Antisense oligonucleotides targeting the CFTR 3849+10 kilobase (KB) c->t splicing mutation

Abstract

The present disclosure provides synthetic oligonucleotides that bind to a region of a cryptic exon in the cystic fibrosis transmembrane conductance regulator (CFTR) pre-mRNA comprising the 3849 + 10Kb C-to-T mutation. These synthetic oligonucleotides can be used to treat cystic fibrosis or suppress the inclusion of a cryptic exon between exon 22 and 23 as a result of the 3849 + 10Kb C-to-T mutation. The present disclosure further provides compositions comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 and uses thereof.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING THE CFTR 3849+10 KILOBASE (KB) C->T SPLICING MUTATIONSEQUENCE LISTING STATEMENT

[0001] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. The Sequence Listing is encoded as XML in UTF-8 text. The electronic document, created on September 4, 2024, is entitled “P-630689-PC_ST26.xml”, and is 100 kilobytes in size.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides synthetic oligonucleotides that bind to a region of a cryptic exon present in the cystic fibrosis transmembrane conductance regulator (CFTR) pre- mRNA comprising the 3849 + 10Kb C-to-T mutation, and methods of using the same to treat cystic fibrosis or suppress the inclusion of a cryptic exon between exon 22 and 23 of the CFTR mRNA.BACKGROUND

[0003] Antisense oligonucleotides (ASOs) are single-strand, small synthetic nucleic acid molecules that bind to specific sequences within target RNA molecules. ASOs are usually 16-25 bases long and are designed to hybridize with RNA through Watson-Crick basepairing. ASOs can have different modes of action, one of which is splice switching. Splice switching ASOs consist of chemically modified nucleotides which ablate RNase H activity and allow interaction with nuclear pre-mRNA during the splicing process. These ASOs can be designed to bind to 5’ or 3’ splice junctions, or to splicing enhancer or silencer sites, blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. The benefit to ASO- mediated therapies is that the targeting occurs at the RNA level instead of at downstream processes. Advances made in the development of splice switching ASO-based drugs led to the marketing approval of 5 splice switching ASO-based drugs for two rare diseases, Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) in the recent years.

[0004] Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. It is caused by the presence of mutations in bothalleles of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is inherited in an autosomal recessive manner, meaning that both parents must be carriers of a CF mutation in order for their child to have the disease. Long-term symptoms of CF include difficulty breathing and coughing up mucus as a result of frequent lung infections. Other signs and symptoms may include sinus infections, poor growth, fatty stool, clubbing of the fingers and toes, and infertility in most males. There is no cure for CF, but there are treatments that can help to manage the symptoms and improve the quality of life for people with the disease. These treatments include bronchodilators, antibiotics, pancreatic enzyme replacement therapy, and nutritional support.

[0005] The 3849+ 10Kb C-to-T (3849) mutation is a splicing mutation leading to the inclusion of a cryptic exon harboring a premature termination codon (PTC) in CFTR mRNA. The PTC leads to degradation of a significant fraction of the mRNA by the nonsense mediated mRNA decay (NMD) mechanism, as well as to the production of truncated nonfunctional CFTR proteins. The 3849 is the 7th and 8th most common CFTR mutation in the US and Europe, respectively, with >1500 patients in the US and EU combined carrying at least one 3849 allele. Currently, there are no approved drugs which specifically target the 3849 mutation. There are two FDA approved CFTR modulator drugs available for CF patients carrying the 3849 mutation - Kalydeco® (VX-770) and Symdeko® (VX-661+VX- 770) - and one approved for CF patients heterozygous for the 3849 mutation and another qualifying mutation - Trikafta® (VX-661+VX-445+VX-770). However, these drugs have limited efficacy on lung function in 3849 CF patients.

[0006] A further challenge for ASO-based CF therapeutics is the delivery of the ASO to its target tissue and to specific cells, while minimizing exposure of other tissues. Ideally, ASOs could be delivered to the lung via inhalation. However, the translocation of oligonucleotide- based drugs across the pulmonary mucosal epithelia is hampered by several biological barriers including the mucus layer, tight epithelial cell junctions, and mucociliary clearance (MCC) mechanisms designed to remove inhaled particulates from the airway.

[0007] There remains a need for therapeutics that can be efficiently applied intranasally and that overcome the numerous mutations in the CFTR gene, the 3849 mutation among them, to restore CFTR function.SUMMARY

[0008] In some embodiments, the present disclosure provides synthetic oligonucleotides that bind to a region of a cryptic exon that is present in the pre-mRNA of cystic fibrosistransmembrane conductance regulator (CFTR) comprising the 3849 + 10Kb C-to-T mutation. In one embodiment, the synthetic oligonucleotide comprises SEQ ID NO: 40, wherein the synthetic oligonucleotide is formulated at a dose of 25, 50, or 100 mg. In another embodiment, the synthetic oligonucleotide comprises SEQ ID NO: 40, wherein the synthetic oligonucleotide is formulated at a dose of 20, 40, 80, or 160 mg.

[0009] In another embodiment, the present disclosure provides a composition comprising the synthetic oligonucleotide disclosed herein (e.g. SEQ ID NO: 40) and a pharmaceutically acceptable carrier.

[0010] In another embodiment, the present disclosure provides a composition comprising the synthetic oligonucleotide disclosed herein (e.g. SEQ ID NO: 40) for use in the treatment of cystic fibrosis in a subject in need thereof.

[0011] In another embodiment, the present disclosure provides a composition comprising the synthetic oligonucleotide disclosed herein (e.g. SEQ ID NO: 40) for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in a subject in need thereof.

[0012] In other embodiments, the present disclosure provides a method for treating cystic fibrosis in a subject, comprising administering to the subject a therapeutically effective amount of a synthetic oligonucleotide comprising SEQ ID NO: 40. In one embodiment, the synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 25, 50, or 100 mg.

[0013] In other embodiments, the present disclosure provides a method for suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject, comprising administering to the subject a therapeutically effective amount of a synthetic oligonucleotide comprising SEQ ID NO: 40. In one embodiment, the synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 25, 50, or 100 mg.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1C provide the mean absolute change from baseline (+SD) in percent predicted spirometry values per cohort (20 mg, 40 mg, 80 mg, or 160 mg) and per treatment arm (active, placebo). Figure 1A: Forced expiratory volume (FEV1) is the volume of air that a subject can exhale during a forced breath in 1 second. Figure IB: Forced vital capacity (FVC) is the amount of air that can be forcibly exhaled from a subject’s lungs. Figure 1C: Forced expiratory flow between 25% and 75% (FEF25-75) is the average forced expiratory flow rate at 25%-75% of the pulmonary volume.

[0015] Figure 2 shows the arithmetic mean (+SD) plasma concentrations of SPL84 following administration to subjects by inhalation. The lower line is Cohort Al, which received a nominal dose of 20 mg (estimated delivered dose of 7 mg). The 2ndline from the bottom is Cohort A2, which received a nominal dose of 40 mg (estimated delivered dose of 14 mg). The third line from the bottom is Cohort A3, which received a nominal dose of 80 mg (estimated delivered dose of 28 mg). The upper line is Cohort A4, which received a nominal dose of 160 mg (estimated delivered dose of 56 mg).DETAILED DESCRIPTION

[0016] In some embodiments, the present disclosure provides compositions and methods for treating a subject having cystic fibrosis (CF).Synthetic Oligonucleotides

[0017] In some embodiments, the present disclosure provides a composition comprising a synthetic oligonucleotide, wherein said synthetic oligonucleotide suppresses the inclusion of intron 22 cryptic exon in the mature cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. In some embodiments, the nucleotide sequence of the synthetic oligonucleotide is complementary to a region of the CFTR gene comprising the 3849 + 10Kb C-to-T mutation. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide is complementary to the region upstream and / or downstream of the CFTR gene comprising the 3849 + 10Kb C-to-T mutation. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide is complementary to the region upstream and / or downstream of the CFTR gene comprising the F508del mutation.

[0018] In some embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-25 and 41-71. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises SEQ ID NO: 37. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence as set forth in Table 2. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence as set forth in SEQ ID NO: 40. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide consists essentially of a nucleotide sequence as set forth in SEQ ID NO: 40. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide consists of a nucleotide sequence as set forth in SEQ ID NO: 40. In other embodiments, the nucleotide sequence of the synthetic oligonucleotide is essentially homologous to SEQ ID NO: 40. Insome embodiments, said essentially homologous sequence comprises at least 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% identity to SEQ ID NO: 40.

[0019] In one embodiment, the present disclosure provides a synthetic oligonucleotide comprising SEQ ID NO: 40, wherein the synthetic oligonucleotide is formulated at a dose of 25, 50, or 100 mg. In another embodiment, the present disclosure provides a synthetic oligonucleotide comprising SEQ ID NO: 40, wherein the synthetic oligonucleotide is formulated at a dose of 20, 40, 80, or 160 mg.

[0020] In some embodiments, the synthetic oligonucleotide disclosed herein comprises a chemical modification of the backbone of the oligonucleotide. In other embodiments, the synthetic oligonucleotide disclosed herein comprises a chemical modification of a sugar of the oligonucleotide. In other embodiments, the synthetic oligonucleotide disclosed herein comprises both a chemical modification of the backbone of the oligonucleotide and a chemical modification of a sugar of the oligonucleotide.

[0021] In some embodiments, the chemical modification comprises a phosphate-ribose backbone. In other embodiments, the chemical modification comprises a phosphatedeoxyribose backbone. In other embodiments, the chemical modification comprises a phosphorothioate-deoxyribose backbone. In other embodiments, the chemical modification comprises a phosphorodiamidate morpholino backbone. In other embodiments, the chemical modification comprises a peptide nucleic acid backbone. In other embodiments, the chemical modification comprises an alternating locked nucleic acid backbone. In other embodiments, the chemical modification comprises a constrained ethyl (cET) backbone. In other embodiments, the chemical modification comprises a phosphorothioate backbone. In other embodiments, the chemical modification comprises N3'-P5' phosphoroamidates, 2'-deoxy-2'- fluoro-P-d-arabino nucleic acid. In other embodiments, the chemical modification comprises a cyclohexene nucleic acid backbone. In other embodiments, the chemical modification comprises tricyclo-DNA (tcDNA) nucleic acid backbone. In other embodiments, the chemical modification comprises a combination thereof of two or more of the above chemical modifications.

[0022] In some embodiments, the chemical modification comprises a 2’-O-Methyl phosphorothioate (2’0MP) modification. In other embodiments, the chemical modification comprises a 2’-Methoxy Ethyl (2’MOE) phosphorothioate modification.

[0023] In some embodiments, the synthetic oligonucleotide disclosed herein comprises a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate- deoxyribose backbone, or a phosphorothioate backbone, or a combination thereof. In someembodiments, the synthetic oligonucleotide disclosed herein, comprises a 2'-O-methyl- phosphorothioate (2’0MP) chemical modification, a phosphorodiamidate morpholino chemical modification, a peptide nucleic acid chemical modification, 2 ’-methoxy ethyl phosphorothioate chemical modification, or an alternating locked nucleic acid chemical modification, or a combination thereof. In some embodiments, the synthetic oligonucleotide disclosed herein, e.g. SEQ ID NO: 40, comprises N3'-P5', 2'-deoxy-2'-fluoro-P-d-arabino nucleic acid, cyclohexene nucleic acid, or tricyclo-DNA (tcDNA) nucleic acid, or a combination thereof.

[0024] In some embodiments, a synthetic oligonucleotide as described herein is referred to as “SPL84” or “SPL84-23-1”. These terms are used interchangeably, and all have the same features and limitations.

[0025] In some embodiments, a composition of the present disclosure comprises a synthetic oligonucleotide as described herein.

[0026] In some embodiments, disclosed herein are synthetic oligonucleotides that modulate the correct expression of CFTR. A skilled artisan would appreciate that CFTR, or cystic fibrosis transmembrane conductance regulator, is a membrane protein and anion channel encoded by the CFTR gene. The CFTR gene has been linked to cystic fibrosis. More than 2000 cystic fibrosis-causing mutations of the CFTR gene have been described, which result in non-functional CFTR proteins. In some embodiments, CFTR is also termed ABC35, ABCC7, CF, CFTR / MRP, MRP7, TNR-CFTR, dJ760C5.1, having all the same features and limitations. In some embodiments, human CFTR protein comprises the amino acid sequence having NCBI Reference Sequence: NP_000483.3. In some embodiments, human CFTR gene comprises the oligonucleotide sequence having NCBI Reference Sequence: NM_000492.4.

[0027] In some embodiments, CFTR sequences that are target sequences of the synthetic oligonucleotides described herein are described in Table 1 below.Table 1. CFTR-related sequences (in sense orientation)

[0028] In some embodiments, the synthetic oligonucleotide as described herein targets a CFTR sequence disclosed in Table 1. In other embodiments, the synthetic oligonucleotide as described herein targets SEQ ID NO: 34. In other embodiments, the synthetic oligonucleotide as described herein targets SEQ ID NO: 35. In other embodiments, the synthetic oligonucleotide as described herein targets SEQ ID NO: 36. In other embodiments, the synthetic oligonucleotide as described herein targets SEQ ID NO: 37. In other embodiments, the synthetic oligonucleotide as described herein targets a portion of SEQ ID NOs: 34-37, which, in one embodiment, comprises a 15-25 nucleotide portion of CFTR and, in another embodiment, comprises an 18-22 nucleotide sequence of CFTR.

[0029] In certain embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence within SEQ ID NO: 37. In some embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence not more than 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 bases upstream of a mutation that increase inclusion of an intron 22 cryptic exon. Each possibility represents a separate embodiment of the invention. In some embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence not more than 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 bases downstream of a mutation that increase inclusion of an intron 22 cryptic exon. Each possibility represents a separate embodiment of the invention.

[0030] In some embodiments, the synthetic oligonucleotide as described herein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementarity to a nucleotide sequence within SEQ ID NO: 37.

[0031] In certain embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence corresponding to pre-mRNA molecule comprising a sequence about 100 base pairs before the intron 22 cryptic exon to about 100 base pairs afterthe intron 22 cryptic exon transcribed from a CFTR gene having a 3849 + 10Kb C-to-T mutation. In certain embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence within SEQ ID NO: 37.

[0032] In some embodiments, the synthetic oligonucleotide as described herein is specific to a CFTR pre-mRNA. As used herein, the term “specific” refers to both base pair specificity and also gene specificity. In some embodiments, the synthetic oligonucleotide is specific to the CFTR gene. In some embodiments, the synthetic oligonucleotide is specific to an intronic sequence of CFTR. In some embodiments, the synthetic oligonucleotide is specific to a cryptic exon in CFTR. In some embodiments, the synthetic oligonucleotide is specific to a nucleic acid sequence of intron 22 of CFTR. In some embodiments, the nucleic acid sequence of intron 22 is intron 22 cryptic exon of CFTR.

[0033] In some embodiments, the synthetic oligonucleotide as described herein binds the CFTR pre-mRNA with perfect complementarity. In some embodiments, the synthetic oligonucleotide does not bind any gene other than CFTR with perfect complementarity. In some embodiments, the synthetic oligonucleotide does not bind any gene other than CFTR with a complementarity of greater than 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, the synthetic oligonucleotide does not bind any gene other than CFTR with a complementarity of greater than 90%. In some embodiments, the synthetic oligonucleotide binds SEQ ID NO: 37 with perfect complementarity. In some embodiments, the synthetic oligonucleotide does not bind any sequence other than SEQ ID NO: 37 with perfect complementarity. In some embodiments, the synthetic oligonucleotide does not bind any sequence other than SEQ ID NO: 37 with complementarity of greater than 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, the synthetic oligonucleotide does not bind any sequence other than SEQ ID NO: 37 with a complementarity of greater than 90%. In some embodiments, the synthetic oligonucleotide does not bind with perfect complementarity to anywhere in the genome of a cell other than within CFTR. In some embodiments, the synthetic oligonucleotide does not bind with complementarity of greater than 70, 75, 80, 85, 90, 95, 97, 99 or 100% to anywhere in the genome of a cell other than within CFTR. Each possibility represents a separate embodiment of the invention. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammal is a human.

[0034] In some embodiments, the synthetic oligonucleotide as described herein modulates splicing of CFTR. In some embodiments, the synthetic oligonucleotide modulates splicing ofintron 22 cryptic exon of CFTR. In some embodiments, the synthetic oligonucleotide suppresses inclusion of intron 22 cryptic exon of CFTR. In some embodiments, the synthetic oligonucleotide does not cause an off-target effect. In some embodiments, an off-target effect is caused by binding of the synthetic oligonucleotide as described herein to a nucleotide sequence target that is not CFTR. In some embodiments, an off-target effect is an effect other than splicing, e.g., suppressing the inclusion of intron 22 cryptic exon of CFTR. In some embodiments, the synthetic oligonucleotide as described herein does not substantially or significantly modulate expression of a gene other than CFTR. In some embodiments, the synthetic oligonucleotide does not substantially or significantly modulate splicing of a gene other than CFTR. In some embodiments, the synthetic oligonucleotide does not substantially or significantly modulate splicing of an exon other than the intron 22 cryptic exon of CFTR. In some embodiments, a substantial modulation of expression is a change in expression of at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%. Each possibility represents a separate embodiment of the disclosure. In some embodiments, substantial modulation of expression is a change in expression of at least 20%.

[0035] In some embodiments, a synthetic oligonucleotide as disclosed herein targets or complements the intron 22 cryptic exon in the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene. In other embodiments, a synthetic oligonucleotide as disclosed herein suppresses the inclusion of the intron 22 cryptic exon in the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene.

[0036] The phrase “modulation of splicing” as used herein refers to affecting a change in the level of any RNA or mRNA variant produced by the CFTR native pre-mRNA. For example, modulation may mean causing an increase or decrease in the level of abnormal CFTR mRNA, causing an increase or decrease in the level of normal, full-length CFTR mRNA, and / or causing an increase or decrease in the level of abnormal CFTR RNA or mRNA comprising a premature termination codon (non-sense codon). It is therefore evident that any change in ratio between certain CFTR splicing variants is also considered to be the result of splicing modulation. Each possibility represents a separate embodiment of the present disclosure. In certain embodiments, modulation means increasing the level of normal, full-length CFTR mRNA and / or decreasing the level of abnormal CFTR mRNA.

[0037] In other embodiments, a synthetic oligonucleotide as disclosed herein does not target or complement CFTR pre-mRNA transcribed from a wild type allele of the CFTR gene. In other embodiments, a synthetic oligonucleotide as disclosed herein does not suppressor otherwise alter splicing modulation of CFTR pre-mRNA transcribed from a wild type allele of the CFTR gene.

[0038] In some embodiments, a synthetic oligonucleotide as disclosed herein targets or complements the intron 22 cryptic exon in the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene, or suppresses the inclusion of intron 22 cryptic exon into the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene at least 2 fold more efficiently, at least 3 fold more efficiently, at least 5 fold more efficiently, at least 7 fold more efficiently, at least 10 fold more efficiently, at least 20 fold more efficiently, at least 50 fold more efficiently, or at least 100 fold more efficiently, compared to the wild type allele of the CFTR gene, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, an oligonucleotide as disclosed herein targets or complements the intron 22 cryptic exon into the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene, or suppresses the inclusion of intron 22 cryptic exon into the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene 2-10 fold more efficiently, 3-50 fold more efficiently, 5-100 fold more efficiently, 7-20 fold more efficiently, 2-40 fold more efficiently, 2-25 fold more efficiently, 50-150 fold more efficiently, or 2-100 fold more efficiently, compared to the wild type allele of the CFTR gene. Each possibility represents a separate embodiment of the invention.

[0039] In some embodiments, a synthetic oligonucleotide as described herein, for example an oligonucleotide comprising SEQ ID NO: 40, fully complements a mutated allele of the CFTR gene. As used herein, the term "fully complements" refers to 100% hybridization, meaning the mutated CFTR allele and the synthetic oligonucleotide represent a reversed and complementary nucleic acid sequence version of one another, as would be apparent to one of ordinary skill in the art of molecular biology. In some embodiments, a synthetic oligonucleotide as described herein partially complements the wild type allele of the CFTR gene. As used herein, the term "partially" refers to any value or range lower than 100%. In some embodiments, the synthetic oligonucleotide and the wild type CFTR allele represent a reversed and complementary nucleic acid sequence version of one another which differ by at least one nucleotide, e.g., comprising at least one mismatched nucleotide.

[0040] In some embodiments, the synthetic oligonucleotide as described herein, and methods of use thereof, cause the exclusion of a cryptic exon from the mature CFTR mRNA transcribed from a mutated allele of the CFTR gene. In some embodiments, the mature mRNA transcribed from the wild type allele is devoid of the cryptic exon. In someembodiments, the cryptic exon is the intron 22 cryptic exon. In some embodiments, the cryptic exon is 80-90 bases long.

[0041] In some embodiments, the synthetic oligonucleotide as described herein comprises an active fragment of any one of SEQ ID NOs: 1-25 and 41-44. In some embodiments, the oligonucleotide comprises an active fragment of any one of SEQ ID NOs: 1-5 and 41.

[0042] As used herein, the term “active fragment” refers to a fragment that is 100% identical to a contiguous portion of the full nucleotide sequence of any one of SEQ ID NOs: 1-25 and 41-44, providing that at least: 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the targeted oligonucleotide sequence is retained, or any value and range therebetween. Each possibility represents a separate embodiment of the present disclosure.

[0043] In some embodiments, a synthetic oligonucleotide of the disclosure is an artificial “anti-sense” polynucleotide able to target and bind predetermined sequences at the pre- mRNA molecule of the CFTR gene, and the binding modulates the splicing of the pre-mRNA molecule into mature mRNA, which subsequently translates into a functional CFTR protein. The targets within a CFTR pre-mRNA molecule are those discovered to be involved in splicing, either indirectly, by affecting the splicing of adjacent as well as remote sequences, or directly, by affecting their own splicing.

[0044] The present disclosure provides, in one aspect, a synthetic oligonucleotide comprising 17-21 consecutive bases that are complementary to a pre-mRNA transcript of a CFTR gene, wherein the synthetic oligonucleotide at least partly suppresses the inclusion of intron 22 cryptic exon in the mature CFTR mRNA. In some embodiments, the synthetic oligonucleotide as described herein increases the percentage of correctly spliced mature CFTR mRNA by at least about 10%. In some embodiments, the synthetic oligonucleotide as described herein decreases the level of aberrantly spliced mature CFTR mRNA by at least about 20%. In some embodiments, said synthetic oligonucleotide is administered in a 20, 25, 40, 50, 80, 100, or 160 mg dose.

[0045] The phrase “suppress intron 22 cryptic exon inclusion” as used herein refers to lowering the occurrence of the addition of 84 nucleotides (SEQ ID NO: 35) found within intron 22 of the CFTR gene to the mature CFTR mRNA.

[0046] In some embodiments, the present disclosure provides synthetic oligonucleotides and compositions comprising same, capable of binding to a CFTR pre-mRNA, thereby modulating splicing and restoring the function of the CFTR gene product, such as an oligonucleotide comprising SEQ ID NO: 40. The present disclosure thus identifies sequences within the CFTR pre-mRNA which are targeted in order to modulate the splicing cascade ofthe CFTR pre-mRNA. Modulating CFTR pre-mRNA splicing, as demonstrated in the present disclosure, can avoid improper recognition of intron sequences as exons. As a result of the modulation of splicing, a functional CFTR protein is produced by an otherwise aberrant CFTR allele.

[0047] The terms “complementary” or “complementarity” refer to the ability of nucleic acids, e.g., oligonucleotide, polynucleotide, etc., to form base pairs with one another. As used herein, a first oligonucleotide is complementary to a second oligonucleotide, when it is capable of binding to said second oligonucleotide. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine. However, when a U is denoted in the context of the present disclosure, the ability to substitute a T is implied, unless otherwise stated.

[0048] In certain embodiments, the synthetic oligonucleotide disclosed herein consists of 18 or 19 consecutive nucleotide bases. In certain embodiments, the synthetic oligonucleotide consists of 18 consecutive nucleotide bases. In certain embodiments, the synthetic oligonucleotide consists of 19 consecutive nucleotide bases. In certain embodiments, the synthetic oligonucleotide consists of 20 consecutive nucleotide bases. In certain embodiments, the synthetic oligonucleotide consists of 21 consecutive nucleotide bases.

[0049] In some embodiments, synthetic oligonucleotides as described herein have the sequences provided in Table 2 below.Table 2. Synthetic Oligonucleotides

[0050] In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 3. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 4. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 6. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 7. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 8. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 9. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 10. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 11. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 12. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 13. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 14. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 15. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 16. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 17. In certain embodiments, the oligonucleotide moleculecomprises or consists of a nucleotide sequence set forth in SEQ ID NO: 18. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 19. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 20. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 21. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 22. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 23. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 24. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 25. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 40. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 41. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 42. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 43. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 44. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 45. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 46. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 47. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 48. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 49. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 50. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 51. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 52. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 53. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 54. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 55. In certain embodiments, theoligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 56. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 57. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 58. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 59. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 60. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 61. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 62. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 64. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 65. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 66. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 67. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 68. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 69. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 70. In certain embodiments, the oligonucleotide molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 71.

[0051] The present disclosure further provides, in another aspect, a synthetic oligonucleotide, consisting of 18-50 consecutive bases that are complementary to a pre- mRNA transcript of a CFTR gene having a 3849 + 10Kb C-to-T mutation and at least partly suppresses the inclusion of intron 22 cryptic exon in the mature CFTR mRNA, comprising a nucleotide sequence set forth in one of SEQ ID NO: 1 to SEQ ID NO: 25, and SEQ ID NO: 41 to SEQ ID NO: 44.

[0052] The present disclosure further provides, in another aspect, a synthetic oligonucleotide, consisting of 17-50 consecutive bases that are complementary to a pre- mRNA transcript of a CFTR gene having a 3849 + 10Kb C-to-T mutation and at least partly suppresses the inclusion of intron 22 cryptic exon in the mature CFTR mRNA, comprising anucleotide sequence set forth in one of SEQ ID NO: 1 to SEQ ID NO: 25 and SEQ ID NO: 40 to SEQ ID NO: 71.

[0053] The present disclosure further provides, in another aspect, a synthetic oligonucleotide sequence consisting of 17, 18, 19, or 20 consecutive bases comprising the sequence GAUGGAAGA (SEQ ID NO: 38), wherein the synthetic oligonucleotide sequence is complementary to a pre-mRNA transcript of a CFTR gene having a 3849 + 10Kb C-to-T mutation.

[0054] In certain embodiments, the synthetic oligonucleotide as described herein is complementary to a nucleotide sequence within SEQ ID NO: 37. In certain embodiments, the synthetic oligonucleotide sequence is selected from the group consisting of: SEQ ID NOs: 1, 2, 4 and 7. In certain embodiments, the synthetic oligonucleotide sequence comprises the sequence CAACAGAUGGAAGA (SEQ ID NO: 39). In certain embodiments, the synthetic oligonucleotide sequence is selected from the group consisting of: SEQ ID NOs: 1, 2 and 4. Pharmaceutical Compositions

[0055] The present disclosure further provides, in another aspect, a pharmaceutical composition comprising a synthetic oligonucleotide as described herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a synthetic oligonucleotide comprising SEQ ID NO: 40 and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition comprises a synthetic oligonucleotide as described herein and a pharmaceutically acceptable carrier.

[0056] The term “pharmaceutically acceptable carrier” as used herein refers to any of the standard pharmaceutical carriers known in the field such as sterile solutions, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acids, or salts thereof, magnesium 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, buffers, inert, nontoxic solids (e.g., mannitol, talc). Compositions comprising such carriers are formulated by well-known conventional methods. Depending on the intended mode of administration and the intended use, the compositions may be in the form of solid, semi-solid, or liquid dosage forms, such, for example, as powders, granules, crystals, liquids, suspensions, liposomes, nanoparticles, nanoemulsions, pastes, creams, salves, etc., and may be in unit-dosage forms suitable for administration of relatively precise dosages.

[0057] In certain embodiments, the pharmaceutical composition is formulated for oraladministration. 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 intra-peritoneal administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0058] 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 understood by a skilled artisan that a pharmaceutical composition intended for administration to a subject should not have off-target effects, i.e., effects other than the intended therapeutic ones. In some embodiments, the pharmaceutical composition is substantially devoid of a substantial effect on a gene other than CFTR. In some embodiments, the pharmaceutical composition is devoid of any substantial effect other than suppressing the inclusion of intron 22 cryptic exon to the mature CFTR. In some embodiments, a substantial effect is one with a phenotypic result. In some embodiments, a substantial effect is a deleterious effect. In some embodiments, deleterious is with respect to the health and / or wellbeing of the subject.

[0059] In some embodiments, the composition is formulated for administration by inhalation. In some embodiments, the composition is an inhalation composition.

[0060] In certain embodiments, the pharmaceutical composition comprises at least about 20 mg / ml of the synthetic oligonucleotide, which, in one embodiment, is prior to dilution. In certain embodiments, the pharmaceutical composition comprises at least about 2.5 mg / ml of the synthetic oligonucleotide which, in one embodiment, is the concentration that is administered to a subject. In certain embodiments, the pharmaceutical composition comprises 1 mg / ml to 100 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 2.5 mg / ml to 20 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 50 mg / ml to 100 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 1 mg / ml to 50 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 25 mg / ml to 75 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 1 mg / ml to 5 mg / ml of the synthetic oligonucleotide. In other embodiments, the pharmaceutical composition comprises 1 mg / ml to 3 mg / ml of the synthetic oligonucleotide.

[0061] The present disclosure further provides, in another aspect, the use of a synthetic oligonucleotide as described above, or of a pharmaceutical composition as described above, in preparing a medicament.Dosage

[0062] Disclosed herein are particularly efficient doses of the synthetic oligonucleotides for treating a cystic fibrosis patient. This dosage has been surprisingly found to be efficient at suppressing the inclusion of intron 22 cryptic exon in the mature CFTR mRNA, while being non immunogenic and non-toxic.

[0063] In some embodiments, the oligonucleotide or synthetic oligonucleotide or ASO as described herein (e.g. as shown in Table 2) is administered at a dose of 25 mg. In other embodiments, the oligonucleotide is administered at a dose of 50 mg. In other embodiments, the oligonucleotide is administered at a dose of 100 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 25 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 50 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 100 mg.

[0064] In some embodiments, the oligonucleotide or synthetic oligonucleotide or ASO as described herein (e.g. as shown in Table 2) is administered at a dose of 20 mg. In other embodiments, the oligonucleotide is administered at a dose of 40 mg. In other embodiments, the oligonucleotide is administered at a dose of 80 mg. In other the oligonucleotide or synthetic oligonucleotide or ASO as described herein is administered at a dose of 160 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 20 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 40 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 80 mg. In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40 is formulated at a dose of 160 mg.

[0065] In other embodiments, the oligonucleotide is administered at a dose of 7 mg. In other embodiments, the oligonucleotide is administered at a dose of 14 mg. In other embodiments, the oligonucleotide is administered at a dose of 28 mg. In other embodiments, the oligonucleotide is administered at a dose of 56 mg. In other embodiments, the oligonucleotide is administered at a dose of 10 mg. In other embodiments, the oligonucleotide is administered at a dose of 30 mg. In other embodiments, the oligonucleotide is administered at a dose of 50 mg. In other embodiments, the oligonucleotide is administered at a dose of 75 mg.

[0066] In other embodiments, the oligonucleotide is administered at a dose of 1-200 mg. In other embodiments, the oligonucleotide is administered at a dose of 20-100 mg. In other embodiments, the oligonucleotide is administered at a dose of 50-100 mg. In other embodiments, the oligonucleotide is administered at a dose of 150-200 mg. In other embodiments, the oligonucleotide is administered at a dose of 7-40 mg. In other embodiments, the oligonucleotide is administered at a dose of 50-150 mg.

[0067] In some embodiments, the oligonucleotide or synthetic oligonucleotide or ASO as described herein is administered weekly. In other embodiments, the oligonucleotide is administered biweekly.

[0068] In some embodiments, a composition as described herein is administered in one to four doses per day. In one embodiment, a composition as described herein is administered once per day. In another embodiment, a composition as described herein is administered twice per day. In another embodiment, a composition as described herein is administered three times per day. In another embodiment, a composition as described herein is administered four times per day. In another embodiment, a composition as described herein is administered once every two days, once every three days, twice a week, once a week, once every 2 weeks, once every 3 weeks.

[0069] In one embodiment, a composition as described herein is administered for 7 days to 28 days. In another embodiment, a composition as described herein is administered for 7 days to 8 weeks. In another embodiment, a composition as described herein is administered for 7 days to 50 days. In another embodiment, a composition as described herein is administered for 7 days to six months. In another embodiment, a composition as described herein is administered for 7 days to one and half years. In another embodiment, a composition as described herein is administered for 14 days to 12 months. In another embodiment, a composition as described herein is administered for 14 days to 3 years. In another embodiment, a composition as described herein is administered for several years. In another embodiment, a composition as described herein is administered for one month to six months.

[0070] In one embodiment, a composition as described herein is administered for 7 days. In another embodiment, a composition as described herein is administered for 14 days. In another embodiment, composition as described herein is administered for 21 days. In another embodiment, composition as described herein is administered for 28 days. In another embodiment, composition as described herein is administered for 50 days. In another embodiment, composition as described herein is administered for 56 days. In another embodiment, composition as described herein is administered for 84 days. In anotherembodiment, a composition as described herein is administered for 90 days. In another embodiment, a composition as described herein is administered for 120 days.

[0071] In some embodiments, the oligonucleotides of the present disclosure, for example an oligonucleotide comprising SEQ ID NO: 40, are administered at a therapeutically effective amount. The term "a therapeutically effective amount" as used herein refers to an amount necessary for improving at least one clinical parameter of cystic fibrosis or reducing the severity of at least one clinical parameter of cystic fibrosis in a patient. The therapeutically effective amounts may differ according to the patient’s status, the synthetic polynucleotide molecule’s administration route, excipient usage and co-usage of other active agents.Chemical Modifications

[0072] As used herein, the term “synthetic oligonucleotide” refers to any short single strand of DNA or RNA, usually synthesized in a laboratory by any of the techniques known in the art, such as solid-phase chemical synthesis. In certain embodiments, the bases of the synthetic oligonucleotides are selected from the group consisting of adenine, guanine, cytosine, uracil and optionally thymine. In other certain embodiments, the base is selected from the group consisting of adenine, guanine, cytosine, and uracil. Each possibility represents a separate embodiment of the present disclosure.

[0073] In some embodiments, the synthetic oligonucleotide is chemically modified. In some embodiments, the chemical modification is a modification of a backbone of the oligonucleotide. In some embodiments, the chemical modification is a modification of a sugar of the oligonucleotide. In some embodiments, the chemical modification is a modification of a nucleobase of the oligonucleotide. In some embodiments, the chemical modification increases stability of the oligonucleotide in a cell. In some embodiments, the chemical modification increases stability of the oligonucleotide in vivo. In some embodiments, the chemical modification increases the oligonucleotide's ability to modulate splicing. In some embodiments, the chemical modification increases the oligonucleotide's ability to induce suppress the inclusion of intron 22 cryptic exon. In some embodiments, the chemical modification increases the halflife of the oligonucleotide. In some embodiments, the chemical modification inhibits polymerase extension from the 3' end of the oligonucleotide. In some embodiments, the chemical modification inhibits recognition of the oligonucleotide by a polymerase. In some embodiments, the chemical modification inhibits double- stranded triggered degradation. In some embodiments, the chemically modified oligonucleotide does not trigger nucleic acid doublestranded degradation upon binding a CFTR pre-mRNA. In some embodiments, the chemical modification inhibits RISC-mediated degradation. In some embodiments, the chemicalmodification inhibits RISC-mediated degradation or any parallel nucleic acid degradation pathway.

[0074] In certain embodiments, the consecutive nucleotide bases are linked by a backbone such as a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a 2'-O-methyl- phosphoro thioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphoro thioate backbone, an alternating locked nucleic acid backbone, constrained ethyl backbone, or a phosphorothioate backbone. Each possibility represents a separate embodiment of the invention.

[0075] In some embodiments, the synthetic oligonucleotide comprises a phosphate-ribose backbone. In some embodiments, the synthetic oligonucleotide comprises a phosphatedeoxyribose backbone. In some embodiments, the synthetic oligonucleotide comprises a phosphorothioate-deoxyribose backbone. In some embodiments, the synthetic oligonucleotide comprises a 2'-O-methyl-phosphorothioate (2’0MP) chemical modification. In some embodiments, the synthetic oligonucleotide comprises a phosphorodiamidate morpholino chemical modification. In some embodiments, the synthetic oligonucleotide comprises a peptide nucleic acid chemical modification. In some embodiments, the synthetic oligonucleotide comprises a 2-methoxyethyl phosphorothioate chemical modification. In some embodiments, the synthetic oligonucleotide comprises an alternating locked nucleic acid chemical modification. In some embodiments, the synthetic oligonucleotide comprises a phosphorothioate backbone.

[0076] In some embodiments, the synthetic oligonucleotide comprises N3 -P5' phosphoroamidates. In some embodiments, the synthetic oligonucleotide comprises 2'-deoxy-2'- fluoro-P-d-arabino nucleic acid. In some embodiments, the synthetic oligonucleotide comprises cyclohexene nucleic acid. In some embodiments, the synthetic oligonucleotide comprises tricyclo-DNA (tcDNA) nucleic acid. In some embodiments, the synthetic oligonucleotide comprises a backbone with a 2’ -Methoxy Ethyl (2’ MOE) modification.

[0077] In some embodiments, the modification is throughout the 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 invention. Administration Routes

[0078] Different routes for delivery of synthetic oligonucleotides have been examined in animal models and applied in clinical trials. Any of these are relevant and can be used toimplement the present disclosure.

[0079] In some embodiments, a composition comprising a synthetic oligonucleotide as described herein is formulated for administration by inhalation. In some embodiments, the composition is formulated for intrapulmonary administration. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated for nasal administration. In some embodiments, the composition is formulated for abdominal administration. In some embodiments, the composition is formulated for subcutaneous administration. In some embodiments, the composition is formulated for intraperitoneal administration. In some embodiments, the composition is formulated for intravenous administration.Further Active Compounds

[0080] Being a long-known and well-studied disease, certain drugs and agents are known in the art as useful for the treatment of cystic fibrosis patients. In some embodiments, administering a composition comprising an oligonucleotide as described herein in a dose of e.g. 25, 50 or 100 mg, with one or more of these drugs or agents may be beneficial in achieving significant therapeutic results. In other embodiments, administering a composition comprising an oligonucleotide as described herein in a dose of e.g. 20, 40, 80, or 160 mg, with one or more of these drugs or agents may be beneficial in achieving significant therapeutic results. In some embodiments, the oligonucleotide is SEQ ID NO: 40.

[0081] In certain embodiments, a pharmaceutical composition comprising a synthetic oligonucleotide disclosed herein further comprises one or more CFTR modulators. Examples and functions of CFTR modulators are described below. In one embodiment, examples of CFTR modulators include, but are not limited to, CFTR-splicing-modulating agents, Translational Read-Through agents, CFTR amplifiers, CFTR potentiators, and CFTR correctors.

[0082] In some embodiments, the CFTR modulator increases the duration of the CFTR gate being open, increases chloride flow through the CFTR gate, increases proper folding of the CFTR protein, increases the number of CFTR anchored to the cell membrane, or any combination thereof. In other embodiments, the CFTR modulator (i) increases the probability that a defective cystic fibrosis transmembrane conductance regulator (CFTR) will be opened, (ii) assists in proper CFTR channel folding, (iii) induces a cell to increase its CFTR protein production rates, or a combination thereof. Each possibility represents a separate embodiment of the invention.

[0083] In some embodiments, the composition comprises a CFTR-splicing-modulating agent, a Translational Read-Through agent, a CFTR amplifier, a CFTR potentiator, a CFTRcorrector, or a combination thereof.

[0084] In certain embodiments, the CFTR-splicing-modulating agent is a synthetic antisense oligonucleotide other than SEQ ID NO: 40 capable of suppressing intron 22 cryptic exon inclusion in the mature CFTR mRNA. In some embodiments, the CFTR-splicing-modulating agent is a synthetic antisense oligonucleotide other than those from Table 2 capable of suppressing intron 22 cryptic exon inclusion in the mature CFTR mRNA. In some embodiments, the CFTR-splicing-modulating agent is a synthetic oligonucleotide that is described in WO / 2020 / 225813, which is incorporated herein by reference in its entirety. In some embodiments, the CFTR-splicing-modulating agent is a synthetic antisense oligonucleotide from Table 2.

[0085] In some embodiments, the translational read-through agent refers to an agent that restores protein function by selectively suppressing translation termination at premature termination codons generated by a mutation. In some embodiments, the translational read- through agent is 3-[5-(2-fluorophenyl)-l,2,4-oxadiazol-3-yl]benzoic acid (Ataluren). In other embodiments, the translational read-through agent is EEX-02. In other embodiments, the translational read-through agent is SRI-41315. In other embodiments, the translational read- through agent is release factor inhibitors. In other embodiments, the translational read-through agent is tRNA. In other embodiments, the translational read-through agent is aminoglycosides. In other embodiments, the translational read-through agent is G418.

[0086] In one embodiment, the term "CFTR amplifier" refers to any agent that induces a cell to increase its CFTR protein production rates or yields, therefore resulting in an increased amount of the CFTR protein. In some embodiments, the CFTR amplifier is PTI-428.

[0087] In one embodiment, the term "CFTR potentiator" refers to any agent that increases the probability that a defective CFTR will be open and therefore allows chloride ions to pass through the channel pore. In some embodiments, the CFTR potentiator is N-(2,4-Di-tert-butyl-5- hydroxyphenyl)-4-oxo-l,4-dihydroquinoline-3-carboxamide (Ivacaftor; VX-770). In other embodiments, the CFTR potentiator is QBW251. In other embodiments, the CFTR potentiator is PTI-808. In other embodiments, the CFTR potentiator is ABBV-191. In other embodiments, the CFTR potentiator is ABBV-3067 (SION-3067). In other embodiments, the CFTR potentiator is GEPG1837. In other embodiments, the CFTR potentiator is VX-561 (deuterated ivacaftor).

[0088] In one embodiment, the term "CFTR corrector" refers to any agent that assists in proper CFTR channel folding so as to enable its trafficking to the cell membrane. In some embodiments, the CFTR corrector is 3-{6-{[l-(2,2-difluoro-l,3-benzodioxol-5- yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid (Eumacaftor; VX-809). Inother embodiments, the CFTR corrector is l-(2,2-difluoro-l,3-benzodioxol-5-yl)—{N]-[l- [(2~ { R} )-2, 3 -dihydroxypropyl] -6-fluoro-2-( 1 -hydroxy -2 -methy lpropan-2-y l)indol-5- y 1] cyclopropane- 1 -carboxamide (Tezacaftor; VX-661). In other embodiments, the CFTR corrector is VX-659. In other embodiments, the CFTR corrector is elexacaftor (VX-445). In other embodiments, the CFTR corrector is VX-121. In other embodiments, the CFTR corrector is VX-152. In other embodiments, the CFTR corrector is VX-440. In other embodiments, the CFTR corrector is FDL169. In other embodiments, the CFTR corrector is ABBV-3221. In other embodiments, the CFTR corrector is ABBV-2222 (SION-2222). In other embodiments, the CFTR corrector is ABBV-119. In other embodiments, the CFTR corrector is ABBV-2851 (SION-2851). In other embodiments, the CFTR corrector is GLPG2737. In other embodiments, the CFTR corrector is SION-638. In other embodiments, the CFTR corrector is SION-719. In other embodiments, the CFTR corrector is SION-451. In other embodiments, the CFTR corrector is SION-109. In other embodiments, the CFTR corrector is PTI-801.

[0089] In certain embodiments, the administration of the therapeutically effective amount of a synthetic polynucleotide molecule of the present disclosure and the administration of the at least one additional anti-cystic fibrosis drug or CFTR modulator are independently oral, nasal, aerosol, inhalational, abdominal, subcutaneous, intra-peritoneal or intravenous administration. Each possibility represents a separate embodiment of the present disclosure. It should be understood that the selection of an administration route depends on the nature of the therapeutic agent and the site of its intended effect, and thus certain agents may be administrated via the same or different administration routes.

[0090] In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises ivacaftor. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises lumacaftor. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises tezacaftor. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises elexacaftor. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises VX-659. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises VX-152. In some embodiments, a composition comprising a synthetic oligonucleotidecomprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, further comprises VX-440.

[0091] In some embodiments, a composition comprising a synthetic oligonucleotide disclosed herein can further comprise a therapeutically effective amount of one or more cystic fibrosis drugs. Examples of cystic fibrosis drugs include, but are not limited to, antibiotic drugs, bronchodilators, corticosteroid, or any combination thereof. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 further comprises an antibiotic drug. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 further comprises a bronchodilator. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 further comprises a corticosteroid.

[0092] In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with an antibiotic drug. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a bronchodilator. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a corticosteroid.

[0093] In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a CFTR-splicing-modulating agent. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a Translational Read-Through agent. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a CFTR amplifier. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a CFTR potentiator. In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 is administered together with a CFTR corrector.

[0094] In some embodiments, a cystic fibrosis drug as described herein comprises a CFTR modulator, Ivacaftor, Lumacaftor, Elexacaftor, Tezacaftor, Symdeco®, Trikafta®, a pancreatic enzyme, Creon®, Pancreaze®, Zenpep®, an antibiotic, cioxacillin, dicloxacillin, cephalosporin, trimethoprim, sulfamethoxazole, erythromycin, amoxicillin, clavulanate, ampicillin, tetracycline, linezolid, tobramycin or aztreonam lysine, fluoroquinolone, gentamicin, and monobactam, a bronchodilators, Albuterol, Salmeterol, Fluticasone, Budesonide, a mucus thinner, Dornase alfa, Hypertonic saline, or Mannitol dry powder.

[0095] In some embodiments, a cystic fibrosis drug as described herein comprises anantibiotic drug, a bronchodilator, a corticosteroid, or any combination thereof. In some embodiments, the antibiotic drug comprises cioxacillin, dicloxacillin, cephalosporin, trimethoprim, sulfamethoxazole, erythromycin, amoxicillin, clavulanate, ampicillin, tetracycline, linezolid, tobramycin or aztreonam lysine, fluoroquinolone, gentamicin, or monobactam with antip seudomonal activity.

[0096] In certain embodiments, the cystic fibrosis drug as described herein is for treating or ameliorating a symptom of cystic fibrosis. In some embodiments, the compositions or methods disclosed herein improves at least one clinical parameter of cystic fibrosis. In some embodiments, the subject after treatment with the compositions or methods disclosed herein exhibits one or more effects such as improving lung function, increasing time to the first pulmonary exacerbation, increasing weight, increasing height, increasing Body Mass Index (BMI), lowering concentration of sweat chloride, lowering number and / or duration and / or frequency of pulmonary exacerbations, decreasing number of days of hospitalization for pulmonary exacerbations, decreasing need for antibiotic therapy or supportive drugs for sinopulmonary signs or symptoms, or a combination thereof.Methods of Treating

[0097] In some embodiments, disclosed herein is a synthetic oligonucleotide comprising SEQ ID NO: 40 for use in the treatment of cystic fibrosis in a subject in need thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting of SEQ ID NO: 40 for use in the treatment of cystic fibrosis in a subject in need thereof. In some embodiments, the synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg.

[0098] In some embodiments, disclosed herein is a synthetic oligonucleotide comprising one of the sequences shown in Table 2 for use in the treatment of cystic fibrosis in a subject in need thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting of one of the sequences shown in Table 2 for use in the treatment of cystic fibrosis in a subject in need thereof. In some embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-25 and 41-44. In some embodiments, the synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg.

[0099] In some embodiments, disclosed herein is a synthetic oligonucleotide comprising SEQ ID NO: 40 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting essentially of SEQ ID NO: 40 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject inneed thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting of SEQ ID NO: 40 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof. In one embodiment, the synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg.

[0100] In some embodiments, disclosed herein is a synthetic oligonucleotide comprising one of the sequences shown in Table 2 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting essentially of one of the sequences shown in Table 2 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof. In some embodiments, disclosed herein is a synthetic oligonucleotide consisting of one of the sequences shown in Table 2 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof. In some embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-25 and 41-44. In one embodiment, the synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg.

[0101] In some embodiments, disclosed herein is a method for treating cystic fibrosis in a subject, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide and wherein said synthetic oligonucleotide is administered at a dose of 20, 25, 40, 50, 80, 100, or 160 mg. In one embodiment, the synthetic oligonucleotide comprises SEQ ID NO: 40. In other embodiments, the synthetic oligonucleotide comprises one of the sequences shown in Table 2. In some embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-25 and 41-44.

[0102] In some embodiments, disclosed herein is a method for suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide and wherein said synthetic oligonucleotide is administered at a dose of 20, 25, 40, 50, 80, 100, or 160 mg. In one embodiment, the synthetic oligonucleotide comprises SEQ ID NO: 40. In other embodiments, the synthetic oligonucleotide comprises one of the sequences shown in Table 2. In some embodiments, the nucleotide sequence of the synthetic oligonucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-25 and 41-44.

[0103] In some embodiments, disclosed herein is a method for treating, suppressing or inhibiting cystic fibrosis in a subject with a low immunogenic response, comprisingadministering to said subject a therapeutically effective amount of a synthetic oligonucleotide disclosed herein. In other embodiments, disclosed herein is a method for treating, suppressing or inhibiting cystic fibrosis in a subject with low toxicity in said subject, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide disclosed herein.

[0104] In some embodiments, the synthetic oligonucleotide used in the above methods, e.g. SEQ ID NO: 40, comprises a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, or a phosphorothioate backbone, or a combination thereof. In some embodiments, the synthetic oligonucleotide used in the above methods, e.g. SEQ ID NO: 40, comprises a 2'-O-methyl-phosphorothioate (2’0MP) chemical modification, a phosphorodiamidate morpholino chemical modification, a peptide nucleic acid chemical modification, 2’ -methoxy ethyl phosphorothioate chemical modification, or an alternating locked nucleic acid chemical modification, or a combination thereof. In some embodiments, the synthetic oligonucleotide used in the above methods, e.g. SEQ ID NO: 40, comprises N3'-P5', 2'-deoxy-2'-fluoro-P-d-arabino nucleic acid, cyclohexene nucleic acid, or tricyclo-DNA (tcDNA) nucleic acid, or a combination thereof.

[0105] In some embodiments, the synthetic oligonucleotide is administered to the subject via inhalation. In other embodiments, the synthetic oligonucleotide is administered to the subject via oral, nasal, abdominal, subcutaneous, intra-peritoneal or intravenous administration. In some embodiments, the synthetic oligonucleotide is in a pharmaceutical composition and said subject is administered said composition comprising said synthetic oligonucleotide. In some embodiments, the subject has a 3849 + 10Kb C-to-T mutation in one or both of the CFTR genes or a combination of a 3849 + 10Kb C-to-T mutation and a F508del mutation in the CFTR gene. In some embodiments, a pathologically thick mucus layer covers the airways of the subject.

[0106] As used herein, the terms “treatment” or “treating” CF encompasses alleviation of at least one CF symptom, a reduction in CF severity, or inhibition of the progression of CF. In some embodiments, the clinical manifestations and / or symptoms of CF can be treated by the oligonucleotides of the present disclosure, for a marked increase and / or improvement in a patient’s clinical status and quality of life. To be an effective treatment, a useful composition herein needs only to reduce the severity of CF, reduce the severity of symptoms associated with CF, or provide improvement to a patient or subject’ s quality of life.

[0107] In some embodiments, the synthetic oligonucleotides described herein, as for example one comprising SEQ ID NO: 40, are useful in treating cystic fibrosis in a subject having the 3849 + 10Kb C-to-T mutation in the CFTR gene, when administered in a dose of 20, 25, 40, 50, 80, 100, or 160 mg, and wherein said synthetic oligonucleotide suppresses theinclusion of intron 22 cryptic exon in the mature CFTR mRNA. In some embodiment, one or more CFTR modulators as described herein are co-administered to the subject. In some embodiments, the subject is heterozygous for the 3849 + 10Kb C-to-T mutation in the CFTR gene. In other embodiments, the subject is homozygous for the 3849 + 10Kb C-to-T mutation in the CFTR gene.

[0108] In other embodiments, the synthetic oligonucleotides described herein, as for example one comprising SEQ ID NO: 40, are useful in suppressing the inclusion of intron 22 cryptic exon in the mature CFTR mRNA in a subject, when administered in a dose of 20, 25, 40, 50, 80, 100, or 160 mg. In some embodiment, one or more CFTR modulators as described herein are co-administered to the subject. In some embodiments, the subject is heterozygous for the 3849 + 10Kb C-to-T mutation in the CFTR gene. In other embodiments, the subject is homozygous for the 3849 + 10Kb C-to-T mutation in the CFTR gene.

[0109] In some embodiments, a CFTR potentiator such as N-(2,4-Di-tert-butyl-5- hydroxyphenyl)-4-oxo-l,4-dihydroquinoline-3-carboxamide (Ivacaftor; VX-770), QBW251, PTI-808, ABBV-191, ABBV-3067 (SION-3067), GLPG1837, or VX-561 (deuterated ivacaftor) is co-administered to the subject.

[0110] In some embodiments, a CFTR corrector such as 3-{6-{[l-(2,2-difluoro-l,3- benzodioxol-5-yl)cyclopropanecarbonyl] amino } -3 -methylpyridin-2-yl (benzoic acid(Lumacaftor; VX-809), l-(2,2-difhioro-l,3-benzodioxol-5-yl)-~ {N]-[l-[(2~{R})-2,3- dihydroxypropyl] -6-fluoro-2-( 1 -hydroxy -2 -methylpropan-2-yl)indol-5-yl]cyclopropane- 1 - carboxamide (Tezacaftor; VX-661), VX-659, elexacaftor (VX-445), VX-121, VX-152, VX-440, FDL169, ABBV-3221, ABBV-2222 (SION-2222), ABBV-119, ABBV-2851 (SION-2851) GLPG2737, SION-638, SION-719, SION-451, SION-109, or PTI-801 is co-administered to the subject.

[0111] In some embodiments, a translational read-through agent such as 3-[5-(2- fluorophenyl)-l,2,4-oxadiazol-3-yl]benzoic acid (Ataluren), ELX-02, SRI-41315, Release factor inhibitors, tRNA, aminoglycosides, or G418 is co-administered to the subject.

[0112] In some embodiments, a CFTR amplifier such as PTI-428 is co-administered to the subject.

[0113] The disclosure further provides, in another aspect, a method for improving at least one clinical parameter of cystic fibrosis in a patient in need thereof, comprising the step of administering a composition comprising a synthetic oligonucleotide, wherein said synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg. In one embodiment, the synthetic oligonucleotide comprises SEQ ID NO: 40.

[0114] In certain embodiments, the method improves one or more clinical parameters of CF such as lung function, time to the first pulmonary exacerbation, change in weight, change in height, a change in Body Mass Index (BMI), change in the concentration of sweat chloride, number and / or duration and / or frequency of pulmonary exacerbations, total number of days of hospitalization for pulmonary exacerbations, and the need for antibiotic therapy or supportive drugs for sinopulmonary signs or symptoms. In some embodiments, treatment of a subject with the synthetic oligonucleotide disclosed herein results in one or more of the following effects: improving lung function, increasing time to the first pulmonary exacerbation, increasing weight, increasing height, increasing Body Mass Index (BMI), lowering concentration of sweat chloride, lowering number and / or duration and / or frequency of pulmonary exacerbations, decreasing number of days of hospitalization for pulmonary exacerbations, and decreasing need for antibiotic therapy or supportive drugs for sinopulmonary signs or symptoms.

[0115] Thus, in some embodiments, the present disclosure provides a method of improving lung function in a subject. In other embodiments, the present disclosure provides a method of increasing weight in a subject. In other embodiments, the present disclosure provides a method of increasing height in a subject. In other embodiments, the present disclosure provides a method of increasing BMI in a subject. In other embodiments, the present disclosure provides a method of lowering the concentration of sweat chloride in a subject. In other embodiments, the present disclosure provides a method of improving pulmonary exacerbations in a subject. According to this aspect and in one embodiment, the present disclosure provides a method of extending the time to a first pulmonary exacerbation, a method of decreasing the number of pulmonary exacerbations, a method of decreasing the duration of pulmonary exacerbations, a method of decreasing the frequency of pulmonary exacerbations, a method of decreasing total number of days of hospitalization for pulmonary exacerbations, or a combination thereof. In other embodiments, the present disclosure provides a decrease in the need for antibiotic therapy or supportive drugs for sinopulmonary signs or symptoms in a subject. Each possibility represents a separate embodiment of the invention.

[0116] In some embodiments, the term “improving” as used herein refers to a favorable change, i.e., an increase or a 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% in a clinical parameter of cystic fibrosis.

[0117] In some embodiments, a composition comprising a synthetic oligonucleotide at a dose of 20, 25, 40, 50, 80, 100, or 160 mg increases the percentage of correctly spliced mature CFTR mRNA. In some embodiments, “the percentage of correctly spliced mature CFTR mRNA” asused herein refers to the percentage of correctly spliced mature CFTR mRNA compared to the total mRNA within the same cells.

[0118] In other embodiments, “the percentage of correctly spliced mature CFTR mRNA” is the percentage of correctly spliced mature CFTR mRNA compared to the amount of correctly spliced mature CFTR mRNA found in a healthy cell or subject not having a mutation in either allele of the CFTR gene. For example, an increase from 1% before treatment by the ASOs provided by the present disclosure to 11% after the treatment by the ASOs provided by the present disclosure is considered an increase of 10%. An increase from 10% before treatment by the ASOs provided by the present disclosure to 11% after the treatment by the ASOs provided by the present disclosure is considered an increase of 1%.

[0119] In some embodiments, the modified oligonucleotide, when administered at a 20, 25, 40, 50, 80, 100, or 160 mg dose, completely restores CFTR function compared to non-mutated CFTR. In other embodiments, the modified oligonucleotide restores at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of CFTR function compared to non-mutated CFTR. Each possibility represents a separate embodiment of the invention.

[0120] In some embodiments, a synthetic oligonucleotide comprising SEQ ID NO: 40, when administered at a 20, 25, 40, 50, 80, 100, or 160 mg dose increases the percentage of correctly spliced mature CFTR mRNA by at least about 10%. 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%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced mature CFTR mRNA by about 70%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced mature CFTR mRNA by about 80%. In certain embodiments, the oligonucleotide molecule increases the percentage of correctly spliced mature CFTR mRNA by about 90%.

[0121] In certain embodiments, the phrase “increases the level of correctly spliced mature CFTR mRNA” as used herein refers to the increase in the level of correctly spliced mature CFTR mRNA after treatment by the ASOs provided by the present disclosure compared to the level before treatment or after mock-treatment. In certain embodiments, the increase in the levelof correctly spliced mature CFTR mRNA is compared to mock- treatment by a control ASO. In certain embodiments, the control ASO consists of the nucleotide sequence in SEQ ID NO: 33. In certain embodiments, the phrase “increases the percentage of correctly spliced mature CFTR mRNA” as used herein refers to the percent increase in the ratio of correctly spliced to aberrantly spliced CFTR mRNA in treated subjects compared to untreated subjects or subjects receiving mock treatment.

[0122] In certain embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40, wherein said synthetic oligonucleotide is formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg, decreases the level of aberrantly spliced mature CFTR mRNA by about 30%. In certain embodiments, the oligonucleotide molecule decreases the level of aberrantly spliced mature CFTR mRNA by about 40%. In certain embodiments, the oligonucleotide molecule decreases the level of aberrantly spliced mature CFTR mRNA by about 50%. In certain embodiments, the oligonucleotide molecule decreases the level of aberrantly spliced mature CFTR mRNA by about 60%. In certain embodiments, the oligonucleotide molecule decreases the level of aberrantly spliced mature CFTR mRNA by about 70%. In certain embodiments, the oligonucleotide molecule decreases the level of aberrantly spliced mature CFTR mRNA by about 80%.

[0123] The phrase “decreases the level of aberrantly spliced mature CFTR mRNA” as used herein refers to the percentage decrease in the level of aberrantly spliced mature CFTR mRNA after treatment by the ASOs provided by the present disclosure compared to the level before treatment or after mock-treatment. In certain embodiments, the decrease in the level of aberrantly spliced mature CFTR mRNA is compared to mock treatment by a control ASO. In certain embodiments, the control ASO consists of the nucleotide sequence in SEQ ID NO: 33.

[0124] In certain embodiments, the use as described herein is for reducing the level of an mRNA molecule comprising the intron 22 cryptic exon. In certain embodiments, the use as described herein is for reducing the level of an mRNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 36. In certain embodiments, the use as described herein is for increasing the level of normal, full-length CFTR mRNA. In certain embodiments, the use as described herein is for increasing the level of an mRNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 34. In certain embodiments, the use as described herein is for correcting or improving chloride transport through the CFTR channel. In certain embodiments, the use as described herein is for increasing the production of functional CFTR protein. Each possibility represents a separate embodiment of the present disclosure.

[0125] It was found that the administration of a composition comprising an oligonucleotidemolecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg is not immunogenic or toxic. In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not increase blood levels of cytokines in vitro. In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not increase blood levels of IFNa, IL-2, IL-8, II- ip, IL-6, TNFa in vitro. In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not increase blood levels of IFNa, IL-2, IL-8, Il-ip, IL-6, TNFa in vitro above a predetermined and safe threshold. In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not increase blood levels of IFNa, IL-2, IL-8, II- 1 p, IL-6, TNFa in vitro above normal values, which, in one embodiment, is the range of concentrations of those markers in healthy subjects, or in another embodiment, in non-CF subjects.

[0126] In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not induce microscopic changes in the lung, tracheo-bronchial and mandibular lymph nodes, kidney and liver. In some embodiments, an oligonucleotide molecule comprising SEQ ID NO: 40 at a dose of 20, 25, 40, 50, 80, 100, or 160 mg does not increase cellularity basophilic granulation in lung, tracheo-bronchial lymph node, or liver.

[0127] As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human. In some embodiments, a subject is a subject diagnosed with CF.

[0128] In some embodiments, the subject is homozygous or heterozygous for the 3849 + 10Kb C-to-T mutation. In some embodiments, the subject is homozygous or heterozygous for the F508del mutation. In some embodiments, a subject treated according to the method of the present disclosure, comprises or is characterized by having a mixture of a wild type full-length and fully functional CFTR protein encoded from the wild type allele and a full-length and fully functional CFTR protein encoded from the pre-mRNA from which the inclusion of intron 22 cryptic exon was suppressed using the oligonucleotide of the present disclosure. In some embodiments, the oligonucleotide of the present disclosure does not reduce the level of the wild type full-length and fully functional CFTR protein in a subject, which, in one embodiment, is a subject heterozygous for the mutation disclosed hereinabove.

[0129] In some embodiments, a thick mucus forms in the airways of CF patients due to an impaired flow of chloride ions and water in and out of cells lining the airway surfaces. This malfunction leads to the excessive absorption of sodium ions and a decrease in water content inthe mucus-producing cells. As a result, the mucus becomes dehydrated and overly viscous, making it difficult to clear from the airways. This thickened mucus can clog the air passages, promote bacterial infections, and contribute to the progressive lung damage characteristic of CF. Thus, a cystic fibrosis drug administered nasally must be capable of crossing a thick mucus barrier.

[0130] In some embodiments, a composition comprising a synthetic oligonucleotide comprising SEQ ID NO: 40 formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg is surprisingly capable of crossing the thick mucus layer covering the lungs of CF patients.Kits

[0131] The present disclosure further provides, in another aspect, a kit comprising a synthetic oligonucleotide as described above. In some embodiments, the kit comprises SEQ ID NO: 40 formulated at a dose of 20, 25, 40, 50, 80, 100, or 160 mg and, optionally, a pharmaceutical carrier.

[0132] In certain embodiments, the kit further comprises an additional anti-cystic fibrosis agent such as an anti-cystic fibrosis drug or CFTR modulator as described herein.

[0133] In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are comprised in one pharmaceutical composition. In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are comprised in different pharmaceutical compositions. In certain embodiments, the synthetic oligonucleotide and the additional anti-cystic fibrosis agent are independently formulated for oral, nasal, inhalation, abdominal, subcutaneous, or intra-peritoneal administration. Each possibility represents a different embodiment of the invention.

[0134] In certain embodiments, the synthetic oligonucleotide in the kit is in a concentration of at least about 2.5 mg / ml. In certain embodiments, the synthetic oligonucleotide in the kit is in a concentration of at least about 2.5 mg / ml. In certain embodiments, the synthetic oligonucleotide in the kit is in a concentration of at least about 20 mg / ml. In certain embodiments, the synthetic oligonucleotide in the kit is in a concentration of 1 mg / ml to 100 mg / ml.

[0135] In one embodiment, the present disclosure provides combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is, at different timepoints and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation.

[0136] In some embodiments, the kit of the invention comprises: at least one oligonucleotide; and at least one of: at least one CFTR modulator; or at least one cystic fibrosis drug, wherein the oligonucleotide is selected from SEQ ID Nos.: 1-25, 40, and 41-44, and wherein the CFTR modulator is selected from: CFTR potentiator, CFTR corrector, and CFTR amplifier.

[0137] In some embodiments, the components of the kit disclosed above are sterile. As used herein, the term "sterile" refers to a state of being free from biological contaminants. Any method of sterilization is applicable and would be apparent to one of ordinary skill in the art.

[0138] In some embodiments, the components of the kit are packaged within a container. In some embodiments, the container is made of a material selected from the group consisting of thin-walled film or plastic (transparent or opaque), paperboard-based foil, rigid plastic, metal (e.g., aluminum), glass, etc.

[0139] In some embodiments, the content of the kit is packaged, as described below, to allow for storage of the components until they are needed. In some embodiments, some or all components of the kit may be packaged in suitable packaging to maintain sterility. In some embodiments, the components of the kit are stored in separate containers within the main kit containment element e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit.

[0140] In some embodiments, the instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. In some embodiments, the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0141] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise.Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application. Unless otherwise indicated, the word "or" in the specification and claims is considered to be the inclusive "or" rather than the exclusive “or”, and indicates at least one of, or any combination of items it conjoins.

[0142] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0143] In the description and claims of the present application, each of the verbs, "comprise", "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0144] 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, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.

[0145] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0146] Additional objects, advantages, and novel features of the present disclosure will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples, which are meant to be construed as non-limiting to the scope of the invention and are to serve merely as illustrative embodiments.

[0147] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments unless the embodiment is inoperative without those elements.EXAMPLES

[0148] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present disclosure include chemical, molecular, biochemical, and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6th Edition) by Leroy "Skip" G Wade; Organic Chemistry by T. W. Graham Solomons and, Craig Fryhle. EXAMPLE 1Phase I Clinical Studies for Safety and Tolerability of SPL84

[0149] The first-in-human, Phase 1 study evaluated the safety, tolerability, and pharmacokinetics (PK) of SPL84 single ascending doses in healthy volunteers. The study was a Phase 1, randomized, double-blind, placebo-controlled study.

[0150] 32 healthy male volunteers received a single dose of SPL84 or placebo administered via inhalation. Four doses were tested: 20, 40, 80, and 160 mg. The ratio of volunteers receiving the active component and placebo was 3:1.Table 3. Dosing Schedule

[0151] The study had a study steering committee to monitor subject safety during the study and make dose escalation decisions. The decision to escalate to the next dose level was made by the study steering committee, which reviewed blinded emerging safety and tolerability data of subjects in a given cohort.

[0152] The study included a screening period, a treatment day, and a follow-up period. All subjects were evaluated throughout the study.

[0153] The study included a 21 -day screening period, a treatment day, and a follow-up period. During the screening period, baseline levels of vital signs, clinical laboratory values, ECG, physical examination, and pulmonary function were ascertained for each subject. On the morning of Day 1, eligible, enrolled subjects received a single dose of study intervention by inhalation according to their assigned cohort and randomization. Subjects were discharged from the Phase 1 unit on Day 2, 24 hours postdose, after collection of blood samples for PK, pulmonary function, and safety evaluations. The subjects returned to the Phase 1 unit on Day 3 and 4 (for collection of blood samples for PK at 48 and 72 hours postdose, respectively). Subjects were followed daily by phone interview (Days 5-6) and returned to the clinic on Day 7 and Day 31 / EOS visit for follow-up safety assessments. For each subject, the study lasted up to 55 days (including screening).

[0154] SPL84 was safe and well tolerated, and no serious adverse events or significant related adverse events were reported. There were no significant changes from baseline in vital signs, clinical laboratory values, ECG, or physical examination. Also, there was no significant or irreversible effect on pulmonary function as measured using spirometry tests.

[0155] Spirometry was used to measure pulmonary function and provides timed measurements of expired lung volumes (Graham, 2019; Stanojevic, 2021). Spirometry was performed locally per ATS / European respiratory society (ERS) Standards and GLI reference equations were used. The following parameters were evaluated:FEV 1 (Forced expiratory volume) - volume exhaled in 1 second (Figure 1A)FVC (Forced vital capacity) - the amount of air that can be forcibly exhaled from the lungs (Figure IB)FEF25-75 (Forced expiratory flow between 25% and 75% of vital capacity) - exhaled vital capacity at 25 and 75% of the pulmonary volume (Figure 1C)

[0156] All changes in the measured parameters were within normal range of pulmonary function (Figures 1A-1C).

[0157] Plasma pharmacokinetic parameters following inhalation administration of a nebulized SPE84 solution are presented in Table 4 below.Table 4. Mean SPL84 Plasma Pharmacokinetic Parameters Following InhalationAdministration of a Nebulized SPL84 Solution

[0158] Systemic exposure to SPE84 was low and tended to be dose dependent (Figure 2). The average area under the curve (AUC) for the 160mg group was 331 ng / ml*hr (Table 4), while the AUC at the NOAEE (no observed adverse effect level) in the mouse toxicity study was ~20X higher than the human exposure at 160 mg.

[0159] Absorption was rapid, with SPE84 Cmax observed at a median Tmax of 1 hr in Cohort 1, 2 hrs in Cohort 2, and 4 hrs in Cohorts 3 and 4.

[0160] There was a slightly high dose-proportional increase in plasma SPL84 exposure with increasing dose, based on Cmax and AUCO-t.

[0161] The terminal elimination phase could be successfully described only for the A3 and A4 and was 3.77 and 5.23 hr, respectively.

[0162] Conclusion: SPL84 was safe and well-tolerated when administered as a single inhaled dose to healthy volunteers up to doses of 160 mg, with minimal systemic exposure. There were no safety issues observed, no serious adverse effects, no significant treatment- related adverse effects, and, importantly, no significant effect on pulmonary function. The independent Study Steering Committee confirmed successful completion of the study and approved moving to multi-dosing of cystic fibrosis patients in a Phase 2 study.

[0163] In animal models, SPL84 demonstrated broad distribution, as well as cell and nucleus penetration of lungs. SPL84 administration overcame hyper-concentrated mucus barriers, thus leading to specific and targeted RNA modulation.

[0164] CF is one of the most common genetic diseases, leading to frequent lung infections, breathing difficulties and reduced life expectancy. Although currently available treatments managed to significantly increase life expectancy in the past few decades, they are focused on symptomatic treatments and there is still a significant unmet medical need for a CF cure. In particular CF patients carrying the 3849 +10 Kb C->T mutation have no specific approved treatment. SPL84 completely restored CFTR activity in the CF gold standard pharmacological model, and the results support its safety in humans.

[0165] Additional data demonstrated broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of the inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL8 through CF patients hyper-concentrated mucus was also demonstrated. The results, supported by a promising preclinical pharmacological effect of full restoration of CFTR channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients.

[0166] Broad lung distribution of SPL84 support the further development of a wider pulmonary pipeline, including treatments for muco-obstructive diseases and idiopathic pulmonary fibrosis.

[0167] The ASO binds specifically to the mutated CFTR RNA in the targeted sequence, leading to the modulation of the mutated region in the mRNA, allowing the cell to produce fully functional CFTR proteins. The ASOs are administered directly and preferentially to the lungs via inhalation which lasts less than 10 minutes and is given weekly or every otherweek. The ASOs were shown to be efficiently taken up by the lung cells and drive the production of corrected CFTR mRNA and eventually fully functional CFTR proteins.EXAMPLE 2Phase II Clinical Studies of SPL84

[0168] The Phase II clinical study is a multiple ascending dose study in CF patients carrying the 3849 + 10Kb C-to-T mutation. The doses are 25 mg, 50 mg, and 100 mg; n=24, dose / week x 9 weeks; 3:1 active:placebo.

[0169] The goal of this clinical trial is to test if various doses of SPL84 are safe for adult patients with cystic fibrosis (CF) and if SPL84 is effective in treating CF patients with a particular CFTR mutation.Inclusion Criteria:

[0170] Diagnosis of CF and two CF causing mutations; 3849+10 Kb C->T mutation on one allele in the CF transmembrane conductance regulator (CFTR) gene (homozygote or compound heterozygote). Source documentation from a certified genetic laboratory is required;

[0171] Body mass index (BMI) of > 17 kg / m2;

[0172] FEV1 40-90% predicted at screening;

[0173] Non-smokers or vapers for at least 180 days (6 months) prior to screening, per participant report.Exclusion Criteria:

[0174] Use of Kalydeco, Orkambi, Symdeko / Symkevi or Trikafta / Kaftrio within 30 days of first dose with study intervention;

[0175] Use of any investigational drug (other than SPL84) or device within 30 days of first dose with study intervention;

[0176] Use of systemic steroids over 3 consecutive months in the last 6 months prior to screening, or use of systemic steroids in the last month prior to screening. Use of inhaled steroids above 1 mg;

[0177] Use of CF medications, e.g. inhaled antibiotics, dornase alfa (Pulmozyme), hypertonic saline and physiotherapy should be on stable regimen for the period 28 days prior to screening; those participants taking inhaled antibiotics for prophylaxis must be on a stable regimen of these drugs for at least 90 days prior to first dose with study intervention;

[0178] Any acute infection including acute upper respiratory or lower respiratory infections, pulmonary exacerbation, changes in therapy for pulmonary disease, or any non CF-related illness which results in the initiation of any new therapy within 14 days prior to first dose with study intervention;

[0179] Hemoptysis of greater than 30 mL within 90 days prior to Day 1, or hospitalization for hemoptysis within 6 months of first dose with study intervention;

[0180] Liver disease characterized by clinically significant cirrhosis and / or documented portal hypertension;

[0181] History of any organ transplantation;

[0182] Documented coronavirus disease (COVID-19) infection within 4 weeks prior to dosing.Primary Outcome Measures

[0183] Safety and Tolerability of SPL84 as evaluated by number of subjects with at least one treatment-related adverse event (AE) or serious adverse event (SAEs)

[0184] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal heart rate

[0185] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal respiratory rate

[0186] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal systolic and diastolic blood pressure

[0187] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal oximetry

[0188] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal temperature

[0189] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal hematology lab test results

[0190] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal biochemistry lab test results

[0191] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal urinalysis lab test results

[0192] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal electrocardiogram (ECG) parameters

[0193] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal physical examination findings

[0194] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal pulmonary function tests results

[0195] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal sputum microbiology results

[0196] Safety and Tolerability of SPL84 as assessed by number of participants with abnormal immunogenicity resultsSecondary Outcome Measures

[0197] Characterization of pharmacokinetics (PK) of SPL84: maximum serum concentration (Cmax)

[0198] Characterization of PK of SPL84: Time to Cmax (Tmax)

[0199] Characterization of PK of SPL84: terminal elimination half-life (t 1 / 2)

[0200] Characterization of PK of SPL84: Area under the curve to the final sample (AUC0- t)

[0201] Characterization of PK of SPL84: Area under the curve to infinity (AUC0-co)

[0202] Characterization of PK of SPL84: Apparent clearance (CL / F)

[0203] Characterization of excretion of SPL84: concentration of SPL84 in urine

[0204] Preliminary efficacy of SPL84 as assessed by change from baseline in percent predicted FEV 1

[0205] Preliminary efficacy of SPL84 as assessed by change from baseline in Cystic Fibrosis Questionnaire-Revised Respiratory Symptom Score

[0206] Preliminary efficacy of SPL84 as assessed by change from baseline in body weight

[0207] Preliminary efficacy of SPL84 as assessed by change from baseline of antibiotic treatment

Claims

CLAIMSWhat is claimed is:

1. A synthetic oligonucleotide comprising SEQ ID NO: 40, wherein said synthetic oligonucleotide is formulated at a dose of 25, 50, or 100 mg.

2. A synthetic oligonucleotide comprising SEQ ID NO: 40, wherein said synthetic oligonucleotide is formulated at a dose of 20, 40, 80, or 160 mg.

3. The synthetic oligonucleotide of claim 1 or claim 2, wherein said synthetic oligonucleotide comprises a chemical modification of the backbone of the oligonucleotide, a chemical modification of a sugar of the oligonucleotide, or a combination thereof.

4. The synthetic oligonucleotide of claim 3, wherein the chemical modification comprises a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, an alternating locked nucleic acid backbone, a constrained ethyl (cET) backbone, a phosphorothioate backbone, N3'-P5' phosphoroamidates, 2'-deoxy-2'-fluoro-P-d-arabino nucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, or a combination thereof.

5. The synthetic oligonucleotide of claim 3, wherein the chemical modification comprises a 2’-O-Methyl phosphorothioate (2’0MP) modification or a 2’ -Methoxy Ethyl (2’MOE) phosphorothioate modification.

6. A composition comprising the synthetic oligonucleotide of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the composition is formulated for administration by inhalation.

8. The composition of claim 6, wherein said composition is administered via oral, nasal,abdominal, subcutaneous, intra-peritoneal or intravenous administration.

9. The composition of any one of claims 6-8, further comprising a cystic fibrosis transmembrane conductance regulator (CFTR)-splicing-modulating agent, a CFTR potentiator, a CFTR corrector, a translational read-through agent, a CFTR amplifier, or a combination thereof.

10. The composition of claim 9, wherein said CFTR potentiator comprises N-(2,4-Di-tert- butyl-5-hydroxyphenyl)-4-oxo- 1 ,4-dihydroquinoline-3-carboxamide (Ivacaftor; VX- 770), QBW251, PTI-808, ABBV-191, ABBV-3067 (SION-3067), GLPG1837, or VX-561 (deuterated ivacaftor); wherein said CFTR corrector comprises 3-{6-{ [l-(2,2-difluoro-l,3-benzodioxol-5- yl)cyclopropanecarbonyl] amino }-3-methylpyridin-2-yl (benzoic acid (Lumacaftor; VX-809), l-(2,2-difluoro-l,3-benzodioxol-5-yl)-~ {N(-[l-[(2~{R})-2,3- dihydroxypropyl] -6-fluoro-2-( 1 -hydroxy-2-methylpropan-2-yl)indol-5 - yl] cyclopropane- 1 -carboxamide (Tezacaftor; VX-661), VX-659, elexacaftor (VX- 445), VX-121, VX-152, VX-440, FDL169, ABBV-3221, ABBV-2222 (SION- 2222), ABBV-119, ABBV-2851 (SION-2851) GLPG2737, SION-638, SION-719, SION-451, SION-109, or PTI-801; wherein said translational read-through agent comprises 3-[5-(2-fluorophenyl)-l,2,4- oxadiazol-3-yl]benzoic acid (Ataluren), ELX-02, SRI-41315, Release factor inhibitors, tRNA, aminoglycosides, or G418; wherein said CFTR amplifier comprises PTI-428, or a combination thereof.

11. The composition of any one of claims 6-10, wherein said composition further comprises a therapeutically effective amount of one or more cystic fibrosis drugs.

12. The composition of claim 11, wherein said cystic fibrosis drug is an antibiotic drug, a bronchodilator, or a corticosteroid, or any combination thereof.

13. The composition of any one of claims 6-12 for use in the treatment of cystic fibrosis in a subject in need thereof.

14. The composition of any one of claims 6-12 for use in suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in a subject in need thereof.

15. A method for treating cystic fibrosis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide comprising SEQ ID NO: 40, wherein said synthetic oligonucleotide is formulated at a dose of 25, 50, or 100 mg.

16. A method for suppressing or inhibiting inclusion of a cryptic exon between exon 22 and 23 of CFTR mRNA in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a synthetic oligonucleotide comprising SEQ ID NO: 40, wherein said synthetic oligonucleotide is formulated at a dose of 25, 50, or 100 mg.

17. The method of claim 15 or claim 16, wherein said synthetic oligonucleotide comprises a chemical modification of the backbone of the oligonucleotide, a chemical modification of a sugar of the oligonucleotide, or a combination thereof.

18. The method of claim 17, wherein the chemical modification comprises a phosphateribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, an alternating locked nucleic acid backbone, a constrained ethyl (cET) backbone, a phosphorothioate backbone, N3'-P5' phosphoroamidates, 2'-deoxy-2'- fluoro-P-d-arabino nucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, or a combination thereof.

19. The method of claim 17, wherein the chemical modification comprises a 2’-O-Methyl phosphorothioate (2’0MP) modification or a 2’-Methoxy Ethyl (2’MOE) phosphorothioate modification.

20. The method of any one of claims 15-19, wherein said composition is administered via inhalation.

21. The method of any one of claims 15-19, wherein said composition is administered via oral, nasal, abdominal, subcutaneous, intra-peritoneal or intravenous administration.

22. The method of any one of claims 15-21, wherein said subject has a 3849 + 10Kb C- to-T mutation in the CFTR gene or a combination of a 3849 + 10Kb C-to-T mutation and a F508del mutation in the CFTR gene.

23. The method of any one of claims 15-22, wherein a pathologically thick mucus layer covers the airways of said subject.

24. The method of any one of claims 15-23, wherein the synthetic oligonucleotide is in a pharmaceutical composition and said subject is administered said composition comprising said synthetic oligonucleotide.

25. The method of any one of claims 15-24, further comprising the step of administering to said subject a compound selected from a CFTR-splicing-modulating agent, a CFTR potentiator, a CFTR corrector, a translational read-through agent, a CFTR amplifier, or a combination thereof.

26. The method of claim 25, wherein said CFTR potentiator comprises N-(2,4-Di-tert- butyl-5-hydroxyphenyl)-4-oxo- 1 ,4-dihydroquinoline-3-carboxamide (Ivacaftor; VX- 770), QBW251, PTI-808, ABBV-191, ABBV-3067 (SION-3067), GLPG1837, or VX-561 (deuterated ivacaftor); wherein said CFTR corrector comprises 3-{6-{ [l-(2,2-difluoro-l,3-benzodioxol-5- yl)cyclopropanecarbonyl] amino }-3-methylpyridin-2-yl (benzoic acid (Lumacaftor; VX-809), l-(2,2-difluoro-l,3-benzodioxol-5-yl)-~{N(-[l-[(2~{R})-2,3- dihydroxypropyl] -6-fluoro-2-( 1 -hydroxy-2-methylpropan-2-yl)indol-5 - yl] cyclopropane- 1 -carboxamide (Tezacaftor; VX-661), VX-659, elexacaftor (VX- 445), VX-121, VX-152, VX-440, FDL169, ABBV-3221, ABBV-2222 (SION- 2222), ABBV-119, ABBV-2851 (SION-2851) GLPG2737, SION-638, SION-719, SION-451, SION-109, or PTI-801; wherein said translational read-through agent comprises 3-[5-(2-fluorophenyl)-l,2,4- oxadiazol-3-yl]benzoic acid (Ataluren), ELX-02, SRI-41315, Release factorinhibitors, tRNA, aminoglycosides, or G418; wherein said CFTR amplifier comprises PTI-428, or a combination thereof.

27. The method of any one of claims 15-26, wherein said treatment with said oligonucleotide has one or more effects on said subject selected from improving lung function, increasing time to the first pulmonary exacerbation, increasing weight, increasing height, increasing Body Mass Index (BMI), lowering concentration of sweat chloride, lowering number and / or duration and / or frequency of pulmonary exacerbations, decreasing number of days of hospitalization for pulmonary exacerbations, and decreasing need for antibiotic therapy or supportive drugs for sinopulmonary signs or symptoms.

28. The method of any one of claims 15-27, wherein said synthetic oligonucleotide is administered weekly or once every 2 weeks.

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