Restoration of the cftr function by splicing modulation

Abstract

The present invention provides oligonucleotides capable of binding to and modulating the splicing of the pre-mRNA of the CFTR gene, including compositions comprising the oligonucleotides, and uses thereof, such as for suppressing the inclusion of a cryptic exon between exon 22 and 23 as a result of the mutation 3849 +10 Kb C-to-T, optionally in combination with additional CF therapeutics.

1 - 31 . (canceled) 
     
     
         32 . A synthetic oligonucleotide molecule characterized by at least partly suppressing the inclusion of intron 22 cryptic exon in the mature mRNA of a CFTR gene having a 3849 +10 Kb C-to-T mutation,
 a) wherein the sequence of said synthetic oligonucleotide molecule comprises the sequence as set forth in any one of SEQ ID NOs: 4, 6, 10, or 17 or an active fragment thereof; or   b) wherein the nucleotide sequence of said synthetic oligonucleotide molecule comprises the sequence as set forth in any one of SEQ ID NOs: 1-3, 5, 8, 40-48, or an active fragment thereof and is characterized by increasing the percentage of correctly spliced mature CFTR mRNA by at least about 10%; and decreasing the level of aberrantly spliced mature CFTR mRNA by at least about 20%,   and wherein said synthetic oligonucleotide molecule or active fragment thereof consists of 17-21 consecutive bases.   
     
     
         33 . The synthetic oligonucleotide molecule of  claim 32 , wherein said active fragment comprises a nucleotide sequence as set forth in SEQ ID NO: 40. 
     
     
         34 . The synthetic oligonucleotide molecule of  claim 32 , comprising the sequence GAUGGAAGA (SEQ ID NO: 38). 
     
     
         35 . The synthetic oligonucleotide molecule of  claim 32 , consisting of 18 or 19 consecutive nucleotide bases. 
     
     
         36 . The synthetic oligonucleotide molecule of  claim 32 , comprising a chemically modified backbone comprising: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a 2′-O-methyl-phosphorothioate (2′OMP) backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, a 2′-Methoxy Ethyl (2′MOE) backbone, an alternating locked nucleic acid backbone, constrained ethyl backbone, a phosphorothioate backbone, N3′-P5′ phosphoroamidates, 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, or a combination thereof. 
     
     
         37 . The synthetic oligonucleotide molecule of  claim 32 , having at least 80% complementarity to a nucleotide sequence within SEQ ID NO: 37. 
     
     
         38 . The synthetic oligonucleotide molecule of  claim 32 , having 100% complementarity to a nucleotide sequence within SEQ ID NO: 37. 
     
     
         39 . A pharmaceutical composition comprising the synthetic oligonucleotide molecule of  claim 32  and a pharmaceutically acceptable carrier. 
     
     
         40 . The pharmaceutical composition of  claim 39 , further comprising a CFTR modifier; a CF drug, or a combination thereof, wherein said CFTR modifier is selected from the group consisting of: CFTR potentiator, CFTR corrector, Translational Read-Through agent, and CFTR amplifier. 
     
     
         41 . The pharmaceutical composition of  claim 40 , wherein said CFTR modifier is ivacaftor, lumacaftor, tezacaftor, elexacaftor, VX-659, VX-152, or VX-440, or any combination thereof. 
     
     
         42 . The pharmaceutical composition of  claim 40 , wherein said CF drug is an antibiotic drug, a bronchodilator, a corticosteroid, or any combination thereof. 
     
     
         43 . The pharmaceutical composition of  claim 39 , formulated for oral, nasal, inhalation, abdominal, subcutaneous, intra-peritoneal or intravenous administration. 
     
     
         44 . A method for treating Cystic Fibrosis (CF) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide that at least partly suppresses the inclusion of intron 22 cryptic exon in the mature CFTR mRNA of a CFTR gene having a 3849 +10 Kb C-to-T mutation,
 a) wherein the sequence of said synthetic oligonucleotide molecule comprises the sequence as set forth in any one of SEQ ID NOs: 4, 6, 10, or 17 or an active fragment thereof; or   b) wherein the nucleotide sequence of said synthetic oligonucleotide molecule comprises the sequence as set forth in any one of SEQ ID NOs: 1-3, 5, 8, 40-48, or an active fragment thereof and increases the percentage of correctly spliced mature CFTR mRNA by at least about 10%; and decreases the level of aberrantly spliced mature CFTR mRNA by at least about 20%,   and wherein said synthetic oligonucleotide molecule or active fragment thereof consists of 17-21 consecutive bases, thereby treating CF in the subject.   
     
     
         45 . The method of  claim 44 , wherein said active fragment comprises a nucleotide sequence as set forth in SEQ ID NO: 40. 
     
     
         46 . The method of  claim 44 , wherein said synthetic oligonucleotide molecule comprises the sequence GAUGGAAGA (SEQ ID NO: 38). 
     
     
         47 . The method of  claim 44 , wherein said synthetic oligonucleotide comprises a backbone selected from the group consisting of: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a 2′-O-methyl-phosphorothioate backbone (2′OMP), a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, a 2′-Methoxy Ethyl (2′MOE) backbone, an alternating locked nucleic acid backbone, a phosphorothioate backbone, N3′-P5′ phosphoroamidates, 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, and a combination thereof. 
     
     
         48 . The method of  claim 44 , wherein said synthetic oligonucleotide molecule has at least 80% complementarity to a sequence within SEQ ID NO: 36 or to a sequence within SEQ ID NO: 37. 
     
     
         49 . The method of  claim 44 , further comprising administering to said subject a therapeutically effective amount of one or more CFTR modifiers comprising a CFTR-splicing-modulating agent, a Translational Read-Through agent, a CFTR amplifier, a CFTR potentiator, or a CFTR corrector. 
     
     
         50 . The method of  claim 44 , wherein said CFTR modifier is selected from the group consisting of: a different synthetic oligonucleotide molecule capable of suppressing intron 22 cryptic exon inclusion in the mature CFTR mRNA, Ataluren, ELX-02, ivacaftor, QBW251, PTI-808, VX-561, lumacaftor, tezacaftor, elexacaftor, VX-659, VX-445, VX-152 and VX-440, GLPG2222, FDL169, PTI-801, and any combination thereof. 
     
     
         51 . The method of  claim 44 , wherein said subject is heterozygous for said 3849 +10 Kb C-to-T mutation. 
     
     
         52 . The method of  claim 44 , wherein said treating comprises improving at least one clinical parameter of CF selected from the group consisting of: lung function, time to the first pulmonary exacerbation, change in weight, change in height, a change in Body Mass Index (BMI), change in the concentration of sweat chloride, number and/or duration of pulmonary exacerbations, total number of days of hospitalization for pulmonary exacerbations, and the need for antibiotic therapy for sinopulmonary signs or symptoms.