Restoration of CFTR function through splicing regulation

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 843,469, filed on May 5, 2019, entitled “RESTORATION OF THE CFTR FUNCTION BY SPLICING MODULATION,” the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present invention relates to oligonucleotides capable of binding to CFTR pre-mRNA and methods of using the same, such as for inhibiting the inclusion of a cryptic exon between exons 22 and 23 due to the mutation 3849+10Kb C-to-T, and for treating cystic fibrosis (CF).

[0004] background

[0005] Cystic fibrosis (CF) is a common, severe autosomal recessive disease caused by mutations in the CFTR gene. The CFTR gene encodes a chloride channel responsible for chloride transport in epithelial cells. The primary manifestation of CF is in the lungs, with more than 90% of deaths related to respiratory disease. Respiratory disease is associated with insufficient CFTR function in the airway epithelium.

[0006] To date, about 2000 different mutations that destroy CFTR function have been identified worldwide, which are divided into five different categories based on their effects on CFTR function. Class I includes mutations that lead to non-functional CFTR (large deletions and stop codon mutations). Class II mutations (including the common ΔF508) lead to abnormally folded CFTR proteins, which are recognized by cell quality control mechanisms and subsequently degraded, resulting in the absence of mature CFTR proteins at the apical cell membrane. Class III mutations lead to full-length CFTR proteins being integrated into the cell membrane, but with defective regulation, so that there is no CFTR function. These three categories usually lead to the classic CF phenotype with pancreatic insufficiency, although there are large differences in the severity of lung disease. CFTR mutations that lead to defective chloride conduction are divided into Class IV. Class V mutations involve abnormal transcriptional regulation, resulting in a reduction in the amount of normal CFTR. The latter two categories are generally associated with milder phenotypes and intact pancreatic function. Specifically, the CFTR produced by Class IV mutations is inserted into the plasma membrane, but due to the reduced probability of chloride penetration and open channels, a reduced single-channel chloride ion conduction is presented. Class V mutations are found in <1% of CF patients to produce normal plasma membrane CFTR. However, the amount is often reduced due to abnormal transcriptional regulation. Class V mutations are often affected by the splicing machinery and produce both abnormally spliced ​​mRNA and correctly spliced ​​mRNA, the levels of which vary between different patients and even between different organs in the same patient. Ultimately, the splicing variants result in a reduced amount of functional CFTR in the plasma membrane.

[0007] About 10%-15% of CFTR mutations affect the correct splicing of gene transcripts. Among them is the splicing mutation 3849+10kb C-to-T, which results in a cryptic exon containing 84 base pairs in the mature messenger RNA (mRNA) (called the "intron 22 cryptic exon inclusion" mutation). This mutation is the 12th most common CFTR mutation in the world, occurring in hundreds of CF patients worldwide (Kerem et al., 1997). Correcting the abnormal splicing of the CFTR gene by "antisense" oligonucleotides (AO) was attempted in Friedman et al., 1999, and recently described in the inventor's international PCT application publication WO 2014 / 045283.

[0008] Administration of antisense oligonucleotides (AO or ASO) is one of the most promising therapeutic approaches for treating genetic disorders caused by splicing mutations. AO is a short synthetic molecule that can anneal to a motif predicted to be involved in pre-mRNA splicing. The method is based on splice switching. AO bound to a selected site is expected to mask the targeted region and promote normal splicing. AO is highly specific to its target and does not affect any other sequence in the cell. Several types of chemically modified AO molecules are commonly used, including: 2'-O-methyl-thiophosphate (2'OMP), phosphorodiamidate morpholino oligomers (PMO), peptide nucleic acids (PNA), 2-methoxyethyl thiophosphate (MOE), constrained ethyl (cET), ligand-conjugated antisense (LICA) and alternating locked nucleic acids (LNA).

[0009] AO modification maintains their stability, improves their target affinity, and provides good pharmacokinetic properties and biostability. It has been shown that in a mouse model of Duchenne muscular dystrophy (DMD), splice switching AO can redirect dystrophin pre-mRNA processing so that exons carrying premature protein termination signals (nonsense mutations) can be excluded from the mature gene transcript, resulting in a shorter but still functional dystrophin isoform. Progress in dystrophin exon skipping has been rapid, with proof-of-concept studies reported and results published for systemic administration of 2OMP and PMO chemicals to patients. In 2016, the FDA granted accelerated approval for the first drug for Duchenne muscular dystrophy (eteplirsen), a PMO AO for exon 51 skipping.

[0010] In addition to induced exon skipping, AOs can also be designed to mask splicing silencer elements that reduce exon recognition and subsequent inclusion in mature mRNA. Spinal muscular atrophy (SMA) is a common autosomal recessive condition caused by deletion of the SMN1 gene as well as a C>T variant in SMN2 exon 7, leading to aberrant splicing in which SMN2 exon 7 is skipped, resulting in a non-functional gene product. AOs have been designed to mask nearby flanking SMN2 splicing silencer elements to promote synthesis of full-length transcripts. Intrathecal administration of morpholino oligomers to neonatal mouse pups with severe SMA was highly successful, significantly prolonging their survival.

[0011] This led to the development of the AO-based drug Spinraza (Nusinersen), which was recently approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The approval follows the highly successful completion of a Phase III clinical trial in patients with SMA that appears in infancy. Treated infants experienced statistically significant improvements in the achievement of motor milestones. The results of this study demonstrate the great potential of AO-based splicing modulation for the treatment of genetic diseases and highlight the importance of local delivery for effective treatment with minimal toxicity.

[0012] In the field of cystic fibrosis treatment, there is a continuing need for novel, effective therapeutic agents designed to overcome the many mutations in the CFTR gene identified to date and to restore CFTR function.

[0013] Overview

[0014] The invention provides compositions comprising oligonucleotides, which can be bound to CFTR precursor mRNA, thereby regulating splicing and recovering or enhancing the function of CFTR gene product. Therefore, the present invention identifies sequences in CFTR precursor mRNA, which are targeted to regulate the splicing cascade of CFTR precursor mRNA. As demonstrated by the present invention, regulating CFTR precursor mRNA splicing can avoid incorrect identification of intron sequences as exons. As a result of splicing regulation, functional CFTR protein is produced with enough levels by originally abnormal CFTR alleles. The present invention also provides oligonucleotides for regulating splicing and recovering or enhancing the modification of CFTR gene product function in some embodiments thereof.

[0015] The present invention is based in part on the discovery that artificial "antisense" oligonucleotide molecules can target and bind to predetermined sequences of the pre-mRNA molecules of the CFTR gene, and that this binding can modulate the splicing of the pre-mRNA molecules into mature mRNAs, which are then translated into functional CFTR proteins at sufficient levels. Targets in the CFTR pre-mRNA molecules are those that are found to be involved in splicing, either indirectly by affecting the splicing of adjacent sequences and more distant sequences, or directly by affecting their own splicing.

[0016] Advantageously, the present invention provides antisense oligonucleotide molecules with improved efficacy. These oligonucleotides are very effective in increasing the percentage of correctly spliced ​​CFTR mRNA. Oligonucleotides are short, having no more than 21 nucleotides, with advantages such as improved cell / tissue penetration.

[0017] According to a first aspect, a synthetic oligonucleotide molecule consisting of 17-21 consecutive bases is provided, which has at least 80% complementarity with a pre-mRNA transcript of a CFTR gene having a 3849+10Kb C-to-T mutation, and is characterized in that it at least partially inhibits the inclusion of the cryptic exon of intron 22 in mature CFTR mRNA, increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%; and reduces the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%.

[0018] According to another aspect, a pharmaceutical composition comprising the synthetic oligonucleotide molecule of the present invention and a pharmaceutically acceptable carrier is provided.

[0019] According to another aspect, a method for treating CF in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a synthetic oligonucleotide, wherein the synthetic oligonucleotide inhibits the inclusion of the cryptic exon of intron 22 in mature CFTR mRNA, increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%; and reduces the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%, thereby treating CF in the subject.

[0020] According to another aspect, a kit is provided comprising: (a) at least one synthetic oligonucleotide molecule; and at least one of: (b) at least one CFTR modifier; or (c) at least one CF drug, wherein the synthetic oligonucleotide is selected from the group consisting of SEQ ID No 1-25 and SEQ ID No 41-44, and wherein the CFTR modifier is selected from the group consisting of: a CFTR potentiator, a CFTR corrector, a translation read-through agent, and a CFTR amplifier.

[0021] In some embodiments, the synthetic oligonucleotide molecule has at least 80% complementarity to the nucleotide sequence within SEQ ID NO:37.

[0022] In some embodiments, the synthetic oligonucleotide molecule has 100% complementarity to the nucleotide sequence within SEQ ID NO:37.

[0023] In some embodiments, the synthetic oligonucleotide molecule consists of 19 consecutive nucleotide bases.

[0024] In some embodiments, the synthetic oligonucleotide molecule consists of 18 consecutive nucleotide bases.

[0025] In some embodiments, the synthetic oligonucleotide molecules comprise a chemically modified backbone, the chemically modified backbones comprising: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a 2'-O-methyl-phosphorothioate backbone, a phosphodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, an alternating locked nucleic acid backbone, a constrained ethyl backbone, a phosphorothioate backbone, an N3'-P5' phosphoamide, a 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid, a cyclohexene nucleic acid backbone, a tricyclic-DNA (tcDNA) nucleic acid backbone, and combinations thereof.

[0026] In some embodiments, the synthetic oligonucleotide molecule comprises a backbone comprising a 2'-O-methylphosphorothioate (2'OMP) modification or a 2'-methoxyethyl (2'MOE) modification.

[0027] In some embodiments, the synthetic oligonucleotide molecule comprises a nucleotide sequence set forth in one of SEQ ID Nos: 1-25 and SEQ ID Nos: 41-44.

[0028] In some embodiments, the synthetic oligonucleotide molecule comprises a nucleotide sequence set forth in one of SEQ ID Nos: 1-5 and SEQ ID No:41.

[0029] In some embodiments, the synthetic oligonucleotide molecule comprises the sequence GAUGGAAGA (SEQ ID NO: 38).

[0030] In some embodiments, the pharmaceutical composition is formulated for oral, nasal, inhaled, abdominal, subcutaneous, intraperitoneal, or intravenous administration.

[0031] In some embodiments, the pharmaceutical composition is used to treat cystic fibrosis (CF) in a subject in need thereof.

[0032] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more CFTR modifying agents.

[0033] In some embodiments, the CFTR modifier is selected from the group consisting of a CFTR splicing regulator, a translational readthrough agent, a CFTR amplifier, a CFTR enhancer, and a CFTR corrector.

[0034] In some embodiments, the CFTR modifier is selected from the group consisting of: different synthetic oligonucleotide molecules capable of inhibiting the inclusion of the cryptic exon of intron 22 in 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.

[0035] In some embodiments, the CFTR modifier is avacaftor, rumacaftor, tezacaftor, elexacaftor, VX-659, VX-152, or VX-440, or any combination thereof.

[0036] In some embodiments, the subject comprises a 3849+10Kb C-to-T mutation in the CFTR gene.

[0037] In some embodiments, the subject is heterozygous for the 3849+10Kb C-to-T mutation.

[0038] In some embodiments, treatment comprises improving at least one clinical parameter of CF selected from the group consisting of: lung function, time to first pulmonary exacerbation, change in weight, change in height, change in body mass index (BMI), change in sweat chloride concentration, number and / or duration of pulmonary exacerbations, total number of days hospitalized for pulmonary exacerbations, and need for antibiotic therapy for sinopulmonary signs or symptoms.

[0039] In some embodiments, the CF medication is an antibiotic medication, a bronchodilator, a corticosteroid, or any combination thereof.

[0040] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only,

[0041] And unexpectedly Figure 1 It is definitely restrictive.

[0042] Other embodiments and the full scope of the applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1is a graph showing the effect of a 3849+10kb C to T mutation in intron 22 of the CFTR gene. Upper panel—When spliced ​​normally, exon 22 and exon 23 become adjacent. Lower panel—A splicing mutation in intron 22 (denoted as "3849+10kb C-to-T" mutation) results in the inclusion of an additional 84 bases in the mature CFTR mRNA (denoted as "intron 22 cryptic exon"). This mutation generates a premature in-frame stop codon, leading to mRNA degradation through a nonsense-mediated mRNA decay (NMD) mechanism.

[0045] Figure 2 is a diagram showing the binding sites (underlined) of oligonucleotides SPL84-1-SPL84-26 within the CFTR allele carrying the 3849+10 kb C to T mutation.

[0046] Figure 3A It is a vertical bar graph showing the changes in the level of abnormally spliced ​​CFTR transcripts in FRT 3849mut cells after antisense oligonucleotide (ASO) transfection (10nM). After 24 hours of treatment with indicated ASO, RNA was extracted and the level of abnormally spliced ​​CFTR transcripts was measured by qRT-PCR. The values ​​shown are the average fold changes (mean ± sem) from 2-7 independent experiments relative to cells treated with control ASO. The values ​​are normalized for the transcripts of the HPRT gene.

[0047] Figure 3B Is a vertical bar graph showing the percentage of correctly spliced ​​CFTR transcripts in FRT 3849mut cells after ASO transfection (10 nM). After 24 hours of treatment with the indicated ASOs, RNA was extracted and the ratio between correctly spliced ​​CFTR transcripts and abnormally spliced ​​CFTR transcripts was measured by RT-PCR. The values ​​shown are the average percentages (mean ± sem) of correctly spliced ​​transcripts from 3-8 independent experiments.

[0048] Figure 4A Is a vertical bar graph showing changes in the levels of abnormally spliced ​​CFTR transcripts in FRT3849mut cells after ASO transfection (2.5 nM) compared to the ASOs described in Friedman et al., 1999. After 24 hours of treatment with the indicated ASOs, RNA was extracted and the levels of abnormally spliced ​​CFTR transcripts were measured by qRT-PCR. The values ​​shown are the average fold changes (mean ± sem) from 3 independent experiments relative to cells treated with control ASOs. Values ​​were normalized for transcripts of the HPRT gene.

[0049] Figure 4BIs a vertical bar graph showing the percentage of correctly spliced ​​CFTR transcripts in FRT3849mut cells after ASO transfection (2.5 nM) compared to the ASOs described in Friedman et al., 1999. After 24 hours of treatment with the indicated ASOs, RNA was extracted and the levels of correctly spliced ​​CFTR transcripts and aberrantly spliced ​​CFTR transcripts were measured by RT-PCR. The values ​​shown are the average percentages of correctly spliced ​​transcripts from 3 independent experiments (mean ± sem).

[0050] Figure 5A Is a vertical bar graph showing changes in the levels of abnormally spliced ​​CFTR transcripts in FRT3849mut cells after ASO transfection (10 nM) compared to the ASOs described in WO 2014 / 045283. After 24 hours of treatment with the indicated ASOs, RNA was extracted and the levels of abnormally spliced ​​CFTR transcripts were measured by qRT-PCR. The values ​​shown are the average fold changes (mean ± sem) from 3 independent experiments relative to cells treated with control ASOs. Values ​​were normalized for transcripts of the HPRT gene.

[0051] Figure 5B Is a vertical bar graph showing the percentage of correctly spliced ​​CFTR transcripts in FRT3849mut cells after ASO transfection (10 nM) compared to the ASOs described in WO 2014 / 045283. After 24 hours of treatment with the indicated ASOs, RNA was extracted and the levels of correctly spliced ​​CFTR transcripts and abnormally spliced ​​CFTR transcripts were measured by RT-PCR. The values ​​shown are the average percentages of correctly spliced ​​transcripts from 3 independent experiments (mean ± sem).

[0052] Figure 6 Is a graph showing the effect of different ASOs at different concentrations on aberrantly spliced ​​CFTR transcripts in FRT3849mut cells treated with a specified ASO at a specified concentration for 24 hours, RNA was extracted, and the levels of aberrantly spliced ​​CFTR transcripts were measured by qRT-PCR. The values ​​shown are the average fold changes (mean ± sem) relative to cells treated with a control ASO. Values ​​were normalized to transcripts of the HPRT gene.

[0053] Figure 7A-7B is a photomicrograph of a Western blot analysis showing the effect of different ASOs on the formation of full-length mature CFTR protein. ( Fig. 7A ) FRT 3849mut cells were transfected with the indicated ASOs. ( Figure 7B) HEK293T cells transiently expressing CFTR cDNA mutated with 3849+10Kb C to T mutation and containing flanking intronic regions were transfected with the indicated ASOs. The M3A7 antibody recognizes the C-terminus of the protein, allowing identification of only full-length CFTR protein.

[0054] Fig. 8A is a graph showing representative fluorescent imaging plate reader (FLIPR) traces demonstrating CFTR activation induced by the action of the ASO SPL84-17 in FRT3849mut cells.

[0055] Figure 8B is a vertical bar graph showing the average effect of certain ASOs on CFTR function. The activity of CFTR channels was analyzed using the FLIPR assay in FRT 3849mut cells after transfection with the indicated ASOs (10 nM). Mean ± sem.

[0056] Fig.9A Included are plots showing the effects of ASOs on CFTR activation in primary human nasal epithelial (HNE) cells from a heterozygous CF patient with the genotype 3849+10Kb C-to-T / F508del. Representative traces of electrophysiological responses in Ussing chambers following free uptake of the indicated ASOs.

[0057] Fig. 9B is with Fig.9A ΔI from Ussing chambers after free uptake of indicated ASOs in the same HNE cells SC CFTRinh172 absolute value (μA / cm 2 ). The thick grey horizontal line indicates the median. ΔI SC CFTRinh172 was calculated for 4 filters treated with SPL84-22, SPL84-23 and SPL84-17 from 2 samples obtained from patients, 2 filters treated with SPL84-2 and 1 filter treated with SPL84-25 (marked with *). The horizontal dashed line indicates the mean ΔI in HNE cultures from healthy WT / WT individuals SC 50% of CFTRinh172 levels.

[0058] Fig. 10A Included are curves showing the effects of ASOs on CFTR activation in primary HNE cells from a patient homozygous for the 3849+10Kb C-to-T mutation. Representative traces of electrophysiological responses in Ussing chambers following treatment with the indicated ASOs.

[0059] Fig. 10B is with Fig. 10A ΔI from Ussing chambers after free uptake of indicated ASOs in the same HNE cells SC CFTRinh172 absolute value (μA / cm 2 ). The thick grey horizontal line indicates the median. ΔI SC CFTRinh172 was calculated for: 2 filters except one filter treated with SPL84-2 (marked with *). The horizontal dashed line indicates the mean ΔI in HNE cultures from healthy WT / WT individuals SC CFTRinh172 levels.

[0060] Fig.11 is a vertical bar graph showing the profile of CFTR activation in heterozygous patients. Mean (± SEM) of % of WT (ΔI SC CFTRinh172-patients*100 / average ΔI SC CFTRinh172-WT) was based on each patient's ΔI SC Median values ​​of CFTRinh172.WT levels were calculated based on the mean ΔI in HNE cultures from healthy WT / WT individuals. SC These values ​​were calculated from 9 filters from 3 patients treated with SPL84-17 and SPL84-22, 24 filters from 5 patients treated with SPL84-23, 5 filters from 2 patients treated with SPL84-2, and 1 filter from 1 patient treated with SPL84-25.

[0061] Figure 12A-12B Included are vertical bar graphs and gel electrophoresis micrographs showing the effect of ASO SPL84-23 on the splicing pattern of the 3849+10kb C>T mutation. HNE cells from patients homozygous (N=1) or heterozygous (N=4) for the 3849+10kb C-to-T mutation were treated with 200 nM SPL84-23. RNA was extracted and the levels of abnormally spliced ​​CFTR transcripts and correctly spliced ​​CFTR transcripts were measured. Fig. 12A qRT-PCR analysis of the effect of SPL84-23 on aberrantly spliced ​​CFTR transcripts is shown. The values ​​shown are the average fold changes (mean ± SEM) relative to cells treated with control ASOs. Values ​​were normalized to transcripts of the GUSb gene. Statistical analysis was performed using the student t-test (1-tailed, paired). ***p<0.001. Fig. 12B RT-PCR of homozygous samples in agarose gel is shown.

[0062] Fig.13is a graph showing the effect of chemical modifications on ASO SPL84-23 on the splicing pattern of the 3849+10kb C>T mutation. ΔI SC CFTRinh172 absolute value (μA / cm 2 ). Horizontal lines mark median values. ΔI was calculated for cells treated with the indicated concentrations of ASO modified with 2'OMP or 2'MOE SC CFTRinh172. The horizontal dashed line indicates the mean ΔI in HNE cultures sampled from healthy WT / WT individuals. SC CFTRinh172 levels.

[0063] Fig.14 is a vertical bar graph showing the optimization of ASO length for aberrantly spliced ​​variants of the CFTR transcript. Short versions of ASO84-23 were screened for their effects on splicing (SPL84-23-1-SPL84-23-9) by free uptake in primary patient HNE cells. On average, SPL84-23-1 (19mer) had an effect comparable to SPL84-23 in the three 3849 heterozygous patients analyzed.

[0064] Figure 15A-15B is a graph showing the optimization of ASO length. Primary HNE cells were obtained from a heterozygous patient (3849 / F508del). In primary HNE cells, the effect of SPL84-23-1 (19mer) on CFTR function was similar to that of the longer SPL84-23 ASO. ( Fig.15A ) represents ΔI SC Absolute value of CFTRinh172 (μA / cm 2 ),and( Fig. 15B ) represents the ratio compared with WT (WT(I SC CFTRinh172)%.

[0065] Figure 16A-16B is a graph showing the optimization of ASO length. Primary HBE cells were obtained from a heterozygous patient (3849 / F508del). In primary HNE cells, the effect of SPL84-23-1 (19mer) on CFTR function was similar to that of the longer SPL84-23 ASO. ( Fig.16A ) represents ΔI SC Absolute value of CFTRinh172 (μA / cm 2 ),and( Fig. 16B ) represents the ratio compared with WT (WT(I SC CFTRinh172)%.

[0066] Detailed Description

[0067] In some embodiments, a synthetic oligonucleotide molecule consisting of 18-21 consecutive bases is provided, which has at least 80% complementarity to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C-to-T mutation, and is characterized by at least partially inhibiting the inclusion of the intron 22 cryptic exon in the mature CFTR mRNA, increasing the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%; and reducing the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%.

[0068] In some embodiments, a method for treating CF in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a synthetic oligonucleotide of the invention, thereby treating CF in the subject.

[0069] In some embodiments, the treatment comprises inhibiting inclusion of the intron 22 cryptic exon in mature CFTR mRNA, increasing the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%, reducing the level of aberrantly spliced ​​mature CFTR mRNA by at least about 20%, or any combination thereof.

[0070] The present invention provides oligonucleotides and compositions comprising the oligonucleotides, which can be bound to CFTR precursor mRNA, thereby regulating splicing and restoring the function of the CFTR gene product. Therefore, the present invention identifies sequences in CFTR precursor mRNA, which are targeted to regulate the splicing cascade of CFTR precursor mRNA. As demonstrated by the present invention, regulating CFTR precursor mRNA splicing can avoid incorrectly identifying intron sequences as exons. As a result of splicing regulation, originally abnormal CFTR alleles produce functional CFTR protein.

[0071] In some embodiments, the oligonucleotides disclosed herein are synthetic oligonucleotides.

[0072] The present invention is derived in part from the discovery that artificial "antisense" polynucleotide molecules can target and bind to predetermined sequences of the pre-mRNA molecules of the CFTR gene, and that the binding modulates the splicing of the pre-mRNA molecules into mature mRNAs, which are then translated into functional CFTR proteins. Targets in the CFTR pre-mRNA molecules are those that have been found to be involved in splicing, either indirectly by affecting the splicing of adjacent sequences as well as distal sequences, or directly by affecting their own splicing.

[0073] In one aspect, the present invention provides a synthetic oligonucleotide molecule consisting of 17-21 consecutive bases that is complementary to the pre-mRNA transcript of the CFTR gene, wherein the synthetic oligonucleotide molecule at least partially inhibits the inclusion of the cryptic exon of intron 22 in the mature CFTR mRNA, increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%; and reduces the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%.

[0074] In another aspect, the present invention provides a synthetic oligonucleotide molecule consisting of 17-21 consecutive bases, which is complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C-to-T mutation, wherein the synthetic oligonucleotide molecule at least partially inhibits the inclusion of the cryptic exon of intron 22 in the mature CFTR mRNA, and reduces the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%.

[0075] In some embodiments, the oligonucleotide increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 12%. In some embodiments, the oligonucleotide increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%.

[0076] In another aspect, the present invention provides a synthetic oligonucleotide molecule consisting of 17-21 consecutive bases, which is complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C-to-T mutation, wherein the synthetic oligonucleotide molecule at least partially inhibits the inclusion of the intron 22 cryptic exon in the mature CFTR mRNA, increases the percentage of correctly spliced ​​mature CFTR mRNA by at least about 10%; and reduces the level of abnormally spliced ​​mature CFTR mRNA by at least about 20%, wherein the oligonucleotide comprises a 2'-O-methyl-phosphorothioate backbone and / or a 2'-methoxyethyl (2'MOE) backbone. As used herein, the phrase "inhibiting the inclusion of the intron 22 cryptic exon" refers to reducing the incidence of the addition of the 84 nucleotides (SEQ ID NO: 35) found in intron 22 of the CFTR gene to the mature CFTR mRNA.

[0077] As used herein, the phrase "percentage of correctly spliced ​​mature CFTR mRNA" refers to the percentage of correctly spliced ​​mature CFTR mRNA compared to 100% of correctly spliced ​​mature CFTR mRNA found in healthy cells or subjects without mutations in any allele of the CFTR gene. For example, an increase from 1% before treatment with an ASO provided by the present invention to 11% after treatment with an ASO provided by the present invention is considered an increase of 10%. An increase from 10% before treatment with an ASO provided by the present invention to 11% after treatment with an ASO provided by the present invention is considered an increase of 1%.

[0078] In certain embodiments, the CFTR transcript comprises a mutation that increases inclusion of the intron 22 cryptic exon. In certain embodiments, the mutation is a 3849+10Kb C to T mutation. In some embodiments, the intron 22 cryptic exon comprises the sequence of SEQ ID NO: 35 or a fragment thereof.

[0079] In certain embodiments, the oligonucleotide molecule is complementary to a nucleotide sequence within SEQ ID NO: 37. In some embodiments, the oligonucleotide molecule is complementary to a nucleotide sequence no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 bases upstream of a mutation that increases the inclusion of the cryptic exon of intron 22. Each possibility represents a separate embodiment of the present invention. In some embodiments, the oligonucleotide molecule is complementary to a nucleotide sequence no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 bases downstream of a mutation that increases the inclusion of the cryptic exon of intron 22. Each possibility represents a separate embodiment of the present invention.

[0080] In some embodiments, the oligonucleotide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% complementarity to the nucleotide sequence within SEQ ID NO:37.

[0081] In certain embodiments, the oligonucleotide molecule is complementary to a nucleotide sequence corresponding to a pre-mRNA molecule comprising a sequence from about 100 base pairs before the cryptic exon of intron 22 to about 100 base pairs after the cryptic exon of intron 22 transcribed from a CFTR gene having a 3849+10Kb C- to-T mutation. In certain embodiments, the oligonucleotide molecule is complementary to a nucleotide sequence within SEQ ID NO:37.

[0082] The term "complementary" or "complementarity" refers to the ability of nucleic acids such as oligonucleotides, polynucleotides, etc. to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can be base-paired in a Watson-Crick manner (e.g., A to T, A to U, C to G) or in any other manner that allows the formation of a duplex. As known to those skilled in the art, when RNA is used instead of DNA, the base that is considered to be complementary to adenosine is uracil instead of thymine. However, when U is represented in the context of the present invention, unless otherwise stated, the ability to replace T is implied.

[0083] In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced ​​mature CFTR mRNA by about 20%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced ​​mature CFTR mRNA by about 30%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced ​​mature CFTR mRNA by about 40%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced ​​mature CFTR mRNA by about 50%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced ​​mature CFTR mRNA by about 60%.

[0084] As used herein, the phrase "increases the level of correctly spliced ​​mature CFTR mRNA" refers to an increase in the level of correctly spliced ​​mature CFTR mRNA after treatment with an ASO provided herein compared to the level before treatment or after mock treatment. In certain embodiments, the increase in the level of correctly spliced ​​mature CFTR mRNA is compared to mock treatment of a control ASO. In certain embodiments, the control ASO consists of the nucleotide sequence in SEQ ID NO: 33.

[0085] In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 30%. In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 40%. In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 50%. In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 60%. In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 70%. In certain embodiments, the oligonucleotide molecules reduce the level of the mature CFTR mRNA of abnormal splicing by about 80%.

[0086] As used herein, the phrase "reduces the level of aberrantly spliced ​​mature CFTR mRNA" refers to the percentage reduction in the level of aberrantly spliced ​​mature CFTR mRNA after treatment with an ASO provided herein compared to the level before treatment or after mock treatment. In certain embodiments, the reduction in the level of aberrantly spliced ​​mature CFTR mRNA is compared to the mock treatment of a control ASO. In certain embodiments, the control ASO consists of the nucleotide sequence in SEQ ID NO: 33. As used herein, the phrase "increases the percentage of correctly spliced ​​mature CFTR mRNA" refers to the percentage increase in the ratio of correctly spliced ​​CFTR mRNA to aberrantly spliced ​​CFTR mRNA compared to untreated or mock treatment.

[0087] In certain embodiments, the oligonucleotide molecule is composed of 18 or 19 continuous nucleotide bases. In certain embodiments, the oligonucleotide molecule is composed of 18 continuous nucleotide bases. In certain embodiments, the oligonucleotide molecule is composed of 19 continuous nucleotide bases. In certain embodiments, the oligonucleotide molecule is composed of 20 continuous nucleotide bases. In certain embodiments, the oligonucleotide molecule is composed of 21 continuous nucleotide bases.

[0088] In certain embodiments, the base is selected from the group consisting of: adenine, guanine, cytosine, uracil and optionally thymine. In certain other embodiments, the base is selected from the group consisting of: adenine, guanine, cytosine and uracil. Each possibility represents a separate embodiment of the present invention.

[0089] In some embodiments, the oligonucleotide is chemically modified. In some embodiments, the chemical modification is a modification of the main chain of the oligonucleotide. In some embodiments, the chemical modification is a modification of the sugar of the oligonucleotide. In some embodiments, the chemical modification is a modification of the nucleobase of the oligonucleotide. In some embodiments, the chemical modification increases the stability of the oligonucleotide in the cell. In some embodiments, the chemical modification increases the stability of the oligonucleotide in vivo. In some embodiments, the chemical modification increases the ability of the oligonucleotide to regulate splicing. In some embodiments, the chemical modification increases the ability of the oligonucleotide to induce the inhibition of the inclusion of the cryptic exon of intron 22. In some embodiments, the chemical modification increases the half-life of the oligonucleotide. In some embodiments, the chemical modification inhibits the extension of the polymerase from the 3' end of the oligonucleotide. In some embodiments, the chemical modification inhibits the recognition of the oligonucleotide by the polymerase. In some embodiments, the chemical modification inhibits the degradation of double-stranded triggering. In some embodiments, the chemically modified oligonucleotide does not trigger the degradation of nucleic acid double strands when binding to CFTR pre-mRNA. In some embodiments, the chemical modification inhibits the degradation mediated by RISC. In some embodiments, the chemical modification inhibits the degradation mediated by RISC or any parallel nucleic acid degradation pathway.

[0090] In some embodiments, oligonucleotide lacks labeling part.In some embodiments, oligonucleotide is not labeled.In some embodiments, oligonucleotide does not send detectable signal or does not include the part that can be identified so that nucleic acid detection (for example, digoxin and fluorescently labeled anti-DIG antibody) can be carried out.In some embodiments, detectable signal includes dye or emission energy, which provides the detection of compound such as polynucleotide in vivo, in vitro or in vitro.In some embodiments, detectable signal includes: fluorescent signal, color signal or radioactive signal.

[0091] In some embodiments, the oligonucleotide lacks a radionucleobase; digoxigenin, streptavidin, biotin, a fluorophore, a hapten label, a CLICK label, an amine label, or a thiol label.

[0092] In certain embodiments, consecutive nucleotide bases are connected by a backbone selected from the group consisting of: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a 2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethylphosphorothioate backbone, an alternating locked nucleic acid backbone, a constrained ethyl backbone, and a phosphorothioate backbone. Each possibility represents a separate embodiment of the present invention.

[0093] According to some embodiments, the oligonucleotide comprises a 2'-O-methyl-phosphorothioate backbone. According to other embodiments, the oligonucleotide comprises a 2'-methoxyethyl (2'MOE) modification.

[0094] In some embodiments, the oligonucleotide comprises a 2'-O-methyl-phosphorothioate modification. In some embodiments, the oligonucleotide comprises a 2'MOE modification. In some embodiments, the modification is throughout the entire molecule. In some embodiments, the modification is at the 3' end of the molecule. In some embodiments, the modification is at the 5' end of the molecule. In some embodiments, the molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 modifications. Each possibility represents a separate embodiment of the present invention.

[0095] According to some embodiments, oligonucleotide modification can fully restore CFTR function compared to unmutated CFTR. According to certain embodiments, oligonucleotide modification can restore at least 70%, 75%, 80%, 85%, 90% or 95% CFTR function compared to unmutated CFTR. Each possibility represents a separate embodiment of the present invention.

[0096] In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 1. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 2. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 3. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 4. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 5. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 6. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 7. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:8 or consists of the nucleotide sequence listed in SEQ ID NO:8. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:9 or consists of the nucleotide sequence listed in SEQ ID NO:9. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:10 or consists of the nucleotide sequence listed in SEQ ID NO:10. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:11 or consists of the nucleotide sequence listed in SEQ ID NO:11. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:12 or consists of the nucleotide sequence listed in SEQ ID NO:12. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:13 or consists of the nucleotide sequence listed in SEQ ID NO:13. In certain embodiments, the oligonucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:14 or consists of the nucleotide sequence listed in SEQ ID NO:14. In certain embodiments, the oligonucleotide molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:15.In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 16. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 17. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 18. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 19. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 20. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 21. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 22. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:23. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:24. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:25. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:40. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:41. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:42. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:43. In certain embodiments, the oligonucleotide molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:44.In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:45 or consists of a nucleotide sequence listed in SEQ ID NO:45. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:46 or consists of a nucleotide sequence listed in SEQ ID NO:46. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:47 or consists of a nucleotide sequence listed in SEQ ID NO:47. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:48 or consists of a nucleotide sequence listed in SEQ ID NO:48. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:49 or consists of a nucleotide sequence listed in SEQ ID NO:49. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:50 or consists of a nucleotide sequence listed in SEQ ID NO:50. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in SEQ ID NO:51 or consists of a nucleotide sequence listed in SEQ ID NO:51. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:52. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:53. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:54. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:55. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:56. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:57. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:58. In certain embodiments, the oligonucleotide molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:59.In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:60. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:61. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:62. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:63. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:64. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:65. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO:66. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 67. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 68. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 69. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 70. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 71.

[0097] In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence set forth in one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 41. In certain embodiments, the oligonucleotide molecule consists of a nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 41. Each possibility represents a separate embodiment of the present invention.

[0098] In some embodiments, the oligonucleotide is specific for CFTR precursor mRNA. As used herein, the term "specificity" refers to both base pair specificity and gene specificity. In some embodiments, the oligonucleotide is specific for the CFTR gene. In some embodiments, the oligonucleotide is specific for the intron sequence of CFTR. In some embodiments, the oligonucleotide is specific for the secret exon in CFTR. In some embodiments, the oligonucleotide is specific for the nucleic acid sequence of intron 22 of CFTR. In some embodiments, the nucleic acid sequence of intron 22 is the intron 22 secret exon of CFTR.

[0099] In some embodiments, the oligonucleotide binds to CFTR pre-mRNA with perfect complementarity. In some embodiments, the oligonucleotide does not bind to any gene other than CFTR with perfect complementarity. In some embodiments, the oligonucleotide does not bind to any gene other than CFTR with greater than 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the oligonucleotide does not bind to any gene other than CFTR with greater than 90% complementarity. In some embodiments, the oligonucleotide binds to SEQ ID NO:37 with perfect complementarity. In some embodiments, the oligonucleotide does not bind to any sequence other than SEQ ID NO:37 with perfect complementarity. In some embodiments, the oligonucleotide does not bind to any sequence other than SEQ ID NO:37 with greater than 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the oligonucleotide does not bind to any sequence other than SEQ ID NO:37 with greater than 90% complementarity. In some embodiments, the oligonucleotide does not bind to any position in the cell genome except CFTR with complete complementarity. In some embodiments, the oligonucleotide does not bind to any position in the cell genome except CFTR with greater than 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammal is a human.

[0100] In some embodiments, the oligonucleotide regulates the expression of CFTR. In some embodiments, the oligonucleotide regulates the splicing of CFTR. In some embodiments, the oligonucleotide regulates the splicing of the intron 22 secret exons of CFTR, such as suppressing the inclusion of the intron 22 secret exons of CFTR. In some embodiments, the oligonucleotide does not cause off-target effects. In some embodiments, off-target is a target other than CFTR. In some embodiments, off-target is a target other than the splicing of the intron 22 secret exons of CFTR (for example, suppressing the inclusion of the intron 22 secret exons of CFTR). In some embodiments, the oligonucleotide does not substantially or significantly regulate the expression of genes other than CFTR. In some embodiments, the oligonucleotide does not substantially or significantly regulate the splicing of genes other than CFTR. In some embodiments, the oligonucleotide does not substantially or significantly regulate the splicing of exons other than the intron 22 secret exons of CFTR. In some embodiments, the substantial modulation of expression is a change of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% expression. Each possibility represents a separate embodiment of the present invention. In some embodiments, the substantial modulation of expression is a change of at least 20% expression.

[0101] In some embodiments, oligonucleotides as disclosed herein are targeted, complementary, inhibited, or any combination thereof for inclusion of intron 22 cryptic exons to mature CFTR mRNA transcribed from the mutant allele of the CFTR gene. In some embodiments, oligonucleotides as disclosed herein are not targeted, complementary, inhibited, or any combination thereof for splicing regulation of CFTR precursor mRNA transcribed from the wild-type allele of the CFTR gene. In some embodiments, oligonucleotides as disclosed herein are targeted, complementary, inhibited, or any combination thereof for inclusion of intron 22 cryptic exons to mature CFTR mRNA transcribed from the mutant allele of the CFTR gene more effectively at least 2 times, more effectively at least 3 times, more effectively at least 5 times, more effectively at least 7 times, more effectively at least 10 times, more effectively at least 20 times, more effectively at least 50 times, or more effectively at least 100 times, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the oligonucleotides as disclosed herein are 2-10 times more effective, 3-50 times more effective, 5-100 times more effective, 7-20 times more effective, 2-40 times more effective, 2-25 times more effective, 50-150 times more effective, or 2-100 times more effective for targeting, complementation, inhibition, or any combination thereof of the inclusion of the intron 22 cryptic exon to mature CFTR mRNA transcribed from a mutant allele of a CFTR gene compared to a wild-type allele of a CFTR gene. Each possibility represents a separate embodiment of the invention.

[0102] In some embodiments, the oligonucleotides of the present invention are fully complementary to the mutant alleles of the CFTR gene. As used herein, the term "fully complementary" refers to 100% hybridization, which means that the mutant CFTR allele and the oligonucleotide represent versions of nucleic acid sequences that are reverse and complementary to each other, as will be apparent to those of ordinary skill in the field of molecular biology. In some embodiments, the oligonucleotides of the present invention are partially complementary to the wild-type alleles of the CFTR gene. As used herein, the term "partially" refers to any value or range less than 100%. In some embodiments, the oligonucleotides of the present invention and the wild-type CFTR allele represent versions of nucleic acid sequences that are reverse and complementary to each other, and they differ by at least one nucleotide, for example, comprising at least one mismatched nucleotide.

[0103] In some embodiments, the oligonucleotides of the invention and methods of use thereof provide for the exclusion of cryptic exons from mature CFTR mRNA transcribed from a mutant allele of a CFTR gene. In some embodiments, the mature mRNA transcribed from the wild-type allele lacks cryptic exons.

[0104] In some embodiments, the cryptic exon is an intron 22 cryptic exon. In some embodiments, the cryptic exon is 80-90 bases in length.

[0105] In some embodiments, the oligonucleotide comprises an active fragment of any one of SEQ ID Nos: 1-25 and SEQ ID Nos: 41-44.

[0106] In some embodiments, the oligonucleotide comprises an active fragment of any one of SEQ ID Nos: 1-5 and SEQ ID No:41.

[0107] As used herein, the term "active fragment" refers to a fragment that is 100% identical to a continuous portion of a complete nucleotide sequence of an oligonucleotide, provided that at least: 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the original oligonucleotide sequence is retained, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0108] In some embodiments, the subject is heterozygous for the 3849+10Kb C-to-T mutation. In some embodiments, the subject treated according to the methods of the invention comprises or is characterized by a mixture of a wild-type full-length and fully functional CFTR protein encoded by a wild-type allele and a full-length and fully functional CFTR protein encoded by a pre-mRNA (wherein the inclusion of the intron 22 cryptic exon is suppressed using the oligonucleotides of the invention). In some embodiments, the oligonucleotides of the invention do not reduce the level of a wild-type full-length and fully functional CFTR protein in a subject (e.g., heterozygous for the mutations disclosed above).

[0109] In another aspect, the present invention also provides a pharmaceutical composition comprising the synthetic oligonucleotide molecule as described above and a pharmaceutically acceptable carrier.

[0110] The term "pharmaceutically acceptable carrier" as used herein refers to any standard pharmaceutical carrier known in the art, such as sterile solutions, tablets, coated tablets and capsules. Typically, such carriers include excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or its salts, magnesium stearate or calcium stearate, talc, vegetable fats or oils, gums, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Examples of pharmaceutically acceptable carriers include, but are not limited to, the following: water, saline, buffer, inert non-toxic solids (e.g., mannitol, talc). Compositions comprising such carriers are prepared by well-known conventional methods. Depending on the intended mode of administration and intended use, the composition may be in the form of a solid, semisolid or liquid dosage form, such as, for example, powders, particles, crystals, liquids, suspensions, liposomes, nanoparticles, nanoemulsions, pastes, creams, ointments, etc., and may be in a unit dosage form suitable for administration of relatively precise doses.

[0111] In certain embodiments, the pharmaceutical composition is formulated for oral administration. In certain embodiments, the pharmaceutical composition is formulated for nasal administration. In certain embodiments, the pharmaceutical composition is formulated for administration by inhalation. In certain embodiments, the pharmaceutical composition is formulated for abdominal administration. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In certain embodiments, the pharmaceutical composition is formulated for intraperitoneal administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0112] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the subject is a human subject. It will be appreciated by the technician that the pharmaceutical composition intended to be administered to the subject should not have an off-target effect, i.e., an effect other than the expected therapeutic effect. In some embodiments, the pharmaceutical composition lacks a substantial effect on genes other than CFTR. In some embodiments, the pharmaceutical composition lacks any substantial effect except that the cryptic exons of intron 22 are included in mature CFTR. In some embodiments, the substantial effect is an effect with phenotypic results. In some embodiments, the substantial effect is a harmful effect. In some embodiments, harmful is for the health and / or happiness of the subject.

[0113] In some embodiments, the composition is administered by inhalation. In some embodiments, the composition is an inhalation composition. In some embodiments, the composition is a pharmaceutical composition.

[0114] As a well-known and well-studied disease, certain drugs and agents are known in the art for treating cystic fibrosis patients. Administering the synthetic polynucleotide molecules according to the present invention together with one or more of these drugs can be beneficial in obtaining significant therapeutic results.

[0115] In certain embodiments, the pharmaceutical composition further comprises one or more CFTR modifying agents.

[0116] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more CFTR modifying agents.

[0117] In some embodiments, the CFTR modifier increases the duration of CFTR gate opening, chloride flux through the CFTR gate, proper folding of the CFTR protein, the amount of CFTR anchored to the cell membrane, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0118] In some embodiments, the modifier is selected from the group consisting of: an enhancer, a corrector, and an amplifier.

[0119] As used herein, the term "enhancing agent" refers to any agent that increases the likelihood that a defective CFTR will open and thereby allow chloride ions to pass through the channel pore.

[0120] As used herein, the term "correcting agent" refers to any agent that aids in proper folding of the CFTR channel so as to enable its transport to the cell membrane.

[0121] As used herein, the term "amplifying agent" refers to any agent that induces a cell to increase its rate of production or production of CFTR protein, thereby resulting in an increase in the amount of CFTR protein.

[0122] In certain embodiments, the CFTR modifier is selected from the group consisting of: a CFTR splicing regulator, a translation read-through agent, a CFTR amplifier, a CFTR enhancer, and a CFTR corrector. In certain embodiments, the CFTR splicing regulator is a CFTR modulator that is capable of inhibiting the expression of mature CFTR. The invention relates to a method for preparing a CFTR enhancer comprising different synthetic oligonucleotide molecules for inclusion of a cryptic exon of intron 22 in mRNA; a translation readthrough agent selected from the group consisting of 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) and ELX-02; a CFTR amplifier is PTI-428; a CFTR enhancer is selected from the group consisting of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (avacaftor), QBW251, PTI-808 and VX-561 (deuterated avacaftor); a CFTR enhancer is N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide ( or a CFTR corrector selected from the group consisting of 3-{6-{[1-(2,2-difluoro-1,3-benzobisoxazol-5-yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid (Lumacato), 1-(2,2-difluoro-1,3-benzobisoxazol-5-yl)-~{N}-[1-[(2~{R})-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropane-2-yl)indol-5-yl]cyclopropane-1-carboxamide (Tezacaftor), VX-659, VX-445, VX-152 and VX-440, GLPG2222, FDL169 and PTI-801.

[0123] In certain embodiments, the pharmaceutical composition comprises at least about 1 nM of a synthetic oligonucleotide molecule. In certain embodiments, the pharmaceutical composition comprises at least about 2.5 nM of a synthetic oligonucleotide molecule. In certain embodiments, the pharmaceutical composition comprises at least about 10 nM of a synthetic oligonucleotide molecule. In certain embodiments, the pharmaceutical composition comprises 2.5 nM to 10 nM of a synthetic oligonucleotide molecule.

[0124] In certain embodiments, the pharmaceutical composition comprises 1 nM to 1 μM of a synthetic oligonucleotide molecule. In certain embodiments, the pharmaceutical composition comprises 1 nM to 0.5 μM of a synthetic oligonucleotide molecule. In certain embodiments, the pharmaceutical composition comprises 1 nM to 100 nM of a synthetic oligonucleotide molecule.

[0125] In another aspect, the present invention also provides a synthetic oligonucleotide molecule as described above, or a pharmaceutical composition as described above, for use in regulating the splicing of CFTR pre-mRNA transcribed from a CFTR gene having a 3849+10Kb C-to-T mutation.

[0126] As used herein, the phrase "regulation of splicing" refers to changes in the level of any RNA or mRNA variant that affects the production of CFTR natural precursor mRNA. For example, regulation can mean, for example, causing an increase or decrease in abnormal CFTR mRNA levels, causing an increase or decrease in normal full-length CFTR mRNA levels, and / or causing an increase or decrease in abnormal CFTR RNA or mRNA levels containing premature stop codons (nonsense codons). It is therefore apparent that any changes in the ratio between certain CFTR splice variants are also considered to be the result of splicing regulation. Each possibility represents a separate embodiment of the present invention. In certain embodiments, regulation means increasing the level of normal full-length CFTR mRNA and / or reducing the level of abnormal CFTR mRNA.

[0127] In certain embodiments, the use is for reducing the level of mRNA molecules comprising the cryptic exon of intron 22. In certain embodiments, the use is for reducing the level of mRNA molecules comprising the nucleotide sequence listed in SEQ ID NO:36. In certain embodiments, the use is for increasing the level of normal full-length CFTR mRNA. In certain embodiments, the use is for increasing the level of mRNA molecules comprising the nucleotide sequence listed in SEQ ID NO:34. In certain embodiments, the use is for correcting or improving chloride transport through CFTR channels. In certain embodiments, the use is for increasing the production of functional CFTR proteins. Each possibility represents a separate embodiment of the present invention.

[0128] In another aspect, the present invention also provides a synthetic oligonucleotide molecule as described above or a pharmaceutical composition as described above for use in a method for improving at least one clinical parameter of cystic fibrosis.

[0129] In another aspect, the present invention also provides a method for improving at least one clinical parameter of cystic fibrosis in a patient in need thereof, the method comprising the step of administering to the patient a therapeutically effective amount of a synthetic polynucleotide molecule as described above.

[0130] As used herein, the term "therapeutically effective amount" refers to the amount necessary to improve at least one clinical parameter of cystic fibrosis in a patient or to reduce the severity of at least one clinical parameter of cystic fibrosis. Therapeutically effective amounts can vary depending on the patient's condition, the route of administration of the synthetic polynucleotide molecule, the use of excipients, and the co-use of other active agents.

[0131] In certain embodiments, the clinical parameter is selected from the group consisting of: lung function, time to first pulmonary exacerbation, change in weight, change in height, change in body mass index (BMI), change in sweat chloride concentration, number and / or duration of pulmonary exacerbations, total number of days hospitalized for pulmonary exacerbations, and requirement for antibiotic therapy for sinopulmonary signs or symptoms. Each possibility represents a separate embodiment of the present invention.

[0132] As used herein, the terms "treatment" or "treating" a disease, disorder, or condition include alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment need not mean that the disease, disorder, or condition is completely cured. To be effective in treatment, the compositions useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0133] As used herein, the term "condition" includes anatomical and physiological deviations from the normal state, which constitute an impairment of the normal state of a living animal or a part thereof, and manifestations of interrupted or altered bodily function.

[0134] As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of therapy, particularly a mammalian subject, such as a human.

[0135] As a genetic disease, cystic fibrosis cannot be cured at present, but its clinical manifestations and / or symptoms can be treated by the oligonucleotides of the present invention to significantly enhance and / or improve the clinical status and quality of life of patients.

[0136] As used herein, the term "improvement" refers to a favorable change in a clinical parameter of cystic fibrosis, i.e., an increase or decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.

[0137] Different AO delivery routes have been examined in animal models and applied to clinical trials, mainly selected according to target tissues. For example, 2OMP is administered to DMD patients (PRO-051) by local intramuscular injection (van Deutekom et al., 2007) and abdominal subcutaneous injection (Goemans et al., 2011). 2OMP is also administered to SMA mouse models (Williams et al., 2009; Hua et al., 2010) by intraventricular injection. PMO is administered to DMD mouse models by intramuscular injection (Gebski, Mann, Fletcher & Wilton, 2003) and repeated weekly intraperitoneal injections (repeated weakly intraperitonealinjection) (Goyenvalle et al., 2010). PMO has also been administered to SMA mouse models by intracerebroventricular injection (Porensky et al., 2012) and to DMD patients (AVI-4658) by local intramuscular injection (Kinali et al., 2009) or intravenous administration (Cirak et al., 2011; Mendell et al., 2013).

[0138] In certain embodiments, the method further comprises administering to the patient at least one additional anti-cystic fibrosis agent. In certain such embodiments, additional anti-cystic fibrosis agents are selected from the group consisting of CFTR splicing regulators, CFTR enhancers, and CFTR correctors. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the administration of the synthetic polynucleotide molecules of the present invention of a therapeutically effective amount and the administration of at least one additional anti-cystic fibrosis agent are independently oral, nasal, aerosol, inhaled, abdominal, subcutaneous, intraperitoneal, or intravenous administration. Each possibility represents a separate embodiment of the present invention. It should be understood that the selection of route of administration depends on the properties of the therapeutic agent and the site of its intended action, and therefore certain agents can be administered via the same or different routes of administration.

[0139] In certain embodiments, administration of the synthetic oligonucleotide molecule or pharmaceutical composition is oral, nasal, inhaled, abdominal, subcutaneous, intraperitoneal, or intravenous.

[0140] In certain embodiments, synthetic oligonucleotide molecule is administered with a concentration of at least about 1nM. In certain embodiments, synthetic oligonucleotide molecule is administered with a concentration of at least about 2.5nM. In certain embodiments, synthetic oligonucleotide molecule is administered with a concentration of at least about 10nM. In certain embodiments, synthetic oligonucleotide molecule is administered with a concentration of 2.5nM to 10nM.

[0141] In another aspect, the present invention also provides use of the synthetic oligonucleotide molecule as described above or the pharmaceutical composition as described above in the preparation of a medicament.

[0142] In certain embodiments, the drug is used to treat or improve the symptoms of cystic fibrosis. In some embodiments, the drug improves at least one clinical parameter of cystic fibrosis. According to some embodiments, the clinical parameter is selected from the group consisting of: lung function, time to first pulmonary exacerbation, change in weight, change in height, change in body mass index (BMI), change in sweat chloride concentration, number and / or duration of pulmonary exacerbations, total days of hospitalization for pulmonary exacerbations, and the need for antibiotic therapy for sinopulmonary signs or symptoms. Each possibility represents a separate embodiment of the present invention.

[0143] In another aspect, the present invention also provides a kit comprising the synthetic oligonucleotide molecule as described above.

[0144] In certain embodiments, the kit further comprises an additional anti-cystic fibrosis agent.

[0145] In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are contained in one pharmaceutical composition. In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are contained in different pharmaceutical compositions. In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are independently formulated for oral, nasal, inhaled, abdominal, subcutaneous or intraperitoneal administration. Each possibility represents a different embodiment of the present invention.

[0146] In certain embodiments, the synthetic oligonucleotide is at a concentration of at least about 1 nM. In certain embodiments, the synthetic oligonucleotide is at a concentration of at least about 2.5 nM. In certain embodiments, the synthetic oligonucleotide is at a concentration of at least about 10 nM. In certain embodiments, the synthetic oligonucleotide molecule is at a concentration of 2.5 nM to 10 nM.

[0147] In another aspect, the present invention also provides a synthetic oligonucleotide molecule consisting of 18-50 consecutive bases, complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C-to-T mutation, and at least partially inhibiting the inclusion of the cryptic exon of intron 22 in the mature CFTR mRNA, comprising the nucleotide sequence listed in one of SEQ ID NO:1 to SEQ ID NO:25 and SEQ ID NO:41 to SEQ ID NO:44.

[0148] In another aspect, the present invention also provides a synthetic oligonucleotide molecule consisting of 17-50 consecutive bases, complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C-to-T mutation, and at least partially inhibiting the inclusion of the cryptic exon of intron 22 in the mature CFTR mRNA, comprising a nucleotide sequence listed in one of SEQ ID NO:1 to SEQ ID NO:25 and SEQ ID NO:40 to SEQ ID NO:71.

[0149] In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in one of SEQ ID NO: 1 to SEQ ID NO: 10. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 41. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 41. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 41. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence listed in SEQ ID NO: 7.

[0150] In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:40 to SEQ ID NO:71. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:40 to SEQ ID NO:48. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:49 to SEQ ID NO:53. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:54 to SEQ ID NO:58. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:59 to SEQ ID NO:63. In certain embodiments, the oligonucleotide molecule comprises a nucleotide sequence listed in one of SEQ ID NO:54 to SEQ ID NO:71.

[0151] In another aspect, the present invention also provides a synthetic oligonucleotide sequence consisting of 18-20 consecutive bases, comprising the sequence GAUGGAAGA (SEQ ID NO: 38), wherein the synthetic oligonucleotide sequence is complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C- to -T mutation.

[0152] In another aspect, the present invention also provides a synthetic oligonucleotide sequence consisting of 17-20 consecutive bases, comprising the sequence GAUGGAAGA (SEQ ID NO: 38), wherein the synthetic oligonucleotide sequence is complementary to the pre-mRNA transcript of the CFTR gene having a 3849+10Kb C- to -T mutation.

[0153] In certain embodiments, the oligonucleotide sequence is complementary to the nucleotide sequence within SEQ ID NO: 37. In certain embodiments, the oligonucleotide sequence is selected from the group consisting of: SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 4, and SEQ ID No: 7. In certain embodiments, the synthesized oligonucleotide sequence comprises the sequence CAACAGAUGGAAGA (SEQ ID NO: 39). In certain embodiments, the sequence is selected from the group consisting of: SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 4.

[0154] In one embodiment, the invention provides a combination product. In one embodiment, a "combination product" particularly defines a "kit of parts", meaning that the combination partner as defined above can be administered independently or by using different fixed combinations with different amounts of combination partners, i.e., simultaneously, jointly, separately or sequentially. In some embodiments, the components in the kit of parts can then be administered, for example, simultaneously or chronologically staggered, i.e., any component in the kit of parts is administered at different time points and at the same or different time intervals. In some embodiments, the ratio of the total amount of the combination partner can be achieved in the combination product.

[0155] In some embodiments, the kit of the present invention comprises: at least one oligonucleotide; and at least one of the following: at least one CFTR modifier; or at least one CF drug, wherein the oligonucleotide is selected from SEQ ID No: 1-25 and SEQ ID No: 41-44, and wherein the CFTR modifier is selected from: a CFTR enhancer, a CFTR corrector, and a CFTR amplifier.

[0156] In some embodiments, the CF medication is an antibiotic medication, a bronchodilator, a corticosteroid, or any combination thereof.

[0157] The types and dosages of CF drugs such as antibiotics, bronchodilators, and corticosteroids will be apparent to those of ordinary skill in the art. Non-limiting examples of CF drugs such as antibiotics include, but are not limited to, cloxacillin, dicloxacillin, cephalosporins, trimethoprim, sulfamethoxazole, erythromycin, amoxicillin, clavulanic acid, ampicillin, tetracycline, linezolid, tobramycin or aztreonam lysine, fluoroquinolones, gentamicin, and monolactams with anti-pseudomonal activity.

[0158] In some embodiments, the components of the kit disclosed above are sterile. As used herein, the term "sterile" refers to a state free of biological contaminants. Any sterilization method is applicable and will be apparent to those of ordinary skill in the art.

[0159] In some embodiments, the components of the kit are packaged within containers.

[0160] In some embodiments, the container is made of a material selected from the group consisting of thin-walled films or plastics (transparent or opaque), paperboard-based foils, rigid plastics, metals (eg, aluminum), glass, and the like.

[0161] In some embodiments, as described below, the contents of the kit are packaged to allow storage of the components until they are needed.

[0162] In some embodiments, some or all of the components of the kit may be packaged in suitable packaging to maintain sterility.

[0163] In some embodiments, the components of the kit are stored in separate containers within the main kit containing element, such as a box or similar structure, which may or may not be airtight containers, such as to further maintain the sterility of some or all components of the kit.

[0164] In some embodiments, the instructions for use can be recorded on a suitable recording medium or substrate. For example, the instructions for use can be printed on a substrate such as paper or plastic.

[0165] In some embodiments, instructions for use can be present in the medicine box as a package insert, present in the label of the container of the medicine box or its component (that is, being associated with packaging or sub-packaging), etc. In other embodiments, instructions for use exist as an electronic storage data file present in a suitable computer-readable storage medium such as a CD-ROM, a floppy disk, etc. In other embodiments, there are no actual instructions for use in the medicine box, but the means for obtaining instructions for use from a remote resource via the Internet are provided. The example of this embodiment is a medicine box comprising a website, and instructions for use can be checked at this website and / or instructions for use can be downloaded from this website. The same as instructions for use, this means for obtaining instructions for use is recorded on a suitable substrate.

[0166] According to some embodiments, a method for producing a compound suitable for treating CF is provided.

[0167] In some embodiments, the method comprises obtaining a compound that binds to intron 22 of CFTR pre-mRNA. In some embodiments, the method comprises obtaining a compound that binds to SEQ ID NO: 37. In some embodiments, the method comprises determining the inclusion of the intron 22 cryptic exon in the mature CFTR mRNA in the presence of the obtained compound, and selecting at least one compound that inhibits the inclusion of the intron 22 cryptic exon in the mature CFTR mRNA, thereby producing a compound suitable for treating CF.

[0168] In some embodiments, the compound is an oligonucleotide. In some embodiments, the oligonucleotide is an oligonucleotide as disclosed and described herein.

[0169] Methods for determining the inclusion of cryptic exons are commonly used. Non-limiting examples of such methods include, but are not limited to, PCR, qPCR, gene sequencing, Northern blot, dot blot, in situ hybridization or others, all of which will be apparent to those of ordinary skill in the art.

[0170] In the discussion, unless otherwise indicated, adjectives such as "substantially" and "about" that modify a condition or relational characteristic of one or more features of an embodiment of the present invention should be understood to mean that the condition or characteristic is defined to be within an acceptable tolerance for the operation of the embodiment (for its intended application). Unless otherwise indicated, the word "or" in the specification and claims is considered to be an inclusive "or", not an exclusive "or", and indicates at least one or any combination of the items it combines.

[0171] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the listed components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless otherwise specifically stated. Therefore, the terms "a", "an" and "at least one" are used interchangeably in this application.

[0172] In order to better understand the present teachings, and in no way limit the scope of the present teachings, unless otherwise indicated, all numbers indicating amounts, percentages or ratios and other numerical values ​​used in the specification and claims should be understood as being modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties attempted to be obtained. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying ordinary rounding techniques.

[0173] In the specification and claims of this application, each of the verbs "comprise," "include," and "have," and their variations, are used to indicate that one or more objects of the verb are not necessarily a complete list of components, elements, or parts of one or more subjects of the verb.

[0174] Other terms as used herein are intended to be defined by their commonly known meanings in the art.

[0175] Unless otherwise stated or obvious from the context, as used herein, the term "or" is to be understood to be inclusive.

[0176] Throughout the present description and claims, the word "comprise" or variations such as "comprises" or "comprising" indicates the inclusion of any listed integer or group of integers but not the exclusion of any other integer or group of integers.

[0177] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that does not materially change the basic or novel property of a specified method, structure, or composition.

[0178] As used herein, the terms "comprises," "comprising," "containing," "having," and the like may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially of" likewise have the meaning ascribed to U.S. patent law, and the terms are open ended, allowing for more than what is recited, as long as the basic or novel properties recited are not altered by the more than what is recited, but excluding prior art embodiments. In one embodiment, the terms "comprises," "comprising," "having," and "consisting" are interchangeable.

[0179] After studying the following examples which are not intended to be limiting, additional objects, advantages and novel features of the present invention will become apparent to those of ordinary skill in the art. In addition, each of the various embodiments and aspects of the present invention as described above and as claimed in the following claims finds experimental support in the following examples.

[0180] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination, or suitably in any other described embodiment of the invention. Certain features described in the context of multiple embodiments are not considered essential features of those embodiments, unless the embodiment does not function without those elements.

[0181] The terms "comprises," "comprising," "includes," "including," "having," and variations thereof mean "including but not limited to." The term encompasses the terms "consisting of" and "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include more than one compound, including mixtures thereof.

[0182] The following examples are intended to be construed as non-limiting to the scope of the present invention and serve merely as illustrative embodiments.

[0183] After studying the following examples which are not intended to be limiting, additional objects, advantages and novel features of the present invention will become apparent to those of ordinary skill in the art. In addition, each of the various embodiments and aspects of the present invention as described above and as claimed in the following claims finds experimental support in the following examples. Example

[0184] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, and cell biological techniques. Such techniques are explained fully in the literature. See, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM ed. (1994); "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE ed. (1994); The Organic Chemistry of Biological Pathways, John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions, Richard Silverman (Academic Press, 2002); Organic Chemistry (6th Edition), Leroy "Skip" G Wade; Organic Chemistry, TW Graham Solomons and Craig Fryhle.

[0185] Table 1. Antisense oligonucleotides (ASOs).

[0186]

[0187]

[0188] Table 2. CFTR-related sequences (all in sense orientation).

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] According to the assembly version used in the 2013 update (UCSC Genome Browser on Human December 2013 (GRCh38 / hg38)), it corresponds to positions 17279930-117280013 in the genome.

[0195] Materials and methods

[0196] Fisher Rat Thyroid (FRT) Cell System

[0197] In order to screen the antisense oligonucleotide (ASO) regulating the splicing of 3849+10kb C-to-T mutation CFTR, a cell screening system is needed. Because the epithelial cells (nasal or bronchial epithelial cells) from patients are difficult to grow, and have limited proliferation capacity, the inventors have produced fisher rat thyroid (FRT) cells that stably express the CFTR cDNA carrying 3849+10kb C-to-T mutation (FRT 3849mut). The cDNA is transfected into FRT cells using the Flp-In system (Invitrogen), which allows integration at a specific genomic position. It is noteworthy that FRT Flp-In cells are widely used in the analysis of CFTR, and can be used for RNA and functional analysis. In order to study the impact of ASO on splicing, cDNA is cloned to include a minigene containing an intron sequence near an exon of interest. As a positive control, FRT cells (FRT 3849WT) containing a cDNA with a minigene carrying a wild-type (WT) sequence (C in the mutation site) are also produced.

[0198] Transfection

[0199] Each ASO was transfected into FRT 3849 mutant cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the lipofectamine 2000 reagent protocol, using the following lipofectamine amounts: 96-well - 0.15 μl, 6-well - 3 μl, 10 mm plate - 15 μl. In each experiment, the effect of the different ASOs was analyzed compared to cells treated with a control ASO.

[0200] RNA extraction

[0201] Twenty-four (24) hours after transfection of FRT 3849 mutant cells, total RNA was extracted using the RNeasy Mini Kit (QIAGEN). RNA concentration was determined using nanodrop. RNA-less and reverse transcriptase-less reactions were used as controls. Complementary DNA (cDNA) synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). cDNA was analyzed by PCR and qPCR.

[0202] Quantitative detection of aberrantly spliced ​​CFTR transcripts (qPCR)

[0203] Real-time PCR is performed on the QuantStudio 12K Flex Real-Time PCR System Rapid Advanced Master Mix (Applied Biosystems) was performed with a TaqMan probe specific for the 84 bp cryptic exon. Expression levels were normalized to the transcript level of HPRT. Two technical duplicates were analyzed for each sample. Analysis was performed using dual ΔCt analysis.

[0204] Determine the ratio between these two transcripts (PCR)

[0205] Using Platinum TM SuperFi TM PCR was performed on a 1:2.5 dilution of cDNA using Green PCR master mix 12359-10 (Invitrogen), with 2 μl of diluted cDNA per reaction. The PCR products were then run on an agarose gel to detect correctly spliced ​​transcripts and abnormally spliced ​​transcripts. The gel was exposed to UV light to visualize the PCR products and an image was generated. The fraction of correctly spliced ​​transcripts was quantified using image J.

[0206] Half maximal effective concentration (EC50) analysis

[0207] FRT cells were transfected with each primary ASO at six concentrations (0.1 nM, 1 nM, 2.5 nM, 10 nM, 50 nM and 100 nM). For concentrations below 100 nM, a control ASO was added to complete a total concentration of 100 nM. In each experiment, the effects of the three different ASOs were analyzed compared to cells treated with 100 nM control ASO.

[0208] CFTR protein analysis by Western blotting

[0209] For protein analysis, FRT 3849 mutant cells were transfected with 10 nM primary ASO or control ASO every 24 hours for 3 days. Twenty-four (24) hours after the last transfection, proteins were extracted using RIPA buffer and analyzed by immunoblotting with CFTR antibodies. 6% polyacrylamide gel was used for protein separation. The gel was transferred to a nitrocellulose membrane and antibody hybridization and chemiluminescence were performed according to standard procedures. The primary antibodies used in this analysis were mouse anti-CFTRM3A7 (Millipore) and rabbit anti-calnexin (Sigma). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.

[0210] CFTR Functional Studies Using Membrane Potential Assays

[0211] FRT 3849 mutant cells were grown in 96-well (black, flat-bottomed; corning) plates. 24h after transfection of different ASOs, as previously described, CFTR channel function was analyzed using FLIPR membrane potential assay. In brief, cells were loaded with blue membrane potential dye (Molecular Devices), which can detect changes in transmembrane potential. The baseline level of the plate was then read in a fluorescent plate reader (BioTek Synergy H1), followed by CFTR stimulation using cAMP agonist forskolin (10 μM; Sigma), and DMSO vehicle was used as a negative control. CFTR-mediated plasma membrane depolarization was detected as an increase in fluorescence, and hyperpolarization (or repolarization) was detected as reduction. In order to terminate functional assays, CFTR inhibitor CFTRinh-172 (10 μM; Cystic Fibrosis Foundation Therapeutics) was added to each well. The change in transmembrane potential was normalized to the value before activation.

[0212] Sampling and culture of primary nasal epithelial cells

[0213] Human nasal endothelial cells (HNE cells) were sampled by nasal brushing of both nostrils. To increase the number of filters available for testing the efficacy of correctors, the inventors used conditionally reprogrammed and redifferentiated HNE cells. The HNE cells were then seeded on porous filters (0.33 cm 2 , Transwell, Corning) and supplemented with culture medium. After 2 days, cells were cultured at the air-liquid interface for 2-4 weeks. ASO dissolved in DDW was added to the basolateral side of HNE during differentiation.

[0214] Ussing Room Research

[0215] After differentiation, CFTR channels of HNE cells were analyzed using the Ussing chamber system as previously described (Pranke et al., 2017). In general, short-circuit current (Isc) was measured under voltage-clamp conditions using an EVC4000 Precision V / I clamp (World Precision Instruments). For all measurements, a transepithelial chloride concentration gradient was applied by varying the composition of basal and apical Ringer's solutions. After the baseline Isc stabilized, inhibitors and activators were added: sodium (Na+) channel blocker amiloride (100 μM) was added to inhibit the apical epithelial Na+ channel (ENaC); cAMP agonist forskolin (10 μM) was added to activate transepithelial cAMP-dependent currents (including Cl- transport through CFTR channels); VX-770 (10 μM) was added to enhance CFTR activity; CFTR inhibitor CFTRinh172 (5 μM) was added to specifically inhibit CFTR; and ATP (100 μM) was added to challenge purinergic calcium-dependent Cl- secretion. Changes after forskolin were used as indicators of CFTR.

[0216] Example 1

[0217] Novel antisense oligonucleotides (ASOs)

[0218] Twenty-six (26) novel antisense oligonucleotides (ASOs) were analyzed for their effects on splicing of CFTR pre-mRNA including the mutation 3849+10Kb C-to-T. The ASOs were modified with 2'-O-methyl phosphorothioate (2'OMP). Briefly, Fischer rat thyroid (FRT) cells were transfected with ASOs, incubated for 24 hours, and then RNA was extracted and the levels of aberrantly spliced ​​CFTR transcripts were measured by qRT-PCR ( Figure 3A In addition, the percentage of correctly spliced ​​CFTR transcripts after incubation with each ASO was determined ( Figure 3B ). The values ​​shown are the average fold change (mean ± sem) relative to cells treated with control ASO from 2-8 independent experiments. The values ​​were normalized to the transcript of the HPRT gene.

[0219] As shown in FIG. 3 , all tested ASOs except one (SPL84-26) were surprisingly found to significantly reduce the levels of aberrantly spliced ​​CFTR mRNA transcripts (to at least 0.6 of the control ASO) and increase the prevalence of correctly spliced ​​CFTR mRNA transcripts to at least 10%, even at concentrations as low as 10 nM, compared to the negligible effect of the control ASO.

[0220] Example 2

[0221] Comparison of novel ASOs and previously published ASOs

[0222] The novel ASOs provided herein were compared with several previously described ASOs (Friedman et al., J. Biol. Chem., 1999). The same method as in Example 1 above was used, except that lower concentrations of ASOs (2.5 nM versus 10 nM) were used to transfect FRT cells in these experiments.

[0223] according to Figure 4A and Figure 4B As becomes apparent from the data presented in the accompanying drawings, all of the ASOs provided herein were surprisingly found to significantly reduce the levels of aberrantly spliced ​​CFTR mRNA transcripts (to at least 80% of the control ASO levels), and increase the prevalence of correctly spliced ​​CFTR mRNA transcripts to at least 15%, even at concentrations as low as 2.5 nM, compared to the negligible effect of the control ASO. More importantly, it should be noted that these results are far superior to those achieved by previously described ASOs in a direct head-to-head comparison.

[0224] These results become even more surprising and promising when considering that the genetically manipulated FRT cells used in the experiments contain a single copy of the CFTR gene with a 3849+10Kb C-to-T mutation, a more extreme condition than the most acute phenotype of human CF, in which both CFTR gene alleles display little to no CFTR activity.

[0225] Example 3

[0226] Comparison of novel ASOs and previously published ASOs

[0227] The novel ASOs provided herein were compared with several ASOs previously described in WO2014 / 045283. The same method as in Example 1 above was used.

[0228] according to Figure 5A and Figure 5B As becomes apparent from the data presented in , all of the ASOs provided herein are comparable in efficacy to the ASOs described in WO2014 / 045283, i.e., in reducing the levels of aberrantly spliced ​​CFTR mRNA transcripts, and / or in increasing the prevalence of correctly spliced ​​CFTR mRNA transcripts, compared to the negligible effect of the control ASO.

[0229] It is surprising to note that despite being shorter than the ASOs previously described in WO2014 / 045283 (18-21 consecutive bases versus 25 consecutive bases (16%-28%)), the novel ASOs provided herein exhibited comparable efficacy in any of the experiments. This was particularly surprising and unpredictable because the ASOs previously described in Friedman et al., 1999, which had 18-19 consecutive bases, showed inferior efficacy in all experiments.

[0230] Based on the data presented in Examples 1, 2 and 3 above, it is apparent that the ASOs provided by the present invention are highly capable of manipulating the splicing of CFTR pre-mRNA transcripts containing the 3849+10Kb C-to-T mutation such that the ratio between correctly spliced ​​CFTR mRNA transcripts and aberrantly spliced ​​CFTR mRNA transcripts becomes much more favorable.

[0231] These results become even more surprising and promising when considering that the genetically manipulated FRT cells used in the experiments contain a single copy of the CFTR gene with a 3849+10Kb C-to-T mutation, a more extreme condition than the most acute phenotype of human CF, in which both CFTR gene alleles display little to no CFTR activity.

[0232] Example 4

[0233] EC50 analysis of novel ASOs

[0234] The novel ASOs SPL84-2, SPL84-17, SPL84-22, SPL84-23 and SPL84-25 provided herein were tested at several concentrations to generate EC50 curves indicating the affinity and efficacy of each ASO. Figure 6 As shown in Table 3 (below), the ASOs were found to have high efficiency and potency.

[0235] Table 3.

[0236]

[0237] Example 5

[0238] Western blot analysis of CFTR mature protein levels

[0239] The CFTR protein in the Western blot can have two forms: immature-unglycosylated protein (B band) and mature-full-length glycosylated protein (C band). In homozygous WT cells, most of the CFTR protein is present in the C band-full length and mature. In FRT cells with 3849+10Kb C-to-T mutation ("FRT mut") and treated with control ASO, all CFTR proteins appear as truncated CFTR proteins in the B band.

[0240] The transfection protocol was as follows: 10 nM of each ASO was transfected into FRT mut cells for three consecutive days. In each experiment, the effects of several ASOs were analyzed compared to cells treated with 10 nM control ASOs. Each ASO was tested in 2 bioassays. CFTR was stained by an antibody that detects only the full-length CFTR protein (αCFTR-M3A7; Figure 7). In cells treated with the control, C bands were not detected, while C bands appeared after ASO transfection, indicating the formation of full-length mature protein.

[0241] like Fig. 7A and Figure 7B As shown in , the level of mature full-length protein increased after transfection with 10 nM ASO. These data provide direct evidence that the ASOs provided by the present invention have significant and beneficial effects not only at the pre-mRNA / mRNA level, but more importantly at the protein level.

[0242] Example 6

[0243] Functional analysis

[0244] FLIPR TM (Fluorescence imaging plate reader) is a functional system for measuring the change of membrane potential by fluorescent indicator. When the activation of CFTR is realized by adding forskolin (FSK), and the specificity for CFTR channel is verified by adding CFTR specific inhibitor (inh-172), FLIPR can be used to test CFTR activation level. Experiments were carried out 24 hours after each ASO of 10nM was transfected into FRT mut cells.

[0245] Fig. 8A is a representative FLIPR trace demonstrating CFTR activation induced by ASO SPL84-17. Figure 8B The average effect of several ASOs on CFTR function is shown (*, p value ≤ 0.05; **, p value ≤ 0.01; ***, p value ≤ 0.001; ****, p value ≤ 0.0001).

[0246] Fig. 8A and Figure 8BThe data presented in clearly demonstrate that the novel ASOs provided by the present invention have significant and beneficial effects not only at the RNA and protein levels, but even more importantly at the functional level.

[0247] Example 7

[0248] Efficacy in primary cells from CF patients

[0249] CFTR channel activity was analyzed in differentiated primary airway epithelial cells from CF patients. Ussing chambers were used to measure transepithelial currents indicative of CFTR channel activity. This measurement is a strong predictor of patient response to treatment (R 2 =0.95).

[0250] Human nasal epithelial (HNE) cells from CF patients carrying at least one CFTR allele with a 3849+10Kb C-to-T mutation were provided. To increase the number of filter assays, conditionally reprogrammed and redifferentiated HNE cells were used. In these experiments, HNE cells were exposed to ASOs for 15-23 days while differentiating on filters without the addition of transfection reagents.

[0251] Ussing chamber experiments were performed on HNE cells from 5 CF patients. Four of them were heterozygous for the 3849 10+Kb C-to-T mutation and the minimal functional mutation: Paris patient #2 and Israeli patient #4 (3849+10kb C-to-T / F508del), Israeli patient #1 (3849+10kb C-to-T / W1282X), and Israeli patient #2 (3849+10Kb C-to-T / 405+1G-to-A). In patients heterozygous for the 3849+10Kb C-to-T mutation, the maximum expected activity was 50% of WT, which is sufficient to confer complete health status. The other patient tested was homozygous for the 3849+10kb C-to-T mutation (Paris patient #3).

[0252] First, five ASOs (SPL84-17, SPL84-22, SPL84-23, SPL84-2, and SPL84-25) were analyzed in cells from two patients (one heterozygous and one homozygous). In Figure 9, the effects of the five ASOs in a compound heterozygous patient (Paris patient #2-3849+10kb C-to-T / F508del) are presented. In order to measure the CFTR activity level in the most accurate way, the changes after adding a specific CFTR inhibitor (inh-172) were used as an indicator. As shown in the representative trajectory ( Fig.9A ) and the median value of CFTR inh-172 ( Fig. 9B), all tested ASOs resulted in significant activation of CFTR compared to the control ASO. In this patient, SPL84-23 had the strongest effect, reaching 55% of WT when cells were exposed to SPL84-23. This level indicates complete restoration of the 3849 allele. The other 4 ASOs also resulted in significant induction of activity, ranging from 34% to 42.5% of WT.

[0253] The effect of each of the five ASOs was also analyzed in HNE from a 3849+10kb C-to-T mutation homozygous patient (Paris #3). Consistent with the heterozygous patient tested first, ASO SPL84-23 showed complete recovery of CFTR activity, reaching the activity level of WT individuals. In this patient, three other ASOs (SPL84-17, SPL84-22, and SPL84-2) also induced complete recovery, indicating that these three ASOs also have the potential for high therapeutic efficacy. Nevertheless, the level of CFTR activity between these ASOs is different, with SPL84-17 and SPL84-22 showing higher levels of CFTR activity (231% and 186% respectively compared to 151%) compared to SPL84-2. SPL84-25 also induced a high level of activation, although lower than the effect of other ASOs (93%) (Figure 10).

[0254] Based on the results with cells from these patients, the inventors chose to further analyze the effects of the following three ASOs that showed the highest levels of CFTR activity: SPL84-17, SPL84-22, and SPL84-23 (Figures 9 and 10).

[0255] Next, these 3 ASOs were tested in two additional heterozygous patients carrying different minimal functional mutations on the second allele (W1282X and 405+1G-to-A). Of note, the use of HNE cells enabled the acquisition of patients with a variety of genotypes but still carrying a 3849+10Kb C-to-T mutation. Consistent with the results from the first heterozygous patient (Paris #2), the 3 ASOs showed activation of CFTR in the additional patients tested, with variable effects between patients.

[0256] The effect of ASO SPL84-23 was tested in cells from another 3849+10kb C-to-T / F508del patient (Israel #4), and ASO SPL84-23 showed the highest effect among the heterozygotes tested. In this patient, exposure to the ASO also resulted in significant activation of the CFTR channel to 38% of WT levels.

[0257] Summarizing the functional results from all heterozygous patients tested, SPL84-23 had the strongest effect in cells from patients with different genotypes, achieving complete restoration of CFTR activity (mean 42.5% of WT, range 23% to 73.5%). The other two ASOs, SPL84-17 and SPL84-22, also showed significant restoration of the 3849+10Kb C-to-T allele, with more moderate effects (mean 30% and 31% of WT, respectively) ( Fig.11 ).

[0258] Various data indicate that only partial restoration of protein / RNA levels is required to avoid CF disease symptoms (e.g., lung function). As an indication, splice site variants of 10%-25% that allow CFTR transcript levels to reach normal levels have been found in individuals who do not suffer from CF lung disease. Therefore, this result provides a reliable indication of the potential of ASOs as drug candidates with significant clinical benefits.

[0259] Example 8

[0260] Effects of ASO on splicing

[0261] To test whether the dramatic activation of CFTR by ASO SPL84-23 treatment resulted from a reduction in the levels of aberrantly spliced ​​CFTR transcripts, the effect of ASO SPL84-23 on splicing patterns was further analyzed by extracting RNA from the same filters used for Ussing chamber measurements. Fig. 12A As can be seen in the Figure 2, in HNE cells of both heterozygous and homozygous patients, the levels of abnormally spliced ​​CFTR transcripts carrying the 84bp cryptic exon were significantly reduced (as measured by qRT-PCR and calculating the relative fold change in the levels of CFTR abnormal transcripts between the control ASO and SPL84-23). ​​In homozygous cells, since all transcripts are derived from the 3849+10kb C-to-T mutant allele, the absolute levels of correctly spliced ​​transcripts and abnormally spliced ​​transcripts can be visualized on agarose gels using RT-PCR. The results demonstrate that SPL84-23 treatment has a significant effect on the splicing pattern. In this patient, untreated cells or cells treated with the control ASO produced primarily abnormally spliced ​​CFTR with very low levels of correctly spliced ​​transcripts. However, in SPL84-23 treated cells, most of the RNA transcripts were correctly spliced ​​( Fig. 12B ).

[0262] Example 9

[0263] Chemical modification of AO enables CFTR function in HNE cells from CF patients

[0264] The effects of ASOs with the same sequence and different chemical modifications were evaluated. 2'OMP is considered a second generation ASO chemistry, and the effects of another second generation modification, 2'-methoxyethyl (2'MOE), were tested. Both modifications have similar properties: (1) slowing degradation by protecting the ASO from nucleases; and (2) having higher affinity for the target.

[0265] The effects of SPL84-23 with 2'OMP or 2'MOE modifications were compared using the Ussing chamber assay described in Example 7 in primary human nasal epithelial cells from a patient carrying the 3849+10 kb C to T mutation (free uptake).

[0266] CFTR function was measured in nasal epithelial cells from patients homozygous for the 3849 mutation after treatment with naked ASO (free uptake, no transfection reagent). Fig.13 ). ASOs were tested at the indicated 2 concentrations. At both concentrations, SPL84-23 2'MOE showed significantly higher effects on CFTR function compared to SPL84-23 2'OMP. Importantly, while SPL84-23 2'MOE fully rescued CFTR function (to WT levels) at both 200 nM and 50 nM ASO concentrations, SPL84-23 2'OMP fully rescued the function only at the higher (200 nM) concentration.

[0267] Example 10

[0268] Optimization of ASOs improves efficacy and penetration

[0269] The inventors further optimized the lead synthetic oligonucleotide candidates in order to improve their efficacy and penetration. The effects of chemistry and ASO length reduction were examined.

[0270] Changing the chemical modification from 2'OMe to IONIS Generation 2 2'MOE, as well as ASO lengths of 17-20 bases were attempted.

[0271] Short versions of ASO84-23 were screened for their effects on splicing (SPL84-23-1-SPL84-23-9) by free uptake in primary patient HNE cells. On average, SPL84-23-1 (19mer) was comparable to SPL84-23 in the three 3849 heterozygous patients analyzed ( Fig.14 ).

[0272] Optimization of ASO length was further examined in primary HNE cells and primary HBE cells (human bronchial epithelial cells) from a heterozygous patient (3849 / F508del).

[0273] In primary HNE cells ( Figure 15A-15B ) and primary HBE cells ( Figure 16A-16B ), SPL84-23-1 (19 mer) was shown to have similar effects on CFTR function as the longer SPL84-23 ASO.

[0274] Although the present invention has been specifically described, it will be appreciated by those skilled in the art that many changes and modifications may be made. Therefore, the present invention should not be interpreted as being limited to the specifically described embodiments, and the scope and concept of the present invention will be more readily understood by referring to the appended claims.

Claims

1. A synthetic oligonucleotide molecule consisting of 17-21 consecutive bases that are completely complementary to the pre-mRNA transcript of the cystic fibrosis transmembrane conductance regulator (CFTR) gene having a 3849+10Kb C- to -T mutation, wherein the sequence of the synthetic oligonucleotide molecule comprises the sequence listed in SEQ ID NO:

39.

2. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

1.

3. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

2.

4. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

4.

5. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

40.

6. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

41.

7. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

42.

8. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

43.

9. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

44.

10. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

45.

11. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

46.

12. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

47.

13. The synthetic oligonucleotide molecule of claim 1, wherein the sequence comprises SEQ ID NO:

48. The synthetic oligonucleotide molecule according to claim 1 , which consists of 19 consecutive nucleotide bases. The synthetic oligonucleotide molecule according to claim 1 , which consists of 18 consecutive nucleotide bases.

16. The synthetic oligonucleotide molecule of claim 1, comprising a chemical modification of the backbone of the oligonucleotide, a chemical modification of the sugar of the oligonucleotide, or a combination thereof.

17. The synthetic oligonucleotide molecule according to claim 16, wherein the chemically modified synthetic oligonucleotide molecule comprises: A phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphodiamidate morpholino backbone, a peptide nucleic acid backbone, an alternating locked nucleic acid backbone, a constrained ethyl (cET) backbone, a phosphorothioate backbone, an N3'-P5' phosphoamide, a 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid, a cyclohexene nucleic acid backbone, a tricyclic-DNA (tcDNA) nucleic acid backbone, or a combination thereof.

18. The synthetic oligonucleotide molecule of claim 16, wherein the chemical modification comprises a 2'-O-methyl phosphorothioate (2'OMP) modification or a 2'-methoxyethyl (2'MOE) phosphorothioate modification.

19. A pharmaceutical composition comprising the synthetic oligonucleotide molecule according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, further comprising a CFTR splicing regulator, a CFTR enhancer, a CFTR corrector, a translation readthrough agent, and a CFTR amplifier, or a combination thereof.

21. The pharmaceutical composition of claim 20, wherein the CFTR enhancer comprises N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (avacafol), QBW251, PTI-808 or VX-561 (deuterated avacafol); wherein the CFTR corrector comprises 3-{6-{[1-(2,2-difluoro-1,3-benzodioxazol-5-yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid (lumacafol), 1-(2,2-difluoro-1,3-benzodioxazol-5-yl) -~{N}-[1-[(2~{R})-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropane-2-yl)indol-5-yl]cyclopropane-1-carboxamide (Tezacaftor), VX-659, VX-445, VX-152, VX-440, GLPG2222, FDL169 or PTI-801; wherein the translation readthrough agent includes 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) or ELX-02; wherein the CFTR amplifier includes PTI-428.

22. The pharmaceutical composition of claim 19, further comprising a therapeutically effective amount of one or more cystic fibrosis drugs selected from the group consisting of antibiotic drugs, bronchodilators, corticosteroids, or any combination thereof.

23. Use of a pharmaceutical composition comprising a synthetic oligonucleotide molecule according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier in the preparation of a medicament for treating cystic fibrosis in a subject having a 3849+10Kb C-to-T mutation in the CFTR gene.

24. The use according to claim 23, wherein the subject is heterozygous for the 3849+10Kb C-to-T mutation.

25. The use according to claim 23, wherein the treatment comprises improving at least one clinical parameter of cystic fibrosis selected from the group consisting of: lung function, time to first pulmonary exacerbation, change in weight, change in height, change in body mass index (BMI), change in sweat chloride concentration, number and / or duration of pulmonary exacerbations, total days of hospitalization for pulmonary exacerbations, and need for antibiotic therapy for sinopulmonary signs or symptoms.

26. The use according to claim 23, wherein the medicament further comprises a CFTR splicing regulator, a CFTR enhancer, a CFTR corrector, a translation read-through agent, a CFTR amplifier, or a combination thereof.

27. The use according to claim 26, wherein the CFTR enhancer comprises N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (avacato), QBW251, PTI-808 or VX-561 (deuterated avacato); wherein the CFTR corrector comprises 3-{6-{[1-(2,2-difluoro-1,3-benzodioxazol-5-yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid (lumacato), 1-(2,2-difluoro-1,3-benzodioxazol-5-yl)-~ {N}-[1-[(2~{R})-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropane-2-yl)indol-5-yl]cyclopropane-1-carboxamide (Tezacaftor), VX-659, VX-445, VX-152, VX-440, GLPG2222, FDL169 or PTI-801; wherein the translation readthrough agent includes 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) or ELX-02; wherein the CFTR amplifier includes PTI-428.

28. The use according to claim 23, wherein the medicament further comprises a therapeutically effective amount of one or more cystic fibrosis drugs selected from antibiotic drugs, bronchodilators, corticosteroids or any combination thereof.

29. The use according to claim 23, wherein the pharmaceutical composition is formulated for oral, nasal, inhaled, abdominal, subcutaneous, intraperitoneal or intravenous administration.

30. A kit comprising at least one synthetic oligonucleotide molecule according to any one of claims 1 to 18 and: a. a cystic fibrosis transmembrane conductance regulator (CFTR) splicing regulator, a CFTR enhancer, a CFTR corrector, a translation read-through agent, a CFTR amplifier, or a combination thereof; b. Cystic fibrosis drugs; c. or a combination thereof.

31. The kit according to claim 30, wherein the CFTR enhancer comprises N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (avacafol), QBW251, PTI-808 or VX-561 (deuterated avacafol); wherein the CFTR corrector comprises 3-{6-{[1-(2,2-difluoro-1,3-benzodioxazol-5-yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid (lumacator), 1-(2,2-difluoro-1,3-benzodioxazol-5-yl)- {N}-[1-[(2~{R})-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropane-2-yl)indol-5-yl]cyclopropane-1-carboxamide (Tezacaftor), VX-659, VX-445, VX-152, VX-440, GLPG2222, FDL169 or PTI-801; wherein the translation readthrough agent includes 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) or ELX-02; wherein the CFTR amplifier includes PTI-428.

32. The kit of claim 30, wherein the cystic fibrosis medication is an antibiotic medication, a bronchodilator, a corticosteroid, or any combination thereof.

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