue, preferably a modification at the 2′ position. The modification at the 2′ position results in the presence of a moiety which is preferably selected from the group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. In a presently most preferred embodiment the moiety at the 2′ position is methoxy (2′-0-methyl).
In preferred embodiments of the invention, alternating ribonucleotides are modified in both the antisense and the sense strands of the compound. In particular the siRNA used in the Examples has been such modified such that a 2′ O-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 the in case of these particular nucleic acids according to the present invention the first stretch is identical to the first strand and the second stretch is identical to the second strand and these nucleic acids are also blunt ended.
In a particularly preferred embodiment the sequence of the siRNA is that of 15 in Table A.
According to one preferred embodiment of the invention, the antisense and the sense strands of the siRNA molecule are both 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 both non-phosphorylated both at the 3′-terminus and also at the 5′-terminus. According to yet another preferred embodiment of the invention, the 1st nucleotide in the 5′ position in the sense strand is specifically modified to abolish any possibility of in vivo 5′-phosphorylation.
In another embodiment of the compound of the invention, 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.
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.
The invention also provides a composition comprising one or more of the compounds of the invention in a carrier, preferably a pharmaceutically acceptable carrier. This composition may comprise a mixture of two or more different siRNAs.
The invention also provides a composition which comprises the above compound of the invention covalently or non-covalently bound to one or more compounds of the invention in an amount effective to inhibit human p53 and a carrier. This composition may be processed intracellularly by endogenous cellular complexes to produce one or more oligoribonucleotides of the invention.
The invention also provides a composition comprising a carrier and one or more of the compounds of the invention in an amount effective to down-regulate expression in a cell of a human p53, which compound comprises a sequence substantially complementary to the sequence of (N)x.
Additionally the invention provides a method of down-regulating the expression of gene p53 by at least 50% as compared to a control comprising contacting an mRNA transcript of gene p53 with one or more of the compounds of the invention.
In one embodiment the oligoribonucleotide is down-regulating p53 or any pro-apoptotic gene, whereby the down-regulation of p53 or any pro-apoptotic gene is selected from the group comprising down-regulation of gene function, down-regulation of polypeptide and down-regulation of mRNA expression.
In one embodiment the compound is down-regulating a pro-apoptotic polypeptide, whereby the down-regulation is selected from the group comprising down-regulation 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), down-regulation of protein (which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia) and down-regulation of mRNA expression (which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia).
The invention also provides a method of treating a patient suffering from a disease accompanied by an elevated level of a pro-apoptotic polypeptide, the method comprising administering to the patient a composition of the invention in a therapeutically effective dose thereby treating the patient. Preferably, the present invention provides a method of treating a patient suffering from a disease in which temporary inhibition of a pro-apoptotic gene is beneficial.
More particularly, the invention provides an oligoribonucleotide wherein one strand comprises consecutive nucleotides having, from 5′ to 3′, the sequence set forth in SEQ ID NOS: 3-25 (Table A, sense strands) or in SEQ ID NOS: 49-119 (Table B, sense strands) or in SEQ ID NOS: 191-253 (Table C, sense strands) or a homolog thereof wherein in up to 2 of the nucleotides in each terminal region a base is altered.
The terminal region of the oligonucleotide refers to bases 1-4 and/or 16-19 in the 19-mer sequence and to bases 1-4 and/or 18-21 in the 21-mer sequence.
Additionally, the invention provides oligoribonucleotides wherein one strand comprises consecutive nucleotides having, from 5′ to 3′, the sequence set forth SEQ ID NOS: 26-48 (Table A, antisense strands) or SEQ ID NOS: 120-190 (Table B, antisense strands) or SEQ ID NOS: 254-316 (Table C, antisense strands) or a homolog thereof wherein in up to 2 of the nucleotides in each terminal region a base is altered.
Preferred list of siRNA (sense and antisense strands) directed to selected pro-apoptotic genes are in Tables A, B, and C.
The preferred oligonucleotides of the invention are human p53 oligonucleotides serial numbers 3, 5, 20 and 23 in Table D and mouse p53 oligonucleotides serial numbers 1 11, 12, 14, 17 and 18 in Table E. These are identical to serial numbers 3, 5, 20 and 23 (human) and also 11, 12, 14, 17 and 18 (mouse) in Table A. The most preferred oligonucleotides of the invention are human p53 oligonucleotides having the sequence of serial number 23 in Table A. Other preferred oligonucleotides of the invention are oligonucleotides, preferably siRNA oligonucleotides, which correspond to pro-apoptotic genes as defined above.
The presently most preferred compound of the invention is a blunt-ended 19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent. The oligonucleotide molecule is either phosphorylated at 3′ termini of both sense and anti-sense strands, or non-phosphorylated at all; or having 1st nucleotide in the 5′ position on the sense strand specifically modified to abolish any possibility of in vivo 5′-phosphorylation. The alternating ribonucleotides are modified at the 2′ position in both the antisense and the sense strands, wherein the moiety at the 2′ position is methoxy (2′-0-methyl) and wherein 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. The presently most preferred such compounds are such modified oligonucleotides comprising the sequences having serial number 23 in Table A.
In one aspect of the invention the oligonucleotide comprises a double-stranded structure, whereby such double-stranded structure comprises

    
    
        a first strand and a second strand, whereby
        the first strand comprises a first stretch of contiguous nucleotides and the second strand comprises a second stretch of contiguous nucleotides, whereby
        the first stretch is either complementary or identical to a nucleic acid sequence coding for p53 and whereby the second stretch is either identical or complementary to a nucleic acid sequence coding for p53.
    
    


In an embodiment the first stretch and/or the second stretch comprises from about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably from about 19 to 27 nucleotides and most preferably from about 19 to 23 nucleotides, in particular from about 19 to 21 nucleotides. In such an embodiment the oligonucleotide may be from 17-40 nucleotides in length.
Additionally, further nucleic acids according to the present invention comprise at least 14 contiguous nucleotides of any one of the polynucleotides in Tables A, B, and C and more preferably 14 contiguous nucleotide base pairs at any end of the double-stranded structure comprised of the first stretch and second stretch as described above.
In an embodiment the first stretch comprises a sequence of at least 14 contiguous nucleotides of an oligonucleotide, whereby such oligonucleotide is selected from the group comprising SEQ. ID. Nos 3-316, preferably from the group comprising the oligoribonucleotides of having the sequence of any of the serial numbers 3, 5, 20 or 23 (human) or having the sequence of any of the serial numbers 11, 12, 14, 17 and 18 (mouse) in Table A, more preferably selected from the group having the sequence of any of the serial numbers 3, 5, 20 or 23 in Table A.
Additionally, further nucleic acids according to the present invention comprise at least 14 contiguous nucleotides of any one of the SEQ. ID. NO. 3 to 316, and more preferably 14 contiguous nucleotide base pairs at any end of the double-stranded structure comprised of the first stretch and second stretch as described above. 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 stretches forming such nucleic acid according to the present invention, some shifts relative to the coding sequence of p53 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.
Delivery: The siRNA molecules of the present invention may be delivered to the target tissue (such as the cochlea) by direct application of the naked molecules admixed with a carrier or a diluent within the cochlea.
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”. However, the siRNA molecules of the invention can also be 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.
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 letters 539: 111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recently been successfully used for inhibition in primates; for further details see Tolentino et al., Retina 24(1) February 2004 I 132-138. Respiratory formulations for siRNA are described in U.S. patent application No. 2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and other steroid and lipid conjugated siRNAs) can been used for delivery see Soutschek et al Nature 432: 173-177 (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs; and Lorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. 
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 patient, 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.
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. 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 addition, under certain circumstances the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration. The patient being treated is a warm-blooded animal and, in particular, mammals including man. 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 are particularly preferred.
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 term “treatment” as used herein refers to administration of a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
In a particular embodiment, the administration comprises intravenous administration. In another particular embodiment the administration comprises topical or local administration
In another aspect of the invention a pharmaceutical composition is provided which comprises any of the above oligoribonucleotides (SEQ ID NOS: 3-316) or vectors and a pharmaceutically acceptable carrier. Another aspect of the invention is the use of a therapeutically effective amount of any of the above oligoribonucleotides (SEQ ID NOS: 3-316) or vectors for the preparation of a medicament for treating a patient suffering from a disorder which is accompanied by an elevated level of p53.
The present invention relates to the use of compounds which down-regulate the expression of the p53 gene, RTP801 or other pro-apoptotic genes particularly to novel small interfering RNAs (siRNAs), in the treatment of hearing impairment. Methods, molecules and compositions which inhibit pro-apoptotic genes are discussed herein at length, and any of said molecules and/or compositions may be beneficially employed in the treatment of a patient suffering from any of said conditions. Preferred lists of siRNA directed to selected pro-apoptotic genes are in Tables A, B, and C. Other siRNA sequences directed to RTP801 to be used in the present invention may be found in co-pending PCT publication number WO06/023544A2 (PCT/US2005/029236) or U.S. application Ser. No. 11/207,119, which are incorporated by reference in their entirety.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an apoptotic-related disorder such as hearing disorder or impairment (or balance impairment), preferably ototoxin-induced or traumatic inner ear hair cells apoptotic damage. Those in need of treatment include those already experiencing a hearing impairment, those prone to having the impairment, and those in which the impairment is to be prevented. Without being bound by theory, the hearing impairment 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, or, in particular, 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.
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, neoplastic 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 and loop diuretics. 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.
Hearing impairment relevant to the invention 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.
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.
In one embodiment, the present invention constitutes a method for treating a mammal having or prone to a hearing (or balance) impairment or treating a mammal prophylactically in conditions where inhibition of expression of p53 or other pro-apoptotic genes is beneficial. The method of the present invention would prevent or reduce the occurrence or severity of a hearing (or balance) impairment that would result from inner ear cell injury, loss, or degeneration, in particular caused by an ototoxic agent. The method of the invention includes administering a therapeutically effective amount of one or more compounds which down-regulate expression of the p53 gene or other pro-apoptotic genes, particularly the novel siRNAs of the present invention, small molecule inhibitors of p53 or other pro-apoptotic genes as described herein or antibodies to p53 polypeptide or other pro-apoptotic proteins.
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.
The methods and compositions of the present invention are also 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.
The methods and compositions of the present invention are also effective when the ototoxic compound is a neoplastic agent such as vincristine, vinblastine, cisplatin and cisplatin-like compounds and taxol and taxol-like compounds.
The methods and compositions of the present invention are also effective in the treatment of accoustic trauma or mechanical trauma, preferably accoustic or mechanical trauma that leads to inner ear hair cell loss or outer ear hair cell loss. Accoustic trauma to be treated in the present invention may be caused by a single exposure to an extremely loud sound of above 120-140 decibels, or following long-term exposure to everyday loud sounds above 85 decibels. The compositions of the present invention are also effective as a preventive treatment in patients expecting an acoustic trauma. Mechanical inner ear trauma to be treated in the present invention is for example the inner ear trauma following an operation for insertion of an electronic device in the inner ear. The compositions of the present invention prevent or minimize the damage to inner ear hair cells associated with this operation.
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 p53 gene or other pro-apoptotic genes, to the patient 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 p53 gene or other pro-apoptotic genes, particularly novel siRNAs are preferably administered locally within the inner ear.
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 patient 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 preferable administered directly to the cochlea as naked siRNA in a vehicle such as PBS or other physiological solutions, but may alternatively be administered with a delivery vehicle as described above.
In another embodiment the methods of treatment are applied to treatment of hearing impairment 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®), tetraplatin, oxaliplatin, aroplatin and transplatin inter alia.
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.
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.
The present invention also provides for a process of preparing a pharmaceutical composition, which comprises:

    
    
        obtaining one or more double stranded compound of the invention; and
        admixing said compound with a pharmaceutically acceptable carrier.
    
    


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.
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.
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.
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.
In embodiments of the invention, the nucleotides can be selected from naturally occurring or synthetically modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of the oligonucleotides 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-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenine, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 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 addition, analogs of nucleotides can be prepared wherein the structures of the nucleotides are fundamentally altered and are better suited as therapeutic or experimental reagents. An example of a nucleotide analog is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. Further, PNAs have been shown to bind more strongly to a complementary DNA sequence than to a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand. Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.
In one embodiment the modification is a modification of the phosphate moiety, whereby the modified phosphate moiety is selected from the group comprising phosphothioate.
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 S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is, among others, described in Eckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules is described in Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 and respective downstream processes are, among others, described in Pingoud A. et. al., in IRL Press 1989 edited by Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 (supra).
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, Nucleic Acids Res., 18, 5433; Wincott et al., 1995, Nucleic Acids Res. 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.
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 PCT publication No. WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
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.
The compounds of the invention can also be synthesized via a tandem synthesis methodology, as described in US patent application publication No. US2004/0019001 (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.
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 Tables A, B, and C, preferably from Table A, ID Nos: 3, 5, 20 and 23 (human sequences) and 11, 12, 14, 17 and 18 (mouse sequences).
In another embodiment, the siRNA molecules are comprised of a double-stranded nucleic acid structure, wherein the first siRNA sequence is selected from Tables A, B, and C, preferably from Table A, ID Nos: 3, 5, 20 and 23 (human p53 sequences) or 11, 12, 14, 17 and 18 (mouse p53 sequences) and the second siRNA molecule targets a pro-apoptotic gene, thereby providing beneficial activity. The tandem double-stranded structure which comprises two or more siRNA sequences is processed intracellularly to form two or more different siRNAs. Such second siRNA molecule is preferably an siRNA molecule that targets a pro-apoptotic gene. Preferred lists of siRNAs directed to selected pro-apoptotic genes are in the Tables A, B, and C.
The siRNA molecules are covalently or non-covalently bound or joined by a linker to form a tandem siRNA molecule. Such tandem siRNA molecules 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 molecule 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.
siRNA molecules that target p53 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 p53 and said additional gene(s) will likely have an additive or synergistic effect for treatment of the diseases disclosed herein.
In a preferred embodiment, the one or more additional siRNA molecules target a pro-apoptotic gene, thus having an additive or synergistic effect with the p53 siRNA. The additional siRNA molecules may target one or more of the pro-apoptotic genes defined above.
In a specific example, the pharmaceutical composition for treatment of the diseases disclosed herein may be comprised of the following compound combinations: 1) p53 siRNA and Fas siRNA; 2) p53 siRNA and Bax siRNA; 3) p53 siRNA and Noxa siRNA; 4) p53 siRNA and Puma siRNA; 5) p53 siRNA and RTP801 siRNA; 6) p53 siRNA and PIDD siRNA; 7) p53 siRNA, Fas siRNA and any of RTP801 siRNA, Bax siRNA, Noxa siRNA or Puma siRNA or PIDD siRNA to form trimers or polymers (i.e., tandem molecules which encode three siRNAs). Other preferred options of pro-apoptotic genes are siRNA combinations of any of p53, TNFα, caspase 2, caspase 3, caspase 9, E2F1, and PARP-1. A preferred combination according to the present invention is p53 siRNA and RTP801 siRNA. (see PCT patent application PCT/US2005/029236).
According to additional embodiments, the present invention relates to a combination of siRNA molecules targeting at least two pro-apoptotic genes in order to prevent or attenuate the apoptotic damage caused by various diseases and medical conditions, in particular various oto-pathologies.
The pro-apoptotic genes to be targeted by the siRNA molecules may be a combination of at least two of: p53, RTP801, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 12, Caspase 14, Apaf-1, Nod1, Nod2, Ipaf, DEFCAP, RAIDD, RICK, Bcl10, ASC, TUCAN, ARC, CLARP, FADD, DEDD, DEDD2, Cryopirin, PYC1, Pyrin, TRADD, UNC5a, UNC5b, UNC5c, ZUD, p84N5, LRDD, CDK1, CDK2, CDK4, CDK5, CDK9, PITSLRE A, CHK2, LATS1, Prk, MAP4K1, MAP4K2, STK4, SLK, GSK3alpha, GSK3beta, MEKK1, MAP3K5 (Ask1), MAP3K7, MAP3K8, MAP3K9, MAP3K10, MAP3K11, MAP3K12, DRP-1, MKK6, p38, JNK3, DAPK1, DRAK1, DRAK2, IRAK, RIP, RIP3, RIP5, PKR, IRE1, MSK1, PKCalpha, PKCbeta, PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta, PKCzeta, CAMK2A, HIPK2, LKB1, BTK, c-Src, FYN, Lck, ABL2, ZAP70, TrkA, TrkC, MYLK, FGFR2, EphA2, AATYK, c-Met, RET, PRKAA2, PLA2G2A, SMPD1, SMPD2, SPP1, FAN, PLCG2, IP6K2, PTEN, SHIP, AIF, AMID, Cytochrome c, Smac, HtrA2, TSAP6, DAP-1, FEM-1, DAP-3, Granzyme B, DIO-1, DAXX, CAD, CIDE-A, CIDE-B, Fsp27, Ape1, ERCC2, ERCC3, BAP31, Bit1, AES, Huntingtin, HIP1, hSir2, PHAP1, GADD45b, GADD34, RAD21, MSH6, ADAR, MBD4, WW45, ATM, mTOR, TIP49, diubiquitin/FAT10, FAF1, p193, Scythe/BAT3, Amida, IGFBP-3, TDAG51, MCG10, PACT, p52/RAP, ALG2, ALG3, presenelin-1, PSAP, AIP1/Alix, ES18, mda-7, p14ARF, ANT1, p33ING1, p33ING2, p53AIP1, p53DINP1, MGC35083, NRAGE, GRIM19, lipocalin 2, glycodelin A, NADE, Porimin, STAG1, DAB2, Galectin-7, Galectin-9, SPRC, FLJ21908, WWOX, XK, DKK-1, Fzd1, Fzd2, SARP2, axin 1, RGS3, DVL1, NFkB2, IkBalpha, NF-ATC1, NF-ATC2, NF-ATC4, zf3/ZNF319, Egr1, Egr2, Egr3, Sp1, TIEG, WT1, Zac1, Icaros, ZNF148, ZK1/ZNF443, ZNF274, WIG1, HIVEP1, HIVEP3, Fliz1, ZPR9, GATA3, TR3, PPARG, CSMF, RXRa, RARa, RARb, RAR9, T3Ra, ERbeta, VDR, GR/GCCR, p53, p73alpha, p63(human [ta alpha, ta beta, ta gamma, da alpha, da beta, da gamma], 53BP2, ASPP1, E2F1, E2F2, E2F3, HIF1 alpha, TCF4, c-Myc, Max, Mad, MITF, Id2, Id3, Id4, c-Jun, c-Fos, ATF3, NF-IL6, CHOP, NRF1, c-Maf, Bach2, Msx2, Csx, Hoxa5, Ets-1, PU1/Spi1, Ets-2, ELK1, TEL1, c-Myb, TBX5, IRF1, IRF3, IRF4, IRF9, AP-2 alpha, FKHR, FOXO1A, FKHRL1, FOXO3a, AFX1, MLLT7, Tip60, BTG1, AUF1, HNRPD, TIA1, NDG1, PCBP4, MCG10, FXR2, TNFR2, LTbR, CD40, CD27, CD30, 4-1BB, TNFRSF19, XEDAR, Fn14, OPG, DcR3, FAS, TNFR1, WSL-1, p75NTR, DR4, DR5, DR6, EDAR, TNF alpha, FAS ligand, TRAIL, Lymphotoxin alpha, Lymphotoxin beta, 4-1BBL, RANKL, TL1, TWEAK, LIGHT, APRIL, IL-1-alpha, IL-1-beta, IL-18, FGF8, IL-2, IL-21, IL-5, IL-4, IL-6, LIF, IL-12, IL-7, IL-10, IL-19, IL-24, IFN alpha, IFN beta, IFN gamma, M-CSF, prolactin, TLR2, TLR3, TLR4, MyD88, TRIF, RIG-1, CD14, TCR alpha, CD3 gamma, CD8, CD4, CD7, CD19, CD28, CTLA4, SEMA3A, SEMA3B, HLA-A, HLA-B, HLA-L, HLA-DMalpha, CD22, CD33, CALL, DCC, ICAM1, ICAM3, CD66a, PVR, CD47, CD2, Thy-1, SIRPa1, CD5, E-cadherin, ITGAM, ITGAV, CD18, ITGB3, CD9, IgE Fc R beta, CD82, CD81, PERP, CD24, CD69, KLRD1, galectin 1, B4GALT1, Clq alpha, C5R1, MIP1alpha, MIP1beta, RANTES, SDF1, XCL1, CCCKR5, OIAS/OAS1, INDO, MxA, IFI16, AIM2, iNOS, HB-EGF, HGF, MIF, TRAF3, TRAF4, TRAF6, PAR-4, IKKGamma, FIP2, TXBP151, FLASH, TRF1, IEX-1S, Dok1, BLNK, CIN85, Bif-1, HEF1, Vav1, RasGRP1, POSH, Rac1, RhoA, RhoB, RhoC, ALG4, SPP1, TRIP, SIVA, TRABID, TSC-22, BRCA1, BARD1, 53BP1, MDC1, Mdm4, Siah-1, Siah-2, RoRet, TRIM35, PML, RFWD1, DIP1, Socs1, PARC, USP7, CYLD, TP53BP2, CYBA, NOX3, HRK, C1QBP, BNIP3, MAPK8, MAPK14, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, GJA1, TYROBP, CTGF, RTN4R, ANXA2, DUOX1, SLC5A 1, SLC2A2, AKR1B1, SORD, SLC2A 1 and MME.
In another aspect of the invention it is envisaged that any siRNA which targets a pro-apoptotic gene may be used in order to prevent or attenuate the apoptotic damage caused by various diseases and medical conditions. In particular, such medical condition is hearing impairment, or other oto-pathologies associated with cell death of inner ear hair cells. Preferred lists of siRNAs directed to selected pro-apoptotic genes are in the Tables A, B, and C.
The pro-apoptotic genes to be targeted by the siRNA molecules may be one or more of: p53, RTP801, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 12, Caspase 14, Apaf-1, Nod1, Nod2, Ipaf, DEFCAP, RAIDD, RICK, Bcl10, ASC, TUCAN, ARC, CLARP, FADD, DEDD, DEDD2, Cryopirin, PYC1, Pyrin, TRADD, UNC5a, UNC5b, UNC5c, ZUD, p84N5, LRDD, CDK1, CDK2, CDK4, CDK5, CDK9, PITSLRE A, CHK2, LATS1, Prk, MAP4K1, MAP4K2, STK4, SLK, GSK3alpha, GSK3beta, MEKK1, MAP3K5 (Ask1), MAP3K7, MAP3K8, MAP3K9, MAP3K10, MAP3K11, MAP3K12, DRP-1, MKK6, p38, JNK3, DAPK1, DRAK1, DRAK2, IRAK, RIP, RIP3, RIP5, PKR, IRE1, MSK1, PKCalpha, PKCbeta, PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta, PKCzeta, CAMK2A, HIPK2, LKB1, BTK, c-Src, FYN, Lck, ABL2, ZAP70, TrkA, TrkC, MYLK, FGFR2, EphA2, AATYK, c-Met, RET, PRKAA2, PLA2G2A, SMPD1, SMPD2, SPP1, FAN, PLCG2, IP6K2, PTEN, SHIP, AIF, AMID, Cytochrome c, Smac, HtrA2, TSAP6, DAP-1, FEM-1, DAP-3, Granzyme B, DIO-1, DAXX, CAD, CIDE-A, CIDE-B, Fsp27, Ape1, ERCC2, ERCC3, BAP31, Bit1, AES, Huntingtin, HIP1, hSir2, PHAP1, GADD45b, GADD34, RAD21, MSH6, ADAR, MBD4, WW45, ATM, mTOR, TIP49, diubiquitin/FAT10, FAF1, p193, Scythe/BAT3, Amida, IGFBP-3, TDAG51, MCG10, PACT, p52/RAP, ALG2, ALG3, presenelin-1, PSAP, AIP1/Alix, ES18, mda-7, p14ARF, ANT1, p33ING1, p33ING2, p53AIP1, p53DINP1, MGC35083, NRAGE, GRIM19, lipocalin 2, glycodelin A, NADE, Porimin, STAG1, DAB2, Galectin-7, Galectin-9, SPRC, FLJ21908, WWOX, XK, DKK-1, Fzd1, Fzd2, SARP2, axin 1, RGS3, DVL1, NFkB2, IkBalpha, NF-ATC1, NF-ATC2, NF-ATC4, zf3/ZNF319, Egr1, Egr2, Egr3, Sp1, TIEG, WT1, Zac1, Icaros, ZNF148, ZK1/ZNF443, ZNF274, WIG1, HIVEP1, HIVEP3, Fliz1, ZPR9, GATA3, TR3, PPARG, CSMF, RXRa, RARa, RARb, RARg, T3Ra, ERbeta, VDR, GR/GCCR, p53, p73alpha, p63(human [ta alpha, ta beta, ta gamma, da alpha, da beta, da gamma], 53BP2, ASPP1, E2F1, E2F2, E2F3, HIF1 alpha, TCF4, c-Myc, Max, Mad, MITF, Id2, Id3, Id4, c-Jun, c-Fos, ATF3, NF-IL6, CHOP, NRF1, c-Maf, Bach2, Msx2, Csx, Hoxa5, Ets-1, PU1/Spi1, Ets-2, ELK1, TEL1, c-Myb, TBX5, IRF1, IRF3, IRF4, IRF9, AP-2 alpha, FKHR, FOXO1A, FKHRL1, FOXO3a, AFX1, MLLT7, Tip60, BTG1, AUF1, HNRPD, TIA1, NDG1, PCBP4, MCG10, FXR2, TNFR2, LTbR, CD40, CD27, CD30, 4-1BB, TNFRSF19, XEDAR, Fn14, OPG, DcR3, FAS, TNFR1, WSL-1, p75NTR, DR4, DR5, DR6, EDAR, TNF alpha, FAS ligand, TRAIL, Lymphotoxin alpha, Lymphotoxin beta, 4-1BBL, RANKL, TL1, TWEAK, LIGHT, APRIL, IL-1-alpha, IL-1-beta, IL-18, FGF8, IL-2, IL-21, IL-5, IL-4, IL-6, LIF, IL-12, IL-7, IL-10, IL-19, IL-24, IFN alpha, IFN beta, IFN gamma, M-CSF, prolactin, TLR2, TLR3, TLR4, MyD88, TRIF, RIG-1, CD14, TCR alpha, CD3 gamma, CD8, CD4, CD7, CD19, CD28, CTLA4, SEMA3A, SEMA3B, HLA-A, HLA-B, HLA-L, HLA-DMalpha, CD22, CD33, CALL, DCC, ICAM1, ICAM3, CD66a, PVR, CD47, CD2, Thy-1, SIRPa1, CD5, E-cadherin, ITGAM, ITGAV, CD18, ITGB3, CD9, IgE Fc R beta, CD82, CD81, PERP, CD24, CD69, KLRD1, galectin 1, B4GALT1, Clq alpha, C5R1, MIP1alpha, MIP1beta, RANTES, SDF1, XCL1, CCCKR5, OIAS/OAS1, INDO, MxA, IFI16, AIM2, iNOS, HB-EGF, HGF, MIF, TRAF3, TRAF4, TRAF6, PAR-4, IKKGamma, FIP2, TXBP151, FLASH, TRF1, IEX-1S, Dok1, BLNK, CIN85, Bif-1, HEF1, Vav1, RasGRP1, POSH, Rac1, RhoA, RhoB, RhoC, ALG4, SPP1, TRIP, SIVA, TRABID, TSC-22, BRCA1, BARD1, 53BP1, MDC1, Mdm4, Siah-1, Siah-2, RoRet, TRIM35, PML, RFWD1, DIP1, Socs1, PARC, USP7, CYLD, TP53BP2, CYBA, NOX3, HRK, C1QBP, BNIP3, MAPK8, MAPK14, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, GJA1, TYROBP, CTGF, RTN4R, ANXA2, DUOX1, SLC5A1, SLC2A2, AKR1B1, SORD, SLC2A1 and MME.
Preferred pro-apoptotic genes to be targeted by the siRNA molecules may be one or more of the genes: tumor protein p53 binding protein, 2 (TP53BP2); leucine-rich repeats and death domain containing (LRDD); cytochrome b-245, alpha polypeptide (CYBA); activating transcription factor 3 (ATF3); caspase 2, apoptosis-related cysteine peptidase (neural precursor cell expressed, developmentally down-regulated 2) (CASP2); NADPH oxidase 3 (NOX3); harakiri, BCL2 interacting protein (contains only BH3 domain) (HRK); complement component 1, q subcomponent binding protein (C1QBP); BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3); mitogen-activated protein kinase 8 (MAPK8); mitogen-activated protein kinase 14 (MAPK14); 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, bone sialoprotein I, early T-lymphocyte activation 1) (SPP1); reticulon 4 receptor-RTN4R; annexin A2 (ANXA2); ras homolog gene family, member A (RHOA); dual oxidase 1 (DUOX1); solute carrier family 5 (sodium/glucose cotransporter), member 1 (SLC5A1); solute carrier family 2 (facilitated glucose transporter), member 2 (SLC2A2); aldo-keto reductase family 1, member B1 (aldose reductase) (AKR1B1); sorbitol dehydrogenase (SORD); solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) and membrane metallo-endopeptidase (neutral endopeptidase, enkephalinase) (MME).
Particular pro-apoptotic genes to be targeted by the siRNA molecules may be one or more of the genes: dual oxidase 1 (DUOX1), cytochrome b-245, alpha polypeptide (CYBA); activating transcription factor 3 (ATF3); NADPH oxidase 3 (NOX3); ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1); and ras homolog gene family, member A (RHOA).
As disclosed herein, aptamers may also be used in the present invention alone or in combination with the novel siRNAs disclosed herein for targeting p53 or any of the pro-apoptotic genes of the invention and for the treatment of any one of the conditions disclosed herein. For example, an aptamer can be used with any one of the siRNAs disclosed herein in combination therapy for the treatment of any one of the conditions disclosed herein. The novel pharmaceutical composition employed for such a combination therapy, which is also part of the present invention, may comprise an siRNA of the present invention covalently or non-covalently attached to an aptamer. Aptamers are RNA or DNA single-strand or double-strand oligonucleic acids which bind to a target protein and do not generally exhibit non-specific effects. Aptamers can be modified for stability or other desired qualities in accordance with any nucleic acid modifications disclosed herein and/or known to one of skill in the art. Modifications to aptamers can be introduced anywhere in the molecule, such as the 5′ or 3′ termini, or at any internally defined modification site. For example, RNA aptamers can be stabilized with 2′-Fluoro or 2′-amino modified pyrimidines. Aptamers can also be linked to reporter molecules or linker chemistries and can be attached to beads or other solid support if necessary (e.g., 5′ or 3′ amino, thiol ester or biotin groups). Thioaptamers are aptamers which contain sulfur modifications at specific internucleoside phosphoryl sites, and may possess enhanced stability, nuclease resistance, target affinity and/or selectivity. Examples of thioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate (S2-ODN) oligodeoxy thioaptamers. For further information on aptamers and thioaptamers see U.S. Pat. Nos. 5,218,088 and 6,423,493.
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 a p53 siRNA molecule or with any other pro-apoptotic siRNA. In another example, an aptamer which can act like a ligand/antibody may be combined (covalently or non-covalently) with a p53 siRNA molecule or with any other pro-apoptotic siRNA.
The term “Covalent bonding” as used herein refers to chemical bonding that is characterized by the sharing of pairs of electrons between atoms.
The term “Noncovalent bonding” as used herein refers to a variety of interactions that are not covalent in nature between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together, usually in a specific orientation or conformation. These noncovalent interactions include: ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces and Dipole-dipole bonds.
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.
In one specific embodiment of this invention, topical, intracochlear, transtympanic and transdermal formulations are particularly preferred. They can be administered by subcutaneous injection. Additionally, they can be administered by implants, in liquid drops to the ear canal, delivered to the scala tympani chamber of the inner ear by transtympanic injection, or provided as a diffusible member of a cochlear hearing implant.
A preferred administration mode is directly to the affected portion of the ear or vestibule, topically as by implant for example, and, preferably to the affected hair cells or their supporting cells, so as to direct the active molecules to the source and minimize its side effects. A preferred administration mode is a topical delivery of the p53 inhibitor(s) onto the round window membrane of the cochlea. Such a method of administration of other compounds is disclosed for example in Tanaka et al. (Hear Res. 2003 March; 177(1-2):21-31).
As noted, the compositions can be injected through chronically implanted cannulas or chronically infused with the help of osmotic minipumps. Subcutaneous pumps are available that deliver active compounds through a small tubing to the appropriate area. Highly sophisticated pumps can be refilled through the skin and their delivery rate can be set without surgical intervention. Examples of suitable administration protocols and delivery systems involving a subcutaneous pump device or continuous infusion through a totally implanted drug delivery system are described for example by Harbaugh, J. Neural Transm. Suppl., 24: 271-277 (1987) and DeYebenes et al., Mov. Disord., 2: 143-158 (1987), the disclosures of which are incorporated herein by reference.
Delivery of therapeutic agents to the inner ear of a subject can be done by contact with the inner ear or through the external auditory canal and middle ear, as by injection or via catheters, or as exemplified in U.S. Pat. No. 5,476,446, which provides a multi-functional apparatus specifically designed for use in treating and/or diagnosing the inner ear of a human subject. The apparatus is capable of delivering therapeutic agents into the inner ear or to middle-inner ear interface tissues. In addition, other systems may be used to deliver the molecules of the present invention including but not limited to an osmotic pump which is described in Kingma, G. G., et al., “Chronic drug infusion into the scala tympani of the guinea pig cochlea”, Journal of Neuroscience Methods, 45:127-134 (1992). An exemplary, commercially-available osmotic pump may be obtained from the Alza Corp. of Palo Alto, Calif. (USA).
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 an p53 gene. In particular, it is envisaged that this oligonucleotide comprises sense and antisense siRNA sequences as depicted in any one of Tables A, B, and C.
As used herein, the term “polypeptide” refers to, in addition to a polypeptide, an oligopeptide, peptide and a full protein.
As used herein, the term “inhibition” of a pro-apoptotic gene means inhibition of the gene expression (transcription or translation) or polypeptide activity.
Although the inhibitor may be an siRNA molecule, other inhibitors contemplated to be used in the methods of the invention to inhibit a pro-apoptopic 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, proteins, polypeptides and peptides including peptido-mimetics 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.
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:

    (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;
    (2) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′2) is a dimer of two Fab fragments held together by two disulfide bonds;
    (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
    (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.

Screening of Inactivation Compounds for Pro-apoptotic Genes:



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 p53 polypeptide. The compounds to be screened comprise inter alia substances such as small chemical molecules and antisense oligonucleotides.
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.
The present invention is illustrated in detail below with reference to the Examples, but is not to be construed as being limited thereto.
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
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
Generation of Sequences for Active siRNA Compounds
Using proprietary algorithms and the known sequence of gene p53 (SEQ ID NO:1), the sequences of many potential siRNAs were generated. Table A shows 23 siRNAs which have so far been selected, chemically synthesized and tested for activity (see Example 2). All these siRNAs are 19-mers.















TABLE A










NM_000546
NM_011640
NM_0309



Number
Index
Sense strand
Antisense strand
Species
(human)
(mouse)
89(rat)























1
Mo3
GUACAUGUGUAAUAGCUCC
GGAGCUAUUACACAUGUAC
mouse
3 mis
1232-1250
2 mis



 


2
Hu2′
GACUCCAGUGGUAAUCUAC
GUAGAUUACCACUGGAGUC
human*
1026-1044
3 mis
2 mis


 


3
QHMo
CAGACCUAUGGAAACUACU
AGUAGUUUCCAUAGGUCUG
hum, mon
310-328
3 mis
4 mis



n1


 


4
QHMo
CUACCUCCCGCCAUAAAAA
UUUUUAUGGCGGGAGGUAG
hum, mon
1378-1396
1 mis
1 mis



n2


 


5
QH1
CCCAAGCAAUGGAUGAUUU
AAAUCAUCCAUUGCUUGGG
human
361-379
No
No


 


6
QH2
CCCGGACGAUAUUGAACAA
UUGUUCAAUAUCGUCCGGG
human
389-407
No
No


 


7
QM1
GAGUCACAGUCGGAUAUCA
UGAUAUCCGACUGUGACUC
mouse
No
552-570
2 mis


 


8
QM2
GGAUGUUGAGGAGUUUUUU
AAAAAACUCCUCAACAUCC
mouse
No
680-698
4 mis


 


9
QM3
CAUCUUUUGUCCCUUCUCA
UGAGAAGGGACAAAAGAUG
mouse
2 mis
808-826
2 mis


 


10
QM6
GGAAUAGGUUGAUAGUUGU
ACAACUAUCAACCUAUUCC
mouse
No
1870-1888
No


 


11
QM4
GGACAGCCAAGUCUGUUAU
AUAACAGACUUGGCUGUCC
mouse, rat
2 mis
877-895
527-545


 


12
QM5
GAAGAAAAUUUCCGCAAAA
UUUUGCGGAAAUUUUCUUC
mouse, rat
3 mis
1383-1401
1033-









1051


 


13
A17
CUGGGACAGCCAAGUCUGU
ACAGACUUGGCUGUCCCAG
hum, mus
598-616
874-892
2 mis


 


14
E2
UCAUCACACUGGAAGACUC
GAGUCUUCCAGUGUGAUGA
hum, mus,
1012-1030
1288-1306
938-956






rat


 


15
E6
CACACUGGAAGACUCCAGU
ACUGGAGUCUUCCAGUGUG
hum, mus,
1016-1034
1292-1310
942-960






rat


 


16
B1
GCGCCAUGGCCAUCUACAA
UUGUAGAUGGCCAUGGCGC
hum,
724-742
1000-1018
652-






mon, mus


668(17)


 


17
B2
CGCCAUGGCCAUCUACAAG
CUUGUAGAUGGCCAUGGCG
hum,
725-743
1001-1019
652-






mon, mus


669(18)


 


18
C1
AGUCACAGCACAUGACGGA
UCCGUCAUGUGCUGUGACU
hum,
745-763
1021-1039
2 mis






mon, mus


 


19
F2
UCCGAGUGGAAGGAAAUUU
AAAUUUCCUUCCACUCGGA
hum,
835-853
1 mis
3 mis






mon, dog


 


20
F3
CCGAGUGGAAGGAAAUUUG
CAAAUUUCCUUCCACUCGG
hum,
836-854
1 mis
3 mis






mon, dog


 


21
G1
GACAGAAACACUUUUCGAC
GUCGAAAAGUGUUUCUGUC
hum,
873-891
No
No






mon, dog


 


22
H2
GUGUGGUGGUGCCCUAUGA
UCAUAGGGCACCACCACAC
hum,
895-913
3 mis
3 mis






mon, dog


 


23
I5
GAGAAUAUUUCACCCUUCA
UGAAGGGUGAAAUAUUCUC
hum,
1225-1243
2 mis
1 mis






mon, dog





Note that in the above Table A, the sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25 respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOS: 26-48 respectively. siRNA compound No 1 (SEQ ID NOS: 3 and 26) is known from the literature (Dirac and Bernards, Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53, J. Biol. Chem.(2003) 278: 11731) and siRNA No 2 (SEQ ID NOS: 4 and 27) is alsoknown from the literature (Brummelkamp et al. Science 2002, 296: 550-553). However, the use of these compounds in the methods of treatment disclosed herein is previously undisclosed and thus novel.






Table B below shows 71 additional 19-mer siRNAs which have been generated by the proprietary algorithms.















TABLE B










gi2689466










gbU48957.1
gi53575emb
gi499622







U48957
X01237
9dbjAB02






gi13097806
(Macaca
.1MMP53R
0761.1






gbBC003596.1
fascicu-
(Mouse
(Canis


No.
Source
Sense
AntiSense
(Homo sapiens)
laris)
mRNA)
familiaris)























1
Human
GUACCACCAUCCACUACAA
UUGUAGUGGAUGGUGGUAC
[806-824]

[835-852]




 


2
Human
GGAAACUACUUCCUGAAAA
UUUUCAGGAAGUAGUUUCC
[188-206]

[234-247]


 


3
Human
AGACUCCAGUGGUAAUCUA
UAGAUUACCACUGGAGUCU
[894-912]

[922-933]


 


4
Human
CCAUCCACUACAACUACAU
AUGUAGUUGUAGUGGAUGG
[812-830]

[840-858]


 


5
Human
CCACCAUCCACUACAACUA
UAGUUGUAGUGGAUGGUGG
[809-827]

[837-852]


 


6
Human
AAACACUUUUCGACAUAGU
ACUAUGUCGAAAAGUGUUU
[747-765]

—


 


7
Human
CAUGAGCGCUGCUCAGAUA
UAUCUGAGCAGCGCUCAUG
[655-673]

[683-696]


 


8
Human
CCAUGGCCAUCUACAAGCA
UGCUUGUAGAUGGCCAUGG
[596-614]

[624-640]


 


9
Human
CCAAGUCUGUGACUUGCAC
GUGCAAGUCACAGACUUGG
[476-494]

—


 


10
Human
AAACUUUGCUGCCAAAAAA
UUUUUUGGCAGCAAAGUUU
[2476-2494]

—


 


11
Human
CCCUCCUUCUCCCUUUUUA
UAAAAAGGGAGAAGGAGGG
[2421-2439]

—


 


12
Human
GCAAGCACAUCUGCAUUUU
AAAAUGCAGAUGUGCUUGC
[2389-2407]

—


 


13
Human
GGGUCAACAUCUUUUACAU
AUGUAAAAGAUGUUGACCC
[2367-2385]

—


 


14
Human
GAAGGGUCAACAUCUUUUA
UAAAAGAUGUUGACCCUUC
[2364-2382]

—


 


15
Human
CUGGAAGGGUCAACAUCUU
AAGAUGUUGACCCUUCCAG
[2361-2379]

—


 


16
Human
CCAGAGUGCUGGGAUUACA
UGUAAUCCCAGCACUCUGG
[2321-2339]

—


 


17
Human
GAUGGGGUCUCACAGUGUU
AACACUGUGAGACCCCAUC
[2249-2267]

—


 


18
Human
GCCAACUUUUGCAUGUUUU
AAAACAUGCAAAAGUUGGC
[2225-2243]

—


 


19
Human
CCAUGGCCAGCCAACUUUU
AAAAGUUGGCUGGCCAUGG
[2216-2234]

—


 


20
Human
AGACCCAGGUCCAGAUGAA
UUCAUCUGGACCUGGGUCU
[288-306]

—


 


21
Human,
CCAUCAUCACACUGGAAGA
UCUUCCAGUGUGAUGAUGG
[878-896]

[906-924]



mouse


 


22
Human,
CAUCACACUGGAAGACUCC
GGAGUCUUCCAGUGUGAUG
[882-900]

[910-928]



mouse


 


23
Human,
CAUCAUCACACUGGAAGAC
GUCUUCCAGUGUGAUGAUG
[879-897]

[907-925]



mouse


 


24
Human,
ACCAUCAUCACACUGGAAG
CUUCCAGUGUGAUGAUGGU
[877-895]

[905-923]



mouse


 


25
Human,
AUCAUCACACUGGAAGACU
AGUCUUCCAGUGUGAUGAU
[880-898]

[908-926]



mouse


 


26
Human,
CACUGGAAGACUCCAGUGG
CCACUGGAGUCUUCCAGUG
[887-905]

[915-933]



mouse


 


27
Human,
ACACUGGAAGACUCCAGUG
CACUGGAGUCUUCCAGUGU
[886-904]
[766-784]
[914-932]



cynomoglus,



mouse


 


28
Human,
UCACACUGGAAGACUCCAG
CUGGAGUCUUCCAGUGUGA
[884-902]
[764-782]
[912-930]



cynomoglus,



mouse


 


29
Human,
AUCACACUGGAAGACUCCA
UGGAGUCUUCCAGUGUGAU
[883-901]
[763-781]
[911-929]



cynomoglus,



mouse


 


30
Human,
CACAGCACAUGACGGAGGU
ACCUCCGUCAUGUGCUGUG
[617-635]
[497-515]
[645-663]



cynomoglus,



mouse


 


31
Human,
CACUGGAAGACUCCAGUGG
CCACUGGAGUCUUCCAGUG
[887-905]
[767-785]
[915-933]



cynomoglus,



mouse


 


32
Human,
UCACAGCACAUGACGGAGG
CCUCCGUCAUGUGCUGUGA
[616-634]
[496-514]
[644-662]



cynomoglus,



mouse


 


33
Human,
GUCACAGCACAUGACGGAG
CUCCGUCAUGUGCUGUGAC
[615-633]
[495-513]
[643-661]



cynomoglus,



mouse


 


34
Human,
CCAUCCACUACAACUACAU
AUGUAGUUGUAGUGGAUGG
[812-830]
[692-710]

[702-720]



cynomoglus,



dog


 


35
Human,
CCACCAUCCACUACAACUA
UAGUUGUAGUGGAUGGUGG
[809-827]
[689-707]

[699-717]



cynomoglus,



dog


 


36
Human,
GAAUAUUUCACCCUUCAGA
UCUGAAGGGUGAAAUAUUC
[1096-1114]
[976-994]

[986-



cynomoglus,





1004]



dog


 


37
Human,
CGAGUGGAAGGAAAUUUGC
GCAAAUUUCCUUCCACUCG
[706-724]
[586-604]

[596-614]



cynomoglus,



dog


 


38
Human,
GAGAAUAUUUCACCCUUCA
UGAAGGGUGAAAUAUUCUC
[1094-1112]
[974-992]

[984-



cynomoglus,





1002]



dog


 


39
Human,
CUACAUGUGUAACAGUUCC
GGAACUGUUACACAUGUAG
[825-843]
[705-723]

[715-733]



cynomoglus,



dog


 


40
Human,
AACUACAUGUGUAACAGUU
AACUGUUACACAUGUAGUU
[823-841]
[703-721]

[713-731]



cynomoglus,



dog


 


41
Human,
CAACUACAUGUGUAACAGU
ACUGUUACACAUGUAGUUG
[822-840]
[702-720]

[712-730]



cynomoglus,



dog


 


42
Human,
CACUACAACUACAUGUGUA
UACACAUGUAGUUGUAGUG
[817-835]
[697-715]

[707-725]



cynomoglus,



dog


 


43
Human,
CCACUACAACUACAUGUGU
ACACAUGUAGUUGUAGUGG
[816-834]
[696-714]

[706-724]



cynomoglus,



dog


 


44
Human,
GACAGAAACACUUUUCGAC
GUCGAAAAGUGUUUCUGUC
[742-760]
[622-640]

[632-650]



cynomoglus,



dog


 


45
Human,
GGAGAAUAUUUCACCCUUC
GAAGGGUGAAAUAUUCUCC
[1093-1111]
[973-991]

[983-



cynomoglus,





1001]



dog


 


46
Human,
GUGUAACAGUUCCUGCAUG
CAUGCAGGAACUGUUACAC
[831-849]
[711-729]

[721-739]



cynomoglus,



dog


 


47
Human,
ACAACUACAUGUGUAACAG
CUGUUACACAUGUAGUUGU
[821-839]
[701-719]

[711-729]



cynomoglus,



dog


 


48
Human,
ACUACAACUACAUGUGUAA
UUACACAUGUAGUUGUAGU
[818-836]
[698-716]

[708-726]



cynomoglus,



dog


 


49
Human,
ACCAUCCACUACAACUACA
UGUAGUUGUAGUGGAUGGU
[811-829]
[691-709]

[701-719]



cynomoglus,



dog


 


50
Human,
ACCACCAUCCACUACAACU
AGUUGUAGUGGAUGGUGGU
[808-826]
[688-706]

[698-716]



cynomoglus,



dog


 


51
Human,
UACCACCAUCCACUACAAC
GUUGUAGUGGAUGGUGGUA
[807-825]
[687-705]

[697-715]



cynomoglus,



dog


 


52
Human,
ACAGAAACACUUUUCGACA
UGUCGAAAAGUGUUUCUGU
[743-761]
[623-641]

[633-651]



cynomoglus,



dog


 


53
Human,
GAGUGGAAGGAAAUUUGCG
CGCAAAUUUCCUUCCACUC
[707-725]
[587-605]

[597-615]



cynomoglus,



dog


 


54
Human,
AUAUUUCACCCUUCAGAUC
GAUCUGAAGGGUGAAAUAU
[1098-1116]
[978-996]

[988-



cynomoglus,





1006]



dog


 


55
Human,
AAUAUUUCACCCUUCAGAU
AUCUGAAGGGUGAAAUAUU
[1097-1115]
[977-995]

[987-



cynomoglus,





1005]



dog


 


56
Human,
AGAAUAUUUCACCCUUCAG
CUGAAGGGUGAAAUAUUCU
[1095-1113]
[975-993]

[985-



cynomoglus,





1003]



dog


 


57
Human,
UGGAGAAUAUUUCACCCUU
AAGGGUGAAAUAUUCUCCA
[1092-1110]
[972-990]

[982-



cynomoglus,





1000]



dog


 


58
Human,
ACAUGUGUAACAGUUCCUG
CAGGAACUGUUACACAUGU
[827-845]
[707-725]

[717-735]



cynomoglus,



dog


 


59
Human,
UACAACUACAUGUGUAACA
UGUUACACAUGUAGUUGUA
[820-838]
[700-718]

[710-728]



cynomoglus,



dog


 


60
Human,
CUACAACUACAUGUGUAAC
GUUACACAUGUAGUUGUAG
[819-837]
[699-717]

[709-727]



cynomoglus,



dog


 


61
Human,
UCCACUACAACUACAUGUG
CACAUGUAGUUGUAGUGGA
[815-833]
[695-713]

[705-723]



cynomoglus,



dog


 


62
Human,
AUCCACUACAACUACAUGU
ACAUGUAGUUGUAGUGGAU
[814-832]
[694-712]

[704-722]



cynomoglus,



dog


 


63
Human,
CAUCCACUACAACUACAUG
CAUGUAGUUGUAGUGGAUG
[813-831]
[693-711]

[703-721]



cynomoglus,



dog


 


64
Human,
CACCAUCCACUACAACUAC
GUAGUUGUAGUGGAUGGUG
[810-828]
[690-708]

[700-718]



cynomoglus,



dog


 


65
Human,
UGUGUAACAGUUCCUGCAU
AUGCAGGAACUGUUACACA
[830-848]
[710-728]

[720-738]



cynomoglus,



dog


 


66
Human,
CAUGUGUAACAGUUCCUGC
GCAGGAACUGUUACACAUG
[828-846]
[708-726]

[718-736]



cynomoglus,



dog


 


67
Human,
UACAUGUGUAACAGUUCCU
AGGAACUGUUACACAUGUA
[826-844]
[706-724]

[716-734]



cynomoglus,



dog


 


68
Human,
ACUACAUGUGUAACAGUUC
GAACUGUUACACAUGUAGU
[824-842]
[704-722]

[714-732]



cynomoglus,



dog


 


69
Human,
AUCCGAGUGGAAGGAAAUU
AAUUUCCUUCCACUCGGAU
[703-721]
[583-601]

[593-611]



cynomoglus,



dog


 


70
Human,
UCACUCCAGCCACCUGAAG
CUUCAGGUGGCUGGAGUGA
[1212-1230]
[1092-1110]

[1102-



cynomoglus,





1120]



dog


 


71
Human,
CUCACUCCAGCCACCUGAA
UUCAGGUGGCUGGAGUGAG
[1211-1229]
[1091-1109]

[1101-



cynomoglus,





1119]



dog





Note that in the above Table B, the sense strands of siRNAs 1-71 have SEQ ID NOS: 49-119 respectively, and the antisense strands of siRNAs 1-71 have SEQ ID NOS: 120-190 respectively.






Table C below shows 63 additional 21-mer siRNAs which have been generated by the proprietary algorithms.















TABLE C










gi2689466










gbU48957.1
gi53575emb






gi13097806
U48957
X01237.1M
gi4996229dbj






gbBC003596.1
(Macaca
MP53R
AB020761.1






(Homo
fascicu-
(Mouse
(Canis


No.
Source
Sense SiRNA
AntiSense SiRNA
sapiens)
laris)
mRNA)
familiaris)























1
Human
GGAAGAGAAUCUCCGCAAGAA
UUCUUGCGGAGAUUCUCUUCC
[975-995]
—
—
—



 


2
Human
GUACCACCAUCCACUACAACU
AGUUGUAGUGGAUGGUGGUAC
[806-826]
[686-706]
[835-852]
[697-716]


 


3
Human
GGACGAUAUUGAACAAUGGUU
AACCAUUGUUCAAUAUCGUCC
[261-281]
—
—
—


 


4
Human
CCAGCCACCUGAAGUCCAAAA
UUUUGGACUUCAGGUGGCUGG
[1217-1237]
[1097-1115]
—
[1107-1120]


 


5
Human
GAGAAUAUUUCACCCUUCAGA
UCUGAAGGGUGAAAUAUUCUC
[1094-1114]
[974-994]
[1122-1137]
 [984-1004]


 


6
Human
AGAAACCACUGGAUGGAGAAU
AUUCUCCAUCCAGUGGUUUCU
[1079-1099]
[959-979]
—
—


 


7
Human
CUACUGGGACGGAACAGCUUU
AAAGCUGUUCCGUCCCAGUAG
[910-930]
[790-810]
—
—


 


8
Human
AGACUCCAGUGGUAAUCUACU
AGUAGAUUACCACUGGAGUCU
[894-914]
[774-794]
[922-933]
[784-795]


 


9
Human
CUGGAAGACUCCAGUGGUAAU
AUUACCACUGGAGUCUUCCAG
[889-909]
[769-789]
[917-933]
[779-795]


 


10
Human
GAAACUACUUCCUGAAAACAA
UUGUUUUCAGGAAGUAGUUUC
[189-209]
[69-87]
[235-247]
[122-135]


 


11
Human
GGAAACUACUUCCUGAAAACA
UGUUUUCAGGAAGUAGUUUCC
[188-208]
[68-87]
[234-247]
[122-134]


 


12
Human
AAACACUUUUCGACAUAGUGU
ACACUAUGUCGAAAAGUGUUU
[747-767]
[627-647]
—
[637-657]


 


13
Human
GGAGUAUUUGGAUGACAGAAA
UUUCUGUCAUCCAAAUACUCC
[729-749]
[609-629]
—
—


 


14
Human
UCAGACCUAUGGAAACUACUU
AAGUAGUUUCCAUAGGUCUGA
[178-198]
[58-78]
[231-244]
—


 


15
Human
CCAUGGCCAUCUACAAGCAGU
ACUGCUUGUAGAUGGCCAUGG
[596-616]
[476-496]
[624-640]
[485-495]


 


16
Human
CCAAGUCUGUGACUUGCACGU
ACGUGCAAGUCACAGACUUGG
[476-496]
[356-376]
—
—


 


17
Human
GGACAGCCAAGUCUGUGACUU
AAGUCACAGACUUGGCUGUCC
[470-490]
[352-370]
[498-513]
[357-377]


 


18
Human
CCCUCCUUCUCCCUUUUUAUA
UAUAAAAAGGGAGAAGGAGGG
[2421-2441]
—
[1721-1731]
—


 


19
Human,
CCAUCCACUACAACUACAUGU
ACAUGUAGUUGUAGUGGAUGG
[812-832]
[692-712]
[840-860]
[702-722]



cyno-



moglus,



dog


 


20
Human,
CCACCAUCCACUACAACUACA
UGUAGUUGUAGUGGAUGGUGG
[809-829]
[689-709]
[837-857]
[699-719]



cyno-



moglus,



dog


 


21
Human,
GAGAAUAUUUCACCCUUCAGA
UCUGAAGGGUGAAAUAUUCUC
[1094-1114]
[974-994]

 [984-1004]



cyno-



moglus,



dog


 


22
Human,
GGAGAAUAUUUCACCCUUCAG
CUGAAGGGUGAAAUAUUCUCC
[1093-1113]
[973-993]

 [983-1003]



cyno-



moglus,



dog


 


23
Human,
CUACAUGUGUAACAGUUCCUG
CAGGAACUGUUACACAUGUAG
[825-845]
[705-725]

[715-735]



cyno-



moglus,



dog


 


24
Human,
ACAACUACAUGUGUAACAGUU
AACUGUUACACAUGUAGUUGU
[821-841]
[701-721]

[711-731]



cyno-



moglus,



dog


 


25
Human,
CCACUACAACUACAUGUGUAA
UUACACAUGUAGUUGUAGUGG
[816-836]
[696-716]

[706-726]



cyno-



moglus,



dog


 


26
Human,
CACCAUCCACUACAACUACAU
AUGUAGUUGUAGUGGAUGGUG
[810-830]
[690-710]

[700-720]



cyno-



moglus,



dog


 


27
Human,
GAAUAUUUCACCCUUCAGAUC
GAUCUGAAGGGUGAAAUAUUC
[1096-1116]
[976-996]

 [986-1006]



cyno-



moglus,



dog


 


28
Human,
AGAAUAUUUCACCCUUCAGAU
AUCUGAAGGGUGAAAUAUUCU
[1095-1115]
[975-995]

 [985-1005]



cyno-



moglus,



dog


 


29
Human,
UACCACCAUCCACUACAACUA
UAGUUGUAGUGGAUGGUGGUA
[807-827]
[687-707]

[697-717]



cyno-



moglus,



dog


 


30
Human,
GAUGGAGAAUAUUUCACCCUU
AAGGGUGAAAUAUUCUCCAUC
[1090-1110]
[970-990]

 [980-1000]



cyno-



moglus,



dog


 


31
Human,
CCGAGUGGAAGGAAAUUUGCG
CGCAAAUUUCCUUCCACUCGG
[705-725]
[585-605]

[595-615]



cyno-



moglus,



dog


 


32
Human,
AACUACAUGUGUAACAGUUCC
GGAACUGUUACACAUGUAGUU
[823-843]
[703-723]

[713-733]



cyno-



moglus,



dog


 


33
Human,
CAACUACAUGUGUAACAGUUC
GAACUGUUACACAUGUAGUUG
[822-842]
[702-722]

[712-732]



cyno-



moglus,



dog


 


34
Human,
ACUACAACUACAUGUGUAACA
UGUUACACAUGUAGUUGUAGU
[818-838]
[698-718]

[708-728]



cyno-



moglus,



dog


 


35
Human,
CACUACAACUACAUGUGUAAC
GUUACACAUGUAGUUGUAGUG
[817-837]
[697-717]

[707-727]



cyno-



moglus,



dog


 


36
Human,
UCCACUACAACUACAUGUGUA
UACACAUGUAGUUGUAGUGGA
[815-835]
[695-715]

[705-725]



cyno-



moglus,



dog


 


37
Human,
CAUCCACUACAACUACAUGUG
CACAUGUAGUUGUAGUGGAUG
[813-833]
[693-713]

[703-723]



cyno-



moglus,



dog


 


38
Human,
ACCAUCCACUACAACUACAUG
CAUGUAGUUGUAGUGGAUGGU
[811-831]
[691-711]

[701-721]



cyno-



moglus,



dog


 


39
Human,
UGGAGAAUAUUUCACCCUUCA
UGAAGGGUGAAAUAUUCUCCA
[1092-1112]
[972-992]

 [982-1002]



cyno-



moglus,



dog


 


40
Human,
AUGUGUAACAGUUCCUGCAUG
CAUGCAGGAACUGUUACACAU
[829-849]
[709-729]

[719-739]



cyno-



moglus,



dog


 


41
Human,
CAUGUGUAACAGUUCCUGCAU
AUGCAGGAACUGUUACACAUG
[828-848]
[708-728]

[718-738]



cyno-



moglus,



dog


 


42
Human,
UACAACUACAUGUGUAACAGU
ACUGUUACACAUGUAGUUGUA
[820-840]
[700-720]

[710-730]



cyno-



moglus,



dog


 


43
Human,
CUACAACUACAUGUGUAACAG
CUGUUACACAUGUAGUUGUAG
[819-839]
[699-719]

[709-729]



cyno-



moglus,



dog


 


44
Human,
AUCCACUACAACUACAUGUGU
ACACAUGUAGUUGUAGUGGAU
[814-834]
[694-714]

[704-724]



cyno-



moglus,



dog


 


45
Human,
ACCACCAUCCACUACAACUAC
GUAGUUGUAGUGGAUGGUGGU
[808-828]
[688-708]

[698-718]



cyno-



moglus,



dog


 


46
Human,
AAUAUUUCACCCUUCAGAUCC
GGAUCUGAAGGGUGAAAUAUU
[1097-1117]
[977-997]

 [987-1007]



cyno-



moglus,



dog


 


47
Human,
ACUACAUGUGUAACAGUUCCU
AGGAACUGUUACACAUGUAGU
[824-844]
[704-724]

[714-734]



cyno-



moglus,



dog


 


48
Human,
AUGGAGAAUAUUUCACCCUUC
GAAGGGUGAAAUAUUCUCCAU
[1091-1111]
[971-991]

 [981-1001]



cyno-



moglus,



dog


 


49
Human,
UGUGUAACAGUUCCUGCAUGG
CCAUGCAGGAACUGUUACACA
[830-850]
[710-730]

[720-740]



cyno-



moglus,



dog


 


50
Human,
UCCGAGUGGAAGGAAAUUUGC
GCAAAUUUCCUUCCACUCGGA
[704-724]
[584-604]

[594-614]



cyno-



moglus,



dog


 


51
Human,
AUCCGAGUGGAAGGAAAUUUG
CAAAUUUCCUUCCACUCGGAU
[703-723]
[583-603]

[593-613]



cyno-



moglus,



dog


 


52
Human,
UCACACUGGAAGACUCCAGUG
CACUGGAGUCUUCCAGUGUGA
[884-904]
[764-784]
[912-932]



cyno-



moglus,



mouse


 


53
Human,
AUCACACUGGAAGACUCCAGU
ACUGGAGUCUUCCAGUGUGAU
[883-903]
[763-783]

[911-931]



cyno-



moglus,



mouse


 


54
Human,
CACACUGGAAGACUCCAGUGG
CCACUGGAGUCUUCCAGUGUG
[885-905]
[765-785]
[913-933]



cyno-



moglus,



mouse


 


55
Human,
UCAUCACACUGGAAGACUCCA
UGGAGUCUUCCAGUGUGAUGA
[881-901]

[909-929]



mouse


 


56
Human,
CCAUCAUCACACUGGAAGACU
AGUCUUCCAGUGUGAUGAUGG
[878-898]

[906-926]



mouse


 


57
Human,
CAUCACACUGGAAGACUCCAG
CUGGAGUCUUCCAGUGUGAUG
[882-902]

[910-930]



mouse


 


58
Human,
CAUCAUCACACUGGAAGACUC
GAGUCUUCCAGUGUGAUGAUG
[879-899]

[907-927]



mouse


 


59
Human,
ACCAUCAUCACACUGGAAGAC
GUCUUCCAGUGUGAUGAUGGU
[877-897]

[905-925]



mouse


 


60
Human,
UCACACUGGAAGACUCCAGUG
CACUGGAGUCUUCCAGUGUGA
[884-904]

[912-932]



mouse


 


61
Human,
AUCACACUGGAAGACUCCAGU
ACUGGAGUCUUCCAGUGUGAU
[883-903]

[911-931]



mouse


 


62
Human,
AUCAUCACACUGGAAGACUCC
GGAGUCUUCCAGUGUGAUGAU
[880-900]

[908-928]



mouse


 


63
Human,
CACACUGGAAGACUCCAGUGG
CCACUGGAGUCUUCCAGUGUG
[885-905]

[913-933]



mouse





Note that in the above Table C, the sense strands of siRNAs 1-63 have SEQ ID NOS: 191-253 respectively, and the antisense strands of siRNAs 1-63 have SEQ ID NOS: 254-316 respectively.






Example 2
Testing the siRNA Compounds for Anti-p53 Activity
Protocols
I. Preparation of the siRNAs (Double-stranded Oligonucleotides)
Lyophilized oligonucleotides were dissolved in RNAse free distilled water to produce a final concentration of 100 uM. The diluted oligonucleotides were kept at room temperature for 15 min and immediately frozen in liquid nitrogen.
The oligonucleotides were stored at −80° C. and diluted before use with PBS.
II. Transfection of siRNA in Human Cells with Lipofectamine-2000 Reagent:
2×105 p53-wt HCT116 or SW480 cells were seeded per well in 6 wells plate. 24 h subsequently, cells were transfected with p53 oligonucleotides using lipofectamine-2000 reagent (obtained from Invitrogen).
The following procedure was performed:

    
    
        1. Before transfection, the cell medium was replaced by 1500 ul fresh medium without antibiotics.
        2. In a sterile, plastic tube, Lipofectamine-2000 reagent (the amount is calculated according to 5 ul per well) was added to 250 ul serum-free medium, and incubated for 5 min at room temperature.
        3. In another tube the human anti-p53 oligonucleotides (varying amounts to fit the desired final concentration per well) were added to 250 ul serum-free medium.
        4. Lipofectamine-2000 complex was combined with the p53 oligonucleotide solution and incubated for 20 min at room temperature.
        5. The resulting mixture was added dropwise to the cells, and the cells were incubated at 37° C.
        6. SW480 cells: 48 hr after transfection the cells were harvested and proteins were extracted using RIPA buffer.
        7. HCT116 cells:
        
            40 h after transfection, 5Fu (Sigma) was added to cells to produce a final concentration of 25 ug/ml. 48 h after cells transfection (8 h after 5Fu treatment), the cells were harvested and proteins were extracted using RIPA buffer.
        
        
        8. p53 expression was determined by Western Blot analysis using monoclonal antibody (Do-1 clone, Santa Cruz). For normalization, blots were examined for Tubulin expression.

III Co-Transfection of Mouse p 53 Gene and Mouse p53 Oligonucleotides into PC3 Cells Using Lipofectamine-2000 Reagent:

    
    


2×105 p53-null PC3 cells were seeded per well in 6 wells plate. 24 h subsequently, cells were Co-transfected with mouse p53 gene and GFP gene and mouse p53 oligonucleotides using lipofectamine-2000 reagent (Invitrogen). The following procedure was performed:

    
    
        1. Before transfection cell medium was replaced by 1500 ul fresh medium without antibiotics.
        2. In sterile, plastic tube, Lipofectamine-2000 reagent (5 ul per well) was added to 250 ul serum-free medium, and incubated for 5 min at room temperature.
        3. In another tube 4 ug DNA (p53gene:GFPgene, 10:1) and human p53 oligonucleotides were added to 250 ul serum free medium.
        4. Lipofectamine-2000 complex was combined with p53 oligonucleotides solution and incubated for 20 min at room temperature.
        5. The mixture solution was added dropwise to the cells, and cells were incubated at 37° C.
        6. 48 h after transfection, cells were harvested and proteins were extracted using RIPA buffer.
        7. p53 expression was determined by Western Blot analysis using monoclonal antibody (Clone240, Chemicon). For normalization, blots were examined for GFP expression.
    
    


Results:
A. Human p53 Oligonucleotides:









TABLE D









Results of Test












Number
oligo
species
source
SW480
HCT116















2
Hu2′
human
literature
(−)
(+)


3
QHMon1
human, monkey
Proprietary
(++)
(+++)


4
QHMon2
human, monkey
Proprietary
(−)
Not tested


5
QH1
human
Proprietary
(+++)
(+++)


6
QH2
human
Proprietary
(−)
Not tested


13
A17
human, mouse
Proprietary
(−)
Not tested


14
E2
human, mouse, rat
Proprietary
(+)
Not tested


15
E6
human, mouse, rat
Proprietary
(−)
Not tested


16
B1
human, mouse, rat
Proprietary
(−)
Not tested


17
B2
human, mouse, rat
Proprietary
(−)
Not tested


18
C1
human, monkey, mouse
Proprietary
(−)
Not tested


19
F2
human, monkey, dog
Proprietary
(−)
Not tested


20
F3
human, monkey, dog
Proprietary
(+++)
(+++)


21
G1
human, monkey, dog
Proprietary
(+++)
Not tested


22
H2
human, monkey, dog
Proprietary
(+)
Not tested


23
I5
human, monkey, dog
Proprietary
(+++)
Not tested





Note:


The numbers in Table D correspond to the numbers used in Table A, where the sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25 respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOS: 26-48 respectively.






As shown in Table D, four human oligonucleotides were tested in two systems SW480 and HT116, according to Protocols II above. Representative results (Western Blot) on which the Results of Test was based are shown in FIG. 3.












TABLE E










Results of Test






PC3 null






cells/exogenous



oligo
species
source
mouse p53




















1
Mo3
mouse
literature
(+++)


7
QM1
mouse
Proprietary
(−)


8
QM2
mouse
Proprietary
(−)


9
QM3
mouse
Proprietary
(−)


10
QM6
mouse
Proprietary
(−)


11
QM4
mouse, rat
Proprietary
(+++)


12
QM5
mouse, rat
Proprietary
(+++)


13
A17
human, mouse
Proprietary
(−)


14
E2
human, mouse, rat
Proprietary
(++)


15
E6
human, mouse, rat
Proprietary
(−)


16
B1
human, monkey, mouse
Proprietary
(−)


17
B2
human, monkey, mouse
Proprietary
(++)


18
C1
human, monkey, mouse
Proprietary
(++)


19
G1
human, monkey, dog
Proprietary
(++)


20
F3
human, monkey, dog
Proprietary
(+++)


21
I5
human, monkey, dog
Proprietary
(−)


22
QHMon1
human, monkey
Proprietary
(++)





Note:


The numbers in Table E (as for Table D) correspond to the numbers used in Table A, where the sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25 respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOS: 26-48 respectively.






Example 3
Distribution of Cy3-PTEN siRNA in the Cochlea Following Local Application to the Round Window of the Ear
A solution of 1 μg/100 μl of Cy3-PTEN siRNA (total of 0.3-0.4 μg) 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 persistance to up to 15 days after the application of the Cy3-PTEN siRNA. These results indicate for the first time that local application of siRNA molecules within the round window leads to significant penetration of the siRNA molecules to the basal, middle and apical turns of the cochlea.
Example 4
The Effect of p53 siRNA Treatment on Carboplatin-induced Hair Cell Death in the Cochlea of Chinchilla
Eight Chinchillas were pre-treated by direct administration of p53 siRNA in saline (QM5 molecule in Table A, 1, 10 and 30 μg) to the left ear of each animal. Saline was given to the right ear of each animal as placebo. Two days following the administration of the p53 siRNA, the animals were treated with carboplatin (75 mg/kg ip). After sacrifice of the chinchillas (two weeks post carboplatin treatment) the % of dead cells of inner hair cells (IHC) and outer hair cells (OHC) was calculated in the left ear (siRNA treated) and in the right ear (saline treated). Since the effect of the siRNA was similar across dose, the data was pooled from the 3 doses. As demonstrated in Table F-1 below, carboplatin preferentially damages the inner hair cells in the chinchilla at the 75 mg/kg dose while the outer hair cells remain intact. Furthermore, the p53 siRNA significantly reduces carboplatin-induced inner hair cells loss in the cochlea (53.5% of inner hair cell loss in the p53 siRNA treated cochlea versus 71.9% of inner hair cell loss in the PBS treated cochlea).







TABLE F-1







QM5 siRNA Significantly Reduces Carboplatin-Induced


IHC Loss in Chinchilla











SIRNA TREATED

CONTROL EAR












Chinchilla #
IHC
OHC
Chin
IHC
OHC















8136L
64.7
0.6
8136R
68.8
1.1


8140L
48.2
0.8
8140R
87.6
1.8


8143L
53.3
1.5
8143R
64.8
2.4


8149L
38.3
1.9
8149r
68.5
3


8153L
59.7
3.1
8153R
58.2
2.1


8197L
50.1
1.2
8197R
61.2
1.5


8200L
45.4
1.7
8200R
82.5
1.5


8202L
68.5
3.0
8202R
83.5
2.6


Mean Treated
53.5
1.7
Mean Control
71.9
2.0









Example 5
The Effect of p53 siRNA Treatment on Acoustic-induced Hair Cell Death in the Cochlea of Chinchilla
The activity of p53 siRNA (QM5) in an acoustic trauma model was studied in chinchilla. A group of 7 animals underwent the acoustic trauma. The animals were 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 was pre-treated (48 h before the acoustic trauma) with 30 μg of siRNA in  ̃10 μL of saline; the right ear was 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 was assessed 2.5 weeks after the acoustic trauma. Specifically, the mean threshold of the compound action potential recorded from the round window was determined 2.5 weeks after the acoustic trauma in order to determine if the thresholds in the siRNA-treated ear were lower (better) than the untreated (saline) ear. In addition, the amount of inner and outer hair cell loss was determined in the siRNA-treated and the control ear. FIG. 5 shows the mean threshold results recorded from the round window of siRNA-treated (filled circle) and saline-treated (open circle) chinchillas 2.5 weeks after the acoustic trauma. As demonstrated in FIG. 5, the mean thresholds were lower in the siRNA-treated ears versus the untreated ears. The difference at 4 kHz was statistically significant (p<0.033). These results indicate that p53 siRNA administered to the round window of the cochlea is capable of reducing the caused by acoustic trauma.
Table F-2 below shows the loss of outer hair cells (OHC) and inner hair cells (IHC) for each animal in the Basal Half of the cochlea (50-100% from the apex). The mean OHC loss in the siRNA-treated ears was significantly less than in the control ears (13.9% OHC loss in the siRNA-treated ear versus 19.6% OHC loss in the control ear, as determined by paired t-test). In general, there was less IHC loss that OHC loss in both siRNA-treated and control ears. The mean IHC loss was 4.5% in control ears and 1.3% in the siRNA-treated ears. This difference was not significant statistically. These results indicate that p53 siRNA administered to the round window of the cochlea is capable of reducing OHC loss in the Basal Half of the cochlea caused by acoustic trauma.







TABLE F-2







QM5 siRNA Significantly Reduces Acoustic-Induced OHC


Loss in the basal half of cochlea in Chinchilla












SiRNA-treated
Control




(left ear
(right ear)














Chin#
IHC
OHC
IHC
OHC



















8146
0.0%
0.1%
6.9%
9.7%




8196
0.7%
3.4%
2.5%
13.4%



8220
6.7%
79.7% 
19.8% 
92.4%



8222
0.0%
2.4%
0.1%
5.0%



8237
1.4%
2.3%
1.8%
7.5%



8238
0.1%
3.6%
0.0%
1.7%



8246
0.3%
6.0%
0.6%
7.4%



Mean
1.3%
13.9% 
4.5%
19.6%



SD
2.4%
29.1% 
7.1%
32.3%










Example 6
The Effect of p53 or 801 siRNA Treatment on Cisplatin-induced Hair Cell Death in the Cochlea of Rats
Male Wistar Rats were tested for basal auditory brainstem response (ABR) thresholds for signals of clicks, 8, 16 and 32 kHz prior to cisplatin treatment. Following the basal auditory brainstem response testing, cisplatin was administered as an intraperitoneal infusion of 13 mg/kg over 30 minutes. Treated ears received either 15 ug/4 microliters of p53 siRNA (QM5 molecule in Table A) in PBS or 801 siRNA in PBS (applied directly to the round window membrane). The 801 siRNA is designated REDD14 and has the following nucleotide sequence in the sense strand: 5′-GUGCCAACCUGAUGCAGCU-3′ (SEQ ID NO: 317) and in the antisense strand: 5′-AGCUGCAUCAGGUUGGCAC-3′ (SEQ ID NO: 318). Control ears were treated with either non-related GFP siRNA or PBS. The siRNA molecules were administered between 3-5 days prior to cisplatin administration in order to permit protective effect on the cochlea.
The auditory brainstem response (ABR) testing was repeated 3 days after cisplatin administration. The auditory brainstem response thresholds were compared between pretreatment and posttreatment and the shift in thresholds is indicated in Table G. Higher shift in thresholds following cisplatin treatment is indicative for more severe hair cells loss in the cochlea. After the repeat of auditory brainstem response testing, animals were sacrificed and cochleae were removed and processed for scanning electron microscopy (SEM) to quantify outer hair cell (OHC) loss in the hook region (high frequency region). The % outer hair cell loss was calculated by dividing the number of missing or severely damaged cells by the total number of outer hair cells in the field of the photograph.
Table G demonstrates the results obtained from four animals that underwent the cisplatin-induced damage and were analysed for outer hair cell loss in the Hook region. As revealed from the results, animals that received the siRNA directed against p53 or 801 exhibited lower outer hair cell loss and smaller shifts in the threshold for signals of 32 kHz. Both parameters indicate that siRNA directed against the p53 or 801 genes (mRNA) is protective against cisplatin-induced damage in the cochlea.







TABLE G







Hair cell loss versus threshold shift


in cisplatin-treated cochlea of rats










Outer hair cell
Auditory brainstem response


Treatment
(OHC) loss
(Threshold shift at 32 KHz)













QC/L P53 siRNA (QM5)
50%
10
dB


QC/R PBS
100% 
30
dB


QF/L P53 siRNA (QM5)
20%
10
dB


QF/R GFP
56%
27.5
dB


QJ/R 801 siRNA
20%
17.5
dB


(REDD14)


QJ/L GFP
100% 
27.5
dB


QN/L 801 siRNA
 0%
10
dB


(REDD14)


QN/R PBS
100% 
17.5
dB









Example 7
Generation of Sequences for Active siRNA Compounds to Pro-Apoptotic Genes
Using proprietary algorithms and the known sequence of the pro-apoptotic genes, the sequences of many potential siRNAs were generated.

## Claims

1. A method for reducing ototoxin-induced hearing loss in an ear of a subject to be treated with an ototoxin which comprises topically applying to the canal of the subject's ear a liquid composition comprising a siRNA oligoribonucleotide in an amount effective to inhibit expression of a p53 gene in the cochlea of the subject's ear to reduce ototoxin-induced hearing loss in the subject's ear, wherein the siRNA comprises (i) a sense strand and (ii) an antisense strand that comprises the sequence set forth in SEQ ID NO: 48, wherein each strand is 19-23nucleotides in length, and wherein the siRNA may be modified in one or more sugar residues, one or more linkages between nucleotides, and/or one or more bases.

2. The method of claim 1, wherein the siRNA oligoribonucleotide comprises at least one nucleotide in which a sugar residue is modified.

3. The method of claim 2, wherein the modified sugar residue comprises a 2′-O-methyl modified sugar residue.

4. The method of claim 1, wherein alternating ribonucleotides in both the antisense strand and the sense strand of the siRNA oligoribonucleotide comprise 2′-O-methyl modified sugar residues.

5. The method of claim 1, wherein the ototoxin is a chemotherapeutic agent selected from the group consisting of cisplatin, an aminoglycoside antibiotic, a salicylate, or a quinine.

6. A method for reducing ototoxin-induced hearing loss in an ear of a subject to be treated with an ototoxin which comprises topically applying to the canal of the subject's ear a liquid composition comprising a polynucleotide inhibitor in an amount effective to inhibit expression of a p53 gene in the cochlea of the subject's ear to reduce ototoxin-induced hearing loss in the subject's ear, wherein the polynucleotide inhibitor is selected from the group consisting of an siRNA, a vector comprising an siRNA, a vector which expresses an siRNA and a molecule which is endogenously processed into an siRNA oligoribonucleotide, and the siRNA comprises (i) a sense strand and (ii) an antisense strand that comprises the sequence set forth in SEQ ID NO: 48, wherein each strand is 19-23 nucleotides in length, and wherein the siRNA oligoribonucleotide may be modified or unmodified in one or more sugar residues, one or more linkages between nucleotides, and/or one or more bases.

7. The method of claim 6, wherein the siRNA oligoribonucleotide comprises at least one nucleotide in which a sugar residue is modified.

8. The method of claim 7, wherein the modified sugar residue comprises a 2′-O-methyl modified sugar residue.

9. The method of claim 6, wherein alternating ribonucleotides in both the antisense strand and the sense strand of the siRNA oligoribonucleotide comprise 2′-O-methyl modified sugar residues.

10. The method of claim 6, wherein the ototoxin is a chemotherapeutic agent selected from the group consisting of cisplatin, an aminoglycoside antibiotic, a salicylate, or a quinine.

11. A method for reducing cisplatin-induced hearing loss in an ear of a subject to be treated with cisplatin comprising topically applying to the canal of the subject's ear a liquid composition comprising an siRNA oligoribonucleotide in an amount effective to inhibit expression of a p53 gene in the cochlea of the subject's ear to reduce cisplatin-induced hearing loss in the subject's ear, wherein the siRNA oligoribonucleotide has the structure:
5′ ugaagggugaaauauucuc 3′ (antisense strand) (SEQ ID NO: 48)
3′ acuucccacuuuauaagag 5′ (sense strand) (SEQ ID NO: 25)
wherein each strand is 19-23 nucleotides in length, wherein each of a, c, u, and g is a ribonucleotide and each consecutive ribonucleotide is joined to the next ribonucleotide by a covalent bond; and wherein alternating ribonucleotides in both the antisense and sense strands are modified in their sugar residues such that a 2′-O-methyl group is present and modified sugar residues are present at the 5′ and 3′ termini of the antisense stand and unmodified sugar residues are present at the 5′ and 3′ termini of the sense strand.

12. The compound of claim 11, wherein the covalent bond is a phosphodiester bond.

13. The compound of claim 11, wherein the antisense and the sense strands are non-phosphorylated at the 3′ and 5′termini.

14. The compound of claim 11 wherein the antisense and the sense stands are phosphorylated at the 3′ termini.