WO2005002498A2

WO2005002498A2 - Methods of treating disorders caused by formation of transcripts carrying nonsense mutations

Info

Publication number: WO2005002498A2
Application number: PCT/IL2004/000556
Authority: WO
Inventors: Batsheva Kerem; Liat Shushi
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 2003-07-01
Filing date: 2004-06-22
Publication date: 2005-01-13
Also published as: WO2005002498A3

Abstract

A method of treating an individual having a disorder caused by formation of an RNA transcript carrying a nonsense mutation is provided. The method is effected by exposing cells of the individual expressing the RNA transcript to treatment selected capable of increasing a half life of the RNA transcript in the cells; and exposing the cells to an aminoglycoside thereby treating the individual having the genetic disease caused by formation of the RNA transcript carrying the nonsense mutation.

Description

METHODS OF TREATING DISORDERS CAUSED BY FORMATION OF TRANSCRIPTS CARRYING NONSENSE MUTATIONS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to methods of treating disorders caused by formation of transcripts carrying nonsense mutations. More particularly, the methodology of the present invention utilizes a novel approach which combines treatment for increasing the half life of transcripts carrying nonsense mutations with treatment which enables read-through translation of the nonsense mutation of such transcripts to thereby restore normal f nction to nonsense alleles. An estimated one-third of mutations underlying human disorders result in premature termination of translation (nonsense/stop mutations) [1]. Nonsense mutations were originally thought to truncate the encoded protein thus leading to dysfunction of the protein. However, it is now apparent that nonsense mutations have additional effects on transcripts carrying nonsense mutations. They can dramatically decrease the half-lives of mutant mRNAs by the nonsense-mediated decay (NMD) pathway as well as alter the pattern of pre-mRNA splicing by the nonsense-associated altered splicing (NAS) pathway. These pathways exhibit widespread coupling in humans [3]. Nonsense-mediated decay NMD causes degradation of nonsense transcripts, leading to low levels or absence of the protein product [1, 4, 5]. NMD is the quality control system by which mRNAs containing premature nonsense codons are selectively eliminated by eukaryotic cells. It is thought that by removing these defective mRNAs, NMD protects cells from potential damage due to inappropriately truncated proteins. The NMD pathway enables the cell to distinguish between normal and premature nonsense codons. In mammalian cells, the authentic stop codons usually are within the last exon of a gene. Hence, nonsense codons lying upstream of an intron are generally recognized as premature [6]. It was found that translation termination process is a key event in triggering NMD (reviewed in reference 7). The protein complex involved in NMD has been identified in a variety of organisms; in human, this complex is composed of four proteins, hUPFl, hUPF2, hUPF3A and hUPF3B (reviewed in references 8 and 9). While NMD decreases the level of the normally spliced transcripts, NAS increases the level of alternatively spliced transcripts, by using alternative splice acceptor and donor sites, leading to skipping on the nonsense mutation region [10, 11], NAS pathway, unlike NMD, can be independent of translation. One suggested mechanism involves disruption of RNA sequences called exonic splicing enhancers

(ESEs). ESEs are target sites for proteins that help to define pre-mRNA splice sites.

Their disruption by almost any type of mutation can cause altered splicing. Therefore, the promotion of alternate splicing by most nonsense mutations is simply due to the destruction of a key recognition element for the splicing machinery. Another suggested mechanism for NAS requires recognition of the nonsense mutations as stop signals during protein synthesis [12, 13]. Recently it has been shown that NAS requires hUPFl, the main factor in the NMD pathway, and is hUPF2-independent [14]. The other NMD proteins have not yet been studied. One important research field in human genetics is the development of mutation specific therapies. A major effort is devoted to the development of a therapy for patients carrying nonsense mutations, causing human genetic diseases. One model disease is Cystic fibrosis (CF), which is a lethal, autosomal recessive disease common among Caucasians. The disease is caused by defects in the cystic fibrosis tra smembrane conductance regulator (CFTR) gene, which encodes a chloride channel regulated by cAMP [15]. The disease is characterized by progressive lung disease, pancreatic dysfunction, elevated sweat electrolytes and male infertility. There are >1000 mutations causing CF. The major mutation, responsible for 70% of the mutated alleles, is a three base pair deletion (DF508). The nonsense mutations in CF account for approximately 5% of the total alleles. In Israel, and among Jews worldwide, 64% of the patients, carry at least one nonsense mutation in the CFTR gene [16-18]. Since the identification of CFTR, therapies for CF are a major research goal, among which are therapies for specific classes of mutations. A specific therapy for nonsense mutations was recently developed aiming to suppress the nonsense codon, allowing synthesis of a full-length protein. It has been known for many years that aminoglycoside antibiotics, in addition to their antimicrobial activity, can increase the frequency of erroneous insertion of the nonsense codon, thereby permitting protein translation to continue to the normal end of the transcript. This has been demonstrated in prokaryotic and eukaryotic cells [19-22]. Most aminoglycosides are highly active against bacterial ribosomes, but do not affect the cytoplasmic ribosomes in human cells. The sensitivity of eukaryotic ribosomes to some aminoglycosides, such as gentamicin, G-418, and a few others, has been viewed as unwanted side effect associated with these antibacterial drugs. But it is precisely that 'side' effect that opened the possibility of using these drugs for the treatment of human genetic diseases. Howard et al. demonstrated that two

CFTR-associated stop mutations could be suppressed by treating cells with low doses of an aminoglycoside antibiotic [23]. Bedwell et al. demonstrated in the bronchial epithelium cell line IB3-1 carrying the nonsense mutation W1282X, that incubation with aminoglycosides, results in expression of functional CFTR at the apical membrane

[24]. Analysis of a transgenic mouse, expressing a human CFTR-G542X cDNA, showed that daily administration of gentamicin restored the expression of CFTR protein in the intestinal tissues [25]. A similar activation of the CFTR chaimel was achieved by gentamicin in human airway cells from a CF patient carrying G542X nonsense mutation [26] and in liver cells from a CF patient carrying the same nonsense mutation [27]. Thus, in vitro, gentamicin can suppress nonsense mutations in the CFTR gene, leading to a full-length protein. Subsequently, aminoglycoside treatment has been applied to a number of other genetic diseases, caused by nonsense mutations: Duchenne muscular dystrophy (DMD) [28], Hurler syndrome [29] cystinosis [30], late infantile neuronal ceroid lipofuscinosis [31] and nonsense mutations in the p53 gene [32], These studies showed suppression of the nonsense mutations by gentamicin, while studies in human cells from X linked retinitis pigmentosa patients carrying a nonsense mutation, revealed no read-through of the protein synthesis [33]. Aminoglycoside therapy was successfully applied to a mouse model for DMD, carrying a nonsense mutation in the dystrophin [28]. In these mice, gentamicin suppressed the nonsense mutation and lead to normal levels of full-length protein and restoration of the dystrophin function. Obviously, the major goal is to develop the aminoglycoside therapy to patients suffering from many inherited diseases. This approach has already been initiated in DMD, Becker muscular dystrophy (BMD) and in CF. Following intravenous gentamicin treatment, two DMD and two BMD patients, carrying different nonsense mutations in the dystrophin gene, did not demonstrate full- length dystrophin protein [34]. The reason for the lack of response is unknown. In a study including five CF patients each carrying a different CFTR nonsense mutation, restoration of the CFTR function was demonstrated in 4/5 patients [26]. Another CF study investigated the potential of gentamicin in treating a group of patients carrying the same nonsense mutation [2]. The patients carried the W1282X mutation, which is the most frequent nonsense mutation among Jewish Ashkenazi CF patients [16]. Following nasal topical application of gentamicin, 7 out of the 9 patients treated demonstrated correction of chloride transport in their nasal epithelial cells. Subsequently, a randomized, double blind, placebo-controlled, crossover trial was performed by applying gentamicin to nasal mucosal cells of 24 CF patients. A significant correction of baseline and chloride transport was observed in these patients. However, the gentamicin effect varied among the patients. While most of the patients responded to treatment, three did not show any correction of the baseline chloride transport (Wilschanski et al., 2003 NEJM, in press). Thus, although gentamicin treatment can potentially restore the function of defective proteins encoded by nonsense alleles, it is clear that some patients do not respond to aminoglycoside treatment. While reducing the present invention to practice, the present inventors have uncovered that the level of a nonsense transcript in an individual directly correlates with the response of the individual to gentamicin treatment indicating that the NMD mechanism might play an important role in modulating the response to aminoglycoside therapy of individuals carrying nonsense mutations responsible for various genetic disorders.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a method of treating an individual having a disorder caused by formation of an RNA transcript carrying a nonsense mutation, the method comprising (a) exposing cells of the individual expressing the RNA transcript to treatment selected capable of increasing a half life of the RNA transcript in the cells; and (b) exposing the cells to an aminoglycoside thereby treating the individual having the genetic disease caused by formation of the RNA transcript carrying a nonsense mutation. According to further features in preferred embodiments of the invention described below, the aminoglycoside is selected from the group consisting of kanamycin, neomycin, seldomycin, tobramycin, kasugamycin, fortimicin, gentamycin, paromomycin, neamine, sisomicin, amikacin and netilmicin. According to still further features in the described preferred embodiments step

(a) is effected by downregulating an activity of a nonsense-mediated decay (NMD) pathway in the cells. According to still further features in the described preferred embodiments the downregulating is effected by administering to the cells an agent capable of downregulating an activity or expression of at least one component of the nonsense- mediated decay pathway. According to still further features in the described preferred embodiments the agent is selected from the group consisting of: (i) a molecule which binds the at least one component of the nonsense-mediated decay pathway; (ii) an enzyme which cleaves the at least one component of the nonsense-mediated decay pathway; (hi) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the at least one component of the nonsense-mediated decay pathway; (iv) a ribozyme which specifically cleaves transcripts of the at least one component of the nonsense- mediated decay pathway; (v) a non-functional analogue of at least a catalytic or binding portion of the at least one component of the nonsense-mediated decay pathway; (vi) an siRNA molecule capable of inducing degradation of transcripts of the at least one component of the nonsense-mediated decay pathway; and (vii) a DNAzyme which specifically cleaves transcripts or DNA of the at least one component of the nonsense-mediated decay pathway. According to still further features in the described preferred embodiments the at least one component of the nonsense-mediated decay pathway is hUPFl, hUPF2, hUPF3A or hUPF3B. According to still further features in the described preferred embodiments steps (a) and (b) are effected concomitantly. According to still further features in the described preferred embodiments the agent and the aminoglycoside form a part of a pharmaceutical composition. According to still further features in the described preferred embodiments step (a) is effected prior to or following step (b). According to still further features in the described preferred embodiments the disorder is selected from the group consisting of Cystic Fibrosis (CF), Duchenne muscular dystrophy (DMD), Hurler syndrome, cystinosis, and late infantile neuronal ceroid lipofuscinosis. According to another aspect of the present invention there is provided a pharmaceutical composition for treatment of a disorder caused by formation of an RNA transcript carrying a nonsense mutation, the pharmaceutical composition comprising an aminoglycoside and an agent capable of increasing a half life of the RNA transcript in eukaryotic cells. According to still further features in the described preferred embodiments the aminoglycoside is selected from the group consisting of kanamycin, neomycin, seldomycin, tobramycin, kasugamycin, fortimicin, gentamycin, paromomycin, ήeamine, sisomicin, amikacin and netilmicin. According to still further features in the described preferred embodiments the agent downregulates an activity of a nonsense-mediated decay (NMD) pathway in the eukaryotic cells. ' According to still further features in the described preferred embodiments the agent is downregulates an activity or expression of at least one component of the nonsense-mediated decay pathway. According to still further features in the described preferred embodiments the agent is selected from the group consisting of: (i) a molecule which binds the at least one component of the nonsense-mediated decay pathway; (ii) an enzyme which cleaves the at least one component of the nonsense-mediated decay pathway; (iii) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the at least one component of the nonsense-mediated decay pathway; (iv) a ribozyme which specifically cleaves transcripts of the at least one component of the nonsense- mediated decay pathway; (v) a non-functional analogue of at least a catalytic or binding portion of the at least one component of the nonsense-mediated decay pathway; (vi) an siRNA molecule capable of inducing degradation of transcripts of the at least one component of the nonsense-mediated decay pathway; and (vii) a DNAzyme which specifically cleaves transcripts or DNA of the at least one component of the nonsense-mediated decay pathway. According to still further features in the described preferred embodiments the at least one component of the nonsense-mediated decay pathway is hUPFl, hUPF2, hUPF3A or hUPF3B. According to still further features in the described preferred embodiments the disorder is selected from the group consisting of Cystic Fibrosis (CF), Duchenne muscular dystrophy (DMD), Hurler syndrome, cystinosis, and late infantile neuronal ceroid lipofuscinosis. The present invention successfully addresses the shortcomings of the presently known configurations by providing a novel approach for treating disorders caused by formation of transcripts carrying nonsense mutations. Unlike prior art approaches, the efficacy of the present approach is not effected by variations in the transcript levels of the mutated allele, and as such it can be used to efficiently treat any disorder caused by formation of transcripts carrying nonsense mutations regardless of the level of the mutated transcript maintained in the cell. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIGs. la-b illustrate RNA levels obtained from nasal epithelial scraped cells of

CF patients carrying the W1282X nonsense mutation. Figure la - Genescan analysis of

RNA from patient 2, an heterozygous for the W1282X/DF508 mutations. The level of the W1282X transcripts in this patient is relatively high, 65% of the DF508 level. Figure lb - Real-time PCR of RNA from patients 8 and 9 and from a non-CF control individual that does not carry CFTR mutations. The level of the W1282X transcript in patients 9 is 20% of the control individual, while that of patient 8, is ~60% of the control. Patients 8 and 9 are sisters, whose parents are first cousins. FIG. 2 illustrates the relative levels of W1282X transcripts in the 10 studied CF patients carrying at least one W1282X allele. Patients 1-8 show relatively high levels of the W1282X transcripts, while patients 9 and 10 show markedly reduced levels. Patients 6, 8 and 9 were homozygous for the W1282X; patient 7 was heterozygous W1282X/G542X and patient 10 was heterozygous W1282X/3849+10kb C->T. FIG. 3 illustrates the change in PD following perfusion of chloride free and isoproterenol solution before (Pre) and after treatment of gentamicin (G) or placebo (P). CF patient 2 (circle) shows a significant correction of the chloride transport following gentamicin treatment, while in patient 10 (rhombus) no correction was observed (left panel). In addition, patient 2 exhibits hyperpolarization due to chloride transport following gentamicin treatment, while no response was observed with patient 10 (right panel). FIG. 4 is an EtBr stained agarose gel showing the RT-PCR products of patients 8 and 9. RT-PCR was performed using primers flanking the W1282X mutation region. Note, that the only visible product is that expected from normal splicing of exon 20. FIG. 5 illustrates the relative levels of cystic fibrosis transmembrane regulator

(CFTR) gene transcripts in CFP15a cells (blue) and CFP15b cells (yellow). FIG. 6 illustrates the effect of gentamicin (G) on the chloride efflux in CFP15a and CFP15b cells. FIG 7 illustrates the effect of cyclohexamide (CHX) on the level of CFTR transcripts in CFP15a cells. FIG. 8 illustrates the effect of siRNA directed against hUPF2 on the level of CFTR transcripts in CFP15a cells. Blue line represents untreated CFP15a cells; purple line represents siRNA transfected CFP15a cells. FIG. 9 illustrates the effect of gentamicin and of gentamicin combined with siRNA directed against hUPF2, on chloride efflux in cultured CFP15a cells. The combined treatment of gentamicin and siRNA was substantially more effective than gentamicin alone, thus indicating that chloride efflux in CF cells can be restored via downregulation of the nonsense mediated decay (NMD) pathway. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of methods which can be used to treat disorders such as cancer which are caused by transcripts carrying a nonsense mutation. Specifically, the present invention can be used to treat genetic diseases such as Cystic Fibrosis (CF), Duchenne muscular dystrophy (DMD), Hurler syndrome, cystinosis, late infantile neuronal ceroid lipofuscinosis and disorders characterized by nonsense mutations in the p53 gene. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Prior art studies have shown that aminoglycoside treatment can be used to treat disorders caused by transcripts carrying nonsense mutations. While most patients participating in such studies responded to the treatment some did not show any correction of the deleterious effects caused by such nonsense mutations. Thus, although aminoglycoside treatment can potentially restore the function of defective proteins encoded by nonsense alleles, it is clear that in some cases such treatment does not have a therapeutic effect. While reducing the present invention to practice, the present inventors have uncovered that the level of a nonsense transcript in an individual directly correlates with the response of the individual to gentamicin treatment indicating that the NMD mechanism plays an important role in modulating the response to aminoglycoside therapy of individuals carrying nonsense alleles implicated in various genetic diseases. In addition, the present inventors uncovered that the downregulation of NMD pathway can result in restoring function of defective proteins such as CFTR and can substantially improve the efficiency of gentamicin treatment. Thus, according to one aspect of the present invention there is provided a method of treating an individual having a disorder caused by formation of an RNA fr-inscript carrying a nonsense mutation. Examples of disorders which are treatable using the present approach include, but are not limited to Cystic Fibrosis (CF) which is caused by mutation in the CFTR gene, Duchenne muscular dystrophy (DMD) which is caused by mutation in the dystrophin gene, Hurler syndrome which is caused by mutation in the gene encoding alpha-L-iduronidase, cystinosis which is caused by mutation in the CTNS gene, late infantile neuronal ceroid lipofuscinosis which is caused by mutation in the CLN6 gene and disorders characterized by nonsense mutations in the p53 gene. The method is effected by exposing cells of the individual expressing the RNA transcript carrying the nonsense mutation to: (a) treatment selected capable of increasing a half life of the RNA transcript in the cells; and to (b) an aminoglycoside such as for example, kanamycin, neomycin, seldomycin, tobramycin, kasugamycin, fortimicin, gentamicin, paromomycin, neamine, sisomicin, amikacin, netilmicin and the like which can suppress nonsense mutations in mammalian cells in vivo. Steps (a) and (b) are preferably effected concomitantly, although it will be appreciated that these step can be carried sequentially in any order. Treating cells of the individual with an aminoglycoside can be effected using various approaches. Detailed guidelines for effecting such treatment including suitable formulation and doses are provided in the prior art (see, for example, references 2 and

26 of the references section hereinbelow). Additional information regarding formulations and administration modes suitable for use with the present invention are provided hereinbelow. As is mentioned hereinabove, aminoglycoside treatment is effected along with administration of an agent selected capable of increasing the half life of mRNA transcripts carrying the nonsense mutation in the cell. The latter is preferably effected by downregulating the expression and/or activity of at least one protein component of the nonsense-mediated decay (NMDD) complex/pathway. Such components include hUPFl (also known as hRENT or

KIAA0221) (GenBank Accession Number NM_002911, GeneCard GC19P019437 at http://www.rzpd.de/cards/index.html), hUPF2, hUPF3A and hUPF3B (see the Background section hereinabove for further detail). Several approaches for at least partially downregulating activity or expression of such proteins are envisaged by the present invention. According to one preferred embodiment of this aspect of the present invention, partial or complete inhibition of protein expression/activity can be accomplished by introducing into the subject an agent which is capable of partially or completely inhibiting protein activity or an agent which is capable of partially or completely inhibiting protein expression (i.e., transcription of the gene encoding this protein or translation of the mRNA transcript thereof). Preferably, introducing is effected via systemic administration of the agent to a subject. Systemic administration may be effected by, for example, injection (e.g. intravenous, intramuscular, peritoneal or subcutaneous), oral administration, iettr-iocular adminisfration, inttanasal administration, transdermal delivery, intravaginal administration or rectal administration. Further description of suitable routes of administration is provided herein below. Agents suitable for inhibiting protein activity in the cell include, but are not limited to, molecules which specifically bind any of the above described components of the NMD (e.g. antibody or preferably an antibody fragment - Fab, ScFv etc), enzymes which cleave NMD components, non-functional nucleotide analogues which are capable of blocking binding of NMD components to RNA or substrate analogues which are capable of competing for the RNA substrate binding or substrate catalytic region. The generation of such agents is well within the capabilities of the ordinary skilled artisan and as such no further description with respect to approaches which can be used to generate such agents is provided herein. Complete or partial inhibition of expression of NMD components can be achieved using antisense oligonucleotides designed to specifically block expression of, for example, the gene encoding hUPFl or a transcript thereof. Design of antisense molecules which can be used to efficiently inhibit expression of NMD components must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof. The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types (see, for example, Luft (1998) J Mol Med 76(2): 75-6; Kronenwett et al. (1998) Blood 91(3): 852-62; Rajur et al. (1997) Biocόnjug Chem 8(6): 935-40; Lavigne et al. (1997) Biochem

Biophys Res Commun 237(3): 566-71 and Aoki et al. (1997) Biochem Biophys Res

Commun 231(3): 540-5). In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the. energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. (1999) Biotechnol Bioeng 65(1): 1-9]. Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligomicleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gpl30) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries. In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al. (1998) Nature Biotechnology 16, 1374 - 1375). Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used (Holmund et al. (1999) Curr Opin Mol Ther l(3):372-85), while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz (1999) Curr Opin Mol Ther l(3):297-306]. More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al. (2001) Cancer Res 61(21):7855-60]. Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation. Antisense polynucleotide which can be used to specifically inhibit expression of

NMD components (e.g. hUPFl) in cells can be readily designed and synthesized using the above provided information and guidelines. Preferably, the oligonucleotides are designed to be specific to a region at, or immediately downstream of, the initiating ATG translation codon of the NMD component gene. This region of the ttanscript has been selected as being the most efficient place for interfering with translation according to antisense algorithms described hereinabove. Antisense sequences can also include a ribozyme sequence fused thereto. Such a ribozyme sequence can be readily synthesized using solid phase oligonucleotide synthesis. RNA interference (RNAi) is yet another approach which can be utilized by the present invention to specifically inhibit expression of one or more NMD components. RNA interference is a two step process, the first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase in family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232 and Bernstein (2001) Nature 409:363-366]. In the effector step, the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232, Hammond et al. (2001) Nat. Rev. Gen. 2:110-119, Sharp (2001) Genes. Dev. 15:485-90]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore (2002) Curr. Opin.

Genetics and Development 12:225-232]. Because of the remarkable potency of RNAi, an amplification step within the

RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed.

Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. (2001) Nat. Rev. Gen. 2:110-119, Sharp (2001)

Genes. Dev. 15:485-90, Hutvagner and Zamore (2002) Curr. Opin. Genetics and

Development 12:225-232]. For more information on RNAi see the following reviews Tuschl (2001) ChemBiochem. 2:239-245, CuHen (2002) Nat. Immunol. 3:597-599 and

Brantl (2002) Biochem. Biophys. Act. 1575:15-25. Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the NMD component mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR- binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techhb/t_t_791/912.html). Second, potential target sites are compared to an appropriate genomic database (e.g., human) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.n_m.nih.gov/BLAST/^"). Putative target sites hich exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for si NA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene. A suitable siRNA molecule of the present invention is preferably capable of inducing degradation of hUPFl, hUPF2, hUPF3A or hUPF3B transcripts, more preferably, capable of inducing degradation of hUPF2 transcripts, such as the nucleic acid sequence set forth in SEQ ID NO: 7. Inhibition of protein expression can also be effected using ribozymes. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., "Expression of ribozymes in gene transfer systems to modulate target RNA levels/' Curr Opin Biotechnol. 1998 Oct;9(5):486-96]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., "Ribozyme gene therapy for hepatitis C virus infection." Clin Diagn Virol. 1998 Jul 15;10(2-3)J63-71.]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonsfrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page). DNAzymes can also be utilized by the present invention. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;943:4262) A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two subsfrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at pur e:pyrimidine junctions

(Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM Curr Opin Mol Ther 2002;4: 119-21). Examples of construction and amplification of synthetic, engineered

DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al , 20002, Abstract 409, Ann Meeting Am Soc Gen Ther wwvy.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL. The aminoglycoside antibiotic and the agent described hereinabove can be administered (separately or as a part of one formulation) to the subject per se or as part [active ingredient(s)] of one or more pharmaceutical compositions. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate admirύstration of a compound to an organism. Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Pubhshing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially fransnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and inttameduUary injections as well as intrathecal, direct inttaventricular, intravenous, inrtaperitoneal, inttanasal, or intraocular injections. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the pharmaceutical composition (i.e. the aminoglycoside antibiotic and/or the agent capable of downregulating expression or activity of at least one component of the NMD complex/pathway) may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penettants appropriate to the barrier to be permeated are used in the formulation. Such penettants are generally known in the art. For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum ttagacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrroHdone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Ah formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tettafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g. antisense oligonucleotide) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., mammary tumor progression) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model, such as the murine Neu model (Muller et al., (1988) Cell 54, 105-115), to achieve a desired concentration or titer.

Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to levels of the active ingredient are sufficient to retard tumor progression (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

MATERIALS AND METHODS Patients Ten CF patients each carrying at least one copy of the W1282X nonsense mutation were studied. Five patients carry the W1282X and the DF508 mutations (patients #1 - #5); five patients are homozygous for two nonsense mutations, of which at lease one is the W1282X, 3 W1282X/W1282X (#6, #8, #9), one W1282X/G542X (#7) and one W1282X/3849+10kb C->T (#10) which can lead to an inclusion of 84-bρ 'exon' that harbors a nonsense mutation [43]. The clinical data of the patients and the gentamicin protocol are described in Wilschanski et al. (Wilschanski et al., NEJM, in press). Nasal potential difference Transepithelial nasal PD was determined by measuring the potential difference between a fluid-filled exploring bridge on the nasal mucosa and a reference bridge (21- gauge needle filled with Ringers solution in 4% agar) inserted into the subcutaneous space of the forearm. Both bridges were linked by calomel electrodes to a high impedance, low-resistance buffer amplifier. Using direct vision with an otoscope, the exploring catheter as advanced through the inferior meatus of both nostrils and PD was recorded at various sites. After consistent baseline PD measurements have been obtained, the effect of amiloride (10^"4 M) superfusion was evaluated for 3 minutes. The resultant change in PD was recorded and expressed as both an absolute change and as a percentage change from the baseline maximum PD value. To study nasal chloride permeability and cAMP activation of chloride permeability, a large chloride chemical gradient across the apical membrane was generated by superfusion of the nasal mucosa with a chloride-free solution containing 10^"4 mol/L amiloride at a rate of 5ml/min for 3 minutes. Following this, the mucosa was perfused with the same solution with the addition of isoproterenol (10^"5 mol/L) for 3 minutes. The change in voltage response over the final 6 minutes served as an index of epithelial chloride transport. RT-PCR Nasal scrapes were obtained from the CF patients, total RNA was extracted using the RNeasy extraction kit (Qiagen, Hϋden, Germany) and RNA levels were analyzed via RT-PCR. RNA samples of patients carrying the W1282X and the DF508 mutations were analyzed via semi-quantitative RT-PCR using primers flanking the DF508 mutation region (5'-GATTATGGGAGAACTGGAGC; SEQ ID NO:l 3'- TTCTTGCTCGTTGACCTCCA; SEQ ID NO:2). The 5' primer was fluorescent labeled with 6-FAM. Since the DF508 is a 3bp deletion, the two alleles could be differentiated by their size. The level of the W1282X RT-PCR product was analyzed relative to that of the DF508, using the ABI377 sequencer and the GeneScan software. For the RNA samples of the patients who are homozygous for two nonsense mutations, real-time PCR reactions were perfomed using a LightCycler with software version 3.5, using a FastStart DNA Master SYBR Green I kit (Roche Diagnostics,

Mannheim, Germany). Ribosomal protein subunit 9 (RPS9), which is expressed at the same level in different individuals was used for normalization. For each pair of primers (CFTR:5'-GAGGGTAAAATTAAGCACAGT; SEQ ID NO:3 3'-

TGCTCGTTGACCTCCA; SEQ ID NO:4 and RPS9: 5'- AGACCCTTCGAGAAATCTCGTCTCG; SEQ ID NO:5 3'-

TGGGTCCTTCTCATCAAGCGTCAGC; SEQ ID NO:6) a standard curve was performed, and annealing temperatures and elongation times were optimized in order to exclude PCR artificats. Chloride efflux measurement Defects in CFTR may result in reduced or absent chloride permeability. Accordingly, restoration of CFTR mediated chloride permeability in CF cells is expected to have therapeutic value for CF. In this study, chloride transport across plasma membranes (chloride efflux) was measured using the fluorescent indicator

MQAE (Molecular Probes) which is responsive to chloride concentration. Fluorescence intensity was measured using FLUOstar galaxy and/or Tecan Magellan fluorometers (BMG LabTechnologies and Tecan, respectively). Statistical analysis Levels of CFTR nonsense transcripts were compared between responders and non-responders by the Mann-Whitney test. A value of PO.05 was considered statistically significant. Data was expressed as median scores.

RESULTS Nasal epithelial cells were obtained from CF patients as part of a double blind placebo-controlled crossover trial (Wilschanski et al., NEJM, in press). RNA samples were available for analysis from five compound heterozygous for the W1282X and the DF508 mutations and five homozygous for nonsense mutations, of which at least one is the W1282X (see materials and methods). According to the protocol, cells were obtained for each patient at three time points (pre-treatment, placebo and at the end of the gentamicin treatment), at which the CFTR function was measured by nasal potential difference (NPD). The level of CFTR mRNA, ttanscribed from the W1282X allele, was analyzed in the heterozygous W1282X/DF508 patients by semi-quantitative RT-PCR using primers flanking the 3 bp deletion of DF508, to distinguish between the transcripts generated from each allele (Figure la). Among these patients the level of the W1282X transcripts at the pre-treatment was 57%-97% of that ttanscribed from the DF508 allele (Figure 2, left panel). In these patients an impressive functional restoration of the CFTR channel was observed following gentamicin administration, as shown by sodium transport (mean baseline PD) and/or chloride transport (change in PD) (Figure 3). For the patients homozygous for nonsense mutations, real-time quantitative RT-PCR was performed on equivalent amounts of RNA and the results were normalized to a control gene (RPS9), which was found to be expressed at the same level in different individuals and is not affected by the gentamicin treatment. The ratio of CFTR to RPS9 mRNA levels, in each patient at the pretteatment time-point, was compared to the ratio obtained for a control individual with no CFTR mutations. A variability in the CFTR mRNA level, between 14% and 84% of that found in non-CF individual was found among the patients (Figure lb and Figure 2, right panel). Three of these patients (Figure 2, patients #6-#8) showed relative high levels (52-84%) of transcripts, similar to the level found among W1282X/DF508 patients (Figure 2, patients #l-#5). These patients showed correction of the sodium and/or chloride transport, indicating a fractional restoration of the CFTR channel. Two patients (Figure 2, patients #9 and #10) exhibited markedly reduced levels (26% and 14% of the level in the control individual respectively). The patients with the markedly reduced CFTR transcripts did not show any correction of the sodium and the chloride transport, indicating that no restoration of the CFTR function was achieved by the gentamicin treatment (Figure 3, (patients #9 and #10)). In addition to the potential of nonsense mutations to decrease the half-lives of nonsense transcripts they also have the potential to skip the mutation by nonsense- associated altered splicing (NAS) [10, 11]. Thus, it was important to investigate the possibility that the W1282X mutation can up-regulate the NAS pathway and thus can lead to altered sphced transcripts that do not cany the nonsense mutation which is the target of the gentamicin read-through. Therefore, RT-PCR was performed on RNA samples from the 10 studied patients to investigate the possibility of alternatively sphced mRNA lacking the W1282X mutation. The analysis revealed no skipping of the W1282X region in ah patients, responders and non-responders (Figure 4) indicating that NAS does not play a role in modulating the response of patients to gentamicin treatment. The above described results show a strong correlation between the levels of the CFTR nonsense transcripts and the response of patients to gentamicin tteatment. A correction of the CFTR function was found only in patients with relatively high CFTR RNA levels (patients #l-#8), while no change in the abnormal CFTR function was found in patients with markedly reduced levels (patients #9 and #10). Hamosh and Dietz previously suggested that no clinical benefit will be derived from aminoglycoside read÷-through therapy in CF patients with markedly reduced levels of nonsense transcripts [35]. Indeed, in the present study the patients with markedly reduced levels did not respond to the tteatment. However, the results presented herein reveal that there might be variability in the levels of transcripts carrying the same CFTR nonsense mutation. Since nonsense transcripts are prone to degradation by the nonsense- mediated decay (NMD) pathway [1, 4, 5] our results suggest that there are variable NMD efficiencies among different individuals. Previously, variable NMD efficiencies were reported, for transcripts carrying different nonsense mutations, known to result from a polar effect, due to the location of the nonsense codon along the transcript. However, since the present study included patients carrying the same nonsense mutation on the same extended haplotype [36], the different NMD efficiencies might result from factors other than the of location of the mutation. Interestingly, two of the W1282X homozygous patients (#8 and #9) are sisters, whose parents are first degree cousins, and thus are homozygous for the same nonsense allele. These sisters differed in the response to gentamicin associated with a difference in the level of their CFTR transcripts (70% and 32% respectively), further supporting the suggestion that factors other than the CFTR allele can contribute to the NMD efficiency resulting in different RNA levels. It is interesting to note that a variability in the level of CFTR transcripts carrying the W1282X mutation [37, 38] was reported previously. While Hamosh et al. reported that W1282X is associated with severely reduced levels of CFTR mRNA in nasal epithelial cells, the present study demonstrates relatively high transcript levels among patients carrying this mutation, a finding which can be a true variability or technical differences between laboratories. Further support to the present hypothesis that no response to gentamicin treatment is expected in patients with markedly reduced levels of nonsense transcripts can be found in the study performed by Bedwell et al. In this study, gentamicin tteatment was applied in vitro, to D33-1 cells, heterozygous for the W1282X and DF508 mutations. In these cells, although the levels of the nonsense transcripts is very low, following the gentamicin tteatment, a significant increase in the level of the nonsense transcripts and an impressive correction of the CFTR function was observed [24]. Variability in the efficiency of read-through nonsense codons by aminoglycoside antibiotics was reported in vivo and in vitro. A variety of explanations for the lack of response were suggested, among which are the identity of the nonsense codon and the immediately downstream nucleotide [40, 41], species and composition of aminoglycoside [32, 42], variable aminoglycoside permeability [29], and variability in gentamicin metabolizing or in its injection [28]. However, in all these studies the level of the nonsense transcripts was not investigated and thus the potential of the

NMD pathway to contribute to the response to gentamicin treatment could not be evaluated. These explanations could not be applied to the variability in the response to gentamicin found in the present study, in which all the patients carried the same nonsense mutation and were treated with the same antibiotic. However, variability in gentamicin uptake and/or distribution among the patients can not be ruled out. The finding in the current study that all the responding patients had relatively high CFTR RNA level, provides a new insight for the implementation of read-through therapy. There is no doubt that aminoglycoside antibiotics have the read-through nonsense mutations in patients at levels that restore physiologically relevant amounts of functional proteins. The present study provides evidence that the NMD mechanism might play a role in modulating the response of patients to gentamicin treatment. Thus, relatively high levels of nonsense transcripts resulting from non-efficient degradation by the NMD pathway, might be required for the success of the aminoglycoside therapy in patients carrying nonsense mutations in different genetic diseases. In order to determine if downregulating NMD activity can result in a restoration of CFTR function, experiments aimed at evaluating the effect of NMD pathway downregulation on CFTR ttanscript levels and chloride efflux were performed. The experiments were performed usmg epithehal cell lines obtamed from two unrelated CF patients. These cell lines, designated CFP15a and CFP15b, were compound heterozygous for the W1282X and the 3849+1 Okb C->T mutation (generates a cryptic splice site leading to aberrant sphced ttanscript carrying a nonsense mutation). Analysis of CFTR mRNA levels in these cells showed a substantial (4.5 fold) higher level of this ttanscript in CFP15a cells, as compared with CFP15b cells (Figure 5). Untreated CFP15a and CFP15b cells showed no forskolin-stimulated chloride efflux, mdicating that the CFTR channels in both cell lines were inactive. Exposing the cells to 50 μg/ml gentamicin resulted in a substantial increase in chloride efflux in CFP15a cells, in which the CFTR mRNA level is relatively high. On the other hand, no chloride efflux increase was observed in CFP15b cells, in which the CFTR mRNA level is relatively low (Figure 6). The effect of NMD pathway downregulation on the level of CFTR mRNA was evaluated by exposing CFP15a cells to cyclohehimide (CHX; an inhibitor of protein synthesis which indirectly inhibits NMD). The results (Figure 7) show a substantial

(3.5 fold) increase in the level of CFTR mRNA in CHX treated cells. The effect of NMD pathway downregulation on the level of CFTR mRNA was further evaluated by ttansfecting CFP15a cells with siRNA directed against hUPF2 which is involved in the NMD pathway (AAGGCTTTTGTCCCAGCCATC,- ,- SEQ ID

NO: 7). The results (Figure 8) show a substantial (3 fold) increase in the level of

CFTR transcripts in the siRNA treated ceUs. The effect of NMD pathway downregulation on CFTR chloride efflux activity was evaluated by exposing wild-type CFP15a cells and CFP15a cells ttansfected with the siRNA directed against hUPF2, to gentamicin. The results (Figure 9) show a substantially higher activity of chloride efflux in the siRNA treated cells, as compared with wild type cells. Thus, the results presented above clearly indicate that downregulation of the

NMD pathway, via, for example, downregulation of expression of one or more of its components can effectively and substantially increase the level of CFTR mRNA and the activity of chloride efflux in CFTR cells, and thereby restore CFTR function. In addition, the results presented herein also show that NMD pathway downregulation can substantiaUy improve the response of CFTR cells to tteatment of aminoglycosides, such as gentamicin.

It is appreciated that certain features of the mvention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a smgle embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a smgle embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent apphcations and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference mto the specification, to the same extent as if each mdividual pubhcation, patent, patent apphcation or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this apphcation shah not be construed as an admission that such reference is available as prior art to the present mvention.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating an mdividual having a disorder caused by formation of an RNA ttanscript carrymg a nonsense mutation, the method comprismg: (a) exposing cells of the individual expressing the RNA transcript to tteatment selected capable of increasing a half life of the RNA ttanscript in said cells; and (b) exposing said cells to an aminoglycoside thereby treating the mdividual having the genetic disease caused by formation of the RNA transcript carrymg a nonsense mutation.

2. The method of claim 1, wherein said aminoglycoside is selected from the group consisting of kanamycm, neomycin, seldomycm, tobramycm, kasugamycm, fortimicin, gentamicin, paromomycin, neamine, sisomicin, amikacin and netilmicin.

3. The method of claim 1, wherein step (a) is effected by downregulating an activity of a nonsense-mediated decay (NMD) pathway in said cells.

4. The method of claim 3, wherein said dowmegulating is effected by administering to said cehs an agent capable of downregulatmg an activity or expression of at least one component of said nonsense-mediated decay pathway.

5. The method of claim 4, wherein said agent is selected from the group consisting of: (a) a molecule which binds said at least one component of said nonsense- mediated decay pathway; (b) an enzyme which cleaves said at least one component of said nonsense- mediated decay pathway; (c) an antisense polynucleotide capable of specifically hybridizing with an mRNA ttanscript encoding said at least one component of said nonsense-mediated decay pathway; (d) a ribozyme which specifically cleaves transcripts of said at least one component of said nonsense-mediated decay pathway; (e) a non-functional analogue of at least a catalytic or binding portion of said at least one component of said nonsense-mediated decay pathway; (f) an siRNA molecule capable of inducing degradation of transcripts of said at least one component of said nonsense-mediated decay pathway; and (g) a DNAzyme which specifically cleaves transcripts or DNA of said at least one component of said nonsense-mediated decay pathway.

6. The method of claim 4, wherein said at least one component of said nonsense-mediated decay pathway is hUPFl, hUPF2, hUPF3A or hUPF3B.

7. The method of claim 1, wherein steps (a) and (b) are effected concomitantly.

8. The method of claim 7, wherein said agent and said ammoglycoside form a part of a pharmaceutical composition.

9. The method of claim 1, wherein step (a) is effected prior to or following step (b).

10. The method of claim 1, wherein the disorder is selected from the group consisting of Cystic Fibrosis (CF), Duchenne muscular dysttophy (DMD), Hurler syndrome, cystinosis, and late infantile neuronal ceroid lipofuscinosis.

11. A pharmaceutical composition for treatment of a disorder caused by formation of an RNA ttanscript carrying a nonsense mutation, the pharmaceutical composition comprising an aminoglycoside and an agent capable of increasing a half life of the RNA transcript in eukaiyotic cells.

12. The pharmaceutical composition of claim 11, wherein said aminoglycoside is selected from the group consisting of kanamycm, neomycin, seldomycin, tobramycin, kasugamycm, fortimicin, gentamicin, paromomycin, neamine, sisomicin, amikacin and netilmicin.

13. The pharmaceutical composition of claim 11, wherein said agent dowmegulates an activity of a nonsense-mediated decay (NMD) pathway in said eukaryotic cells.

14. The pharmaceutical composition of claim 11, wherein said agent is downregulates an activity or expression of at least one component of said nonsense- mediated decay pathway.

15. The pharmaceutical composition of claim 14, wherein said agent is selected from the group consisting of: (a) a molecule which binds said at least one component of said nonsense- mediated decay pathway; (b) an enzyme which cleaves said at least one component of said nonsense- mediated decay pathway; (c) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding said at least one component of said nonsense-mediated decay pathway; (d) a ribozyme which specifically cleaves transcripts of said at least one component of said nonsense-mediated decay pathway; (e) a non-functional analogue of at least a catalytic or binding portion of said at least one component of said nonsense-mediated decay pathway; (f) an siRNA molecule capable of inducing degradation of transcripts of said at least one component of said nonsense-mediated decay pathway; and (g) a DNAzyme which specifically cleaves transcripts or DNA of said at least one component of said nonsense-mediated decay pathway.

16. The pharmaceutical composition of claim 14, wherein said at least one component of said nonsense-mediated decay pathway is hUPFl, hUPF2, hUPF3A or hUPF3B.

17. The pharmaceutical composition of claim 11, wherein the disorder is selected from the group consisting of Cystic Fibrosis (CF), Duchenne muscular dystrophy (DMD), Hurler syndrome, cystinosis, and late infantile neuronal ceroid lipofuscinosis.