WO2023152369A1 - Nucleic acid mir-9 inhibitor for the treatment of cystic fibrosis - Google Patents

Nucleic acid mir-9 inhibitor for the treatment of cystic fibrosis Download PDF

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WO2023152369A1
WO2023152369A1 PCT/EP2023/053501 EP2023053501W WO2023152369A1 WO 2023152369 A1 WO2023152369 A1 WO 2023152369A1 EP 2023053501 W EP2023053501 W EP 2023053501W WO 2023152369 A1 WO2023152369 A1 WO 2023152369A1
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cftr
nucleic acid
mir
class
inhibitor
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French (fr)
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Olivier Tabary
Florence SONNEVILLE
Christie MITRI
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Sorbonne Université
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of cystic fibrosis.
  • Cystic fibrosis is an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene (1). More than 2,100 different mutations have been described affecting protein production or activity.
  • the CFTR chloride channel present in many epithelia, regulates transepithelial salt and water fluxes.
  • impaired chloride ion efflux coupled with sodium ion hyperabsorption results in epithelial surface dehydration and thick and tenacious mucus obstructing the lungs, pancreatic ducts, the biliary tract, the intestine, and the male reproductive tract.
  • Chronic lung infections are CF’s most common clinical manifestation, leading to pulmonary inflammation, progressive tissue destruction and loss of function, and elevated morbidity and mortality.
  • TMEM16a (anoctamin-1) was identified by three different groups as the gene encoding the airway Ca2+-activated Cl- channel (CaCC) (5-7).
  • TMEM16a like CFTR, is located in airway epithelia, participates in the homeostasis of the airway surface fluid, and has been proposed as an alternative strategy to compensate for CFTR deficiency (8).
  • this protein is downregulated in CF bronchia (9).
  • a strategy increasing TMEM16a could bypass CFTR deficiency and be applied to all patients with CF, particularly those who cannot benefit from current curative treatments.
  • the inventors first demonstrated the inhibitory role of microRNA-9 (miR-9) in the regulation of TMEM16a (9), which other groups have since confirmed in different pathologies (10-13). Then, they developed a strategy based on antisense oligonucleotide (ASO) to inhibit the fixation of miR-9 on the 3’UTR of TMEM16a by competitive interaction and consequently increase its expression (9).
  • ASO antisense oligonucleotide
  • TMEM16a 3’UTR acts as a corrector to increase the expression at the apical membrane. They report that this strategy could apply to all patients with CF to correct chloride efflux and mucociliary clearance without inducing inflammation or toxicity. Consequently, they demonstrated that this strategy could increase the life expectancy of CF mice.
  • TMEM16a chloride activity could represent a therapeutic potential for drug development for all patients with CF.
  • the present invention relates to methods and pharmaceutical compositions for the treatment of cystic fibrosis.
  • the present invention is defined by the claims.
  • CF cystic fibrosis
  • the aims here is to study the effects of TMEM16a potentiation in vitro and in vivo and prepare for preclinical studies by assessing the best administration route and the TMEM16a oligonucleotide's toxicity and specificity.
  • the experiments are performed on cell lines and primary cells with different mutation classes.
  • the CF mouse model is used to study the different administration routes, complete survival data, and study long-term effects.
  • TMEM16a ASO4 The oligonucleotide (TMEM16a ASO4) potentiates the activity of TMEM16a and increases chloride efflux in CFBE41o- cells and mucociliary clearance in human bronchial epithelial cells (hBEC) from patients with CF with different mutations.
  • TMEM16a ASO4 is detectable 30 days after subcutaneous injection in CF mice.
  • TMEM16a ASO4 significantly increases the mice's lifespan. Recovery in CF male mice fertility was noticed. Acute administration of 50 times the effective dose did not show behavioral changes nor macroscopic or pathological changes.
  • TMEM16a ASO4 is very specific and neither induces inflammation nor alters intracellular calcium mobilization or cell proliferation.
  • the first object of the present invention relates to a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO:1).
  • a second object of the present invention relates to the method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1).
  • a subject or “patient” denote a mammal, such as a rodent, a feline, a canine, a pig, a ferret, a rat, mice, a rabbit and a primate.
  • a subject refers to any subject (preferably human) afflicted or at risk of being afflicted with cystic fibrosis.
  • the method of the invention may be performed for any type of cystic fibrosis, such as revised in the World Health Organization Classification of cystic fibrosis and selected from the E84 group: mucoviscidosis, Cystic fibrosis with pulmonary manifestations, Cystic fibrosis with intestinal manifestations and Cystic fibrosis with other manifestations.
  • the subject of the present is sterile.
  • the subject, according to the invention is a human.
  • the subject, according to the invention is a girl or a boy.
  • the subject, according to the invention is an adult.
  • the subject is a child (a human being between the stages of birth and puberty), a teenager (a human being between the stages of puberty to adulthood), or an older person (a human being after puberty).
  • the subject is less than 15 years old. In some embodiments, the subject is less than 10 years old. In some embodiments, the subject is less than 7 years old. In some embodiments, the subject is less than 5 years old. In some embodiments, the subject is less than 3 years old. In some embodiments, the subject is more than 15 years old. In some embodiments, the subject is more than 20 years old. In some embodiments, the subject is more than 25 years old. In some embodiments, the subject is more than 30 years old. In some embodiments, the subject is more than 35 years old. In some embodiments, the subject is more than 50 years old. In some embodiments, the subject is more than 60 years old. In some embodiments, the subject is more than 65 years old, including the elderly.
  • CF Cerastic fibrosis
  • CF cystic fibrosis transmembrane conductance regulator
  • CFTR protein refers to the CFTR protein of 1480 amino acids, also called Cystic Fibrosis Transmembrane conductance Regulator.
  • the CFTR protein is a chloride (C1-) channel found in the membranes of secretory tissues such as intestinal and respiratory mucosa.
  • the CFTR protein is represented by the NCBI reference sequence: P13569.3 (SEQ ID NO: 2)
  • CFTR gene refers to the CFTR gene located on chromosome 7 and may be found in NCBI GenBank locus AC000111 and AC000061, the contents of which are incorporated herein in their entirety by reference.
  • the cDNA for the CFTR gene is found in Audrezet et al., Hum. Mutat. (2004) 23 (4), 343-357.
  • a nucleic acid sequence for human CFTR is represented by SEQ ID NO: 3(NCBI Reference Sequence: NM_000492.3).
  • AN01 belongs to a protein family composed of 10 members whose primary sequence and predicted structure (eight transmembrane domains) have no similarity with those of other anion channels.
  • An exemplary human nucleic acid sequence is the NCBI Reference Sequence NM_018043.5 (SEQ ID NO:4).
  • the term "gene” has its general meaning in the art and refers to means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
  • allele has its general meaning in the art and refers to an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated, results in functional or dysfunctional (including non-existent) gene products.
  • mutation has its general meaning in the art and refers to any detectable change in genetic material, e.g., DNA, RNA, cDNA, or any process, mechanism, or result of such a change.
  • Mutations include the deletion, insertion, or substitution of one or more nucleotides.
  • the mutation may occur in the coding region of a gene (i.e., in exons), in the introns, or in the regulatory regions (e.g., enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, promoters) of the gene.
  • a mutation is identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population.
  • the mutation may be a “missense” mutation, where it replaces one amino acid with another in the gene product, or a “non-sense” mutation, where it replaces an amino acid codon with a stop codon.
  • a mutation may also occur in a splicing site where it creates or destroys signals for exon-intron splicing, thereby leading to a gene product of altered structure.
  • a mutation in the genetic material may also be “silent”, i.e., the mutation does not result in an alteration of the amino acid sequence of the expression product.
  • mutations identified in the CFTR gene or protein are designated pursuant to the nomenclature of Dunnen and Antonarakis (2000).
  • “>” indicates a substitution at the DNA level; (underscore) indicates a range of affected residues, separating the first and last residue affected; “del” indicates a deletion, “dup” indicates a duplication; “ins” indicates an insertion, “inv” indicates an inversion and “con” indicates a conversion. More particularly, “X” denotes that an amino acid is changed to a stop codon (X).
  • the term “homozygous” refers to an individual possessing two copies of the same allele.
  • the term “homozygous mutant” refers to an individual possessing two copies of the same allele, such allele being characterized as the mutant form of a gene.
  • heterozygous refers to an individual possessing two different alleles of the same gene, i.e., an individual possessing two different copies of an allele, such alleles are characterized as mutant forms of a gene.
  • the subject harbors at least one mutation in the CFTR gene.
  • the CFTR gene mutations were classified into six classes according to their resulting damaging effect on the protein (Elborn JS, 2016):
  • Class I mutations are responsible for defects in the early steps of biosynthesis and mainly include premature termination codons or stop codons, often causing mRNA degradation. In class I, some mutations (class la) do not allow mRNA synthesis; they are sometimes called class VII. (De Boeck & Amaral, 2016). Class I is the most challenging CFTR defect to address since no CFTR protein is produced. Nonsense and frameshift mutations account for 8.39% and 16.21% of the total mutations (cftr2.org), respectively. Exemples of class I mutations: W1282X, 1717-1G->A, G542X, R553X, 2183AA>G, 2184delA
  • Class II mutations disrupt protein maturation and trafficking to the plasma membrane (PM).
  • the protein is either absent or present in reduced quantity at the apical membrane.
  • the F508del mutation represents around 70% of all associated alleles; thus, given the recessive inheritance of the disease, approximately 90% of patients with CF carry at least one class II mutation.
  • Class III mutations are most often located in the adenosine-5’ -triphosphate (ATP) binding domains and disrupt the regulation of the CFTR channel requiring ATP hydrolysis at nucleotide-binding domains and cAMP-dependent phosphorylation at the cytoplasmic domain for activation. Hence, the abnormal protein can reach the PM but is not activated by ATP or cAMP.
  • ATP adenosine-5’ -triphosphate
  • Class IV mutations are missense mutations located in the transmembrane channel and affect the conductance of the CFTR channel. Some transmembrane domain segments participate in ionic pore formation. Missense mutations in these regions produce a correctly positioned protein with cAMP-dependent Cl- activity. Nevertheless, these channels’ characteristics differ from those of the endogenous CFTR channel, with decreased ion flux and altered selectivity, making the protein partially functional.
  • class IV mutations R117H, R334W
  • Class V mutations correspond to molecular anomalies that affect transcription (quantitative defect). They alter the mRNA stability; thus, the protein is functional, but the density of CFTR protein expressed at the membrane is low. Exemples of class V mutations: 2789+5G->A, A455E
  • Class VI mutations include some infrequent mutations resulting in a mature functional protein but with an altered stability. Exemples of class VI mutations: 4326delTC, Glnl412X, 4279insA
  • CFTR mutations include, but are not limited to 124del23bp CFTR, CFTRdelel CFTR, M1V CFTR, Q2X CFT, S4X CFTR, P5L CFTR, S13F CFTR, L15P CFTR, 182delT CFTR, CFTRdele2 CFTR, CFTRdele2-4 CFTR, 185+1G->T CFTR, CFTRdele2,3 CFTR, W19X CFTR, G27R CFTR, G27X CFTR, Q30X CFTR, R31C CFTR, R31L CFTR, Q39X CFTR, A46D CFTR, 296+lG->A CFTR, 296+lG->T CFTR, CFTRdele3-10,14b-16 CFTR, 296+28A->G CFTR, 296+2T->C CFTR, 296+3insT CFTR, 297-3
  • the subject harbors at least one allelic mutation selected from class I, class II, class III, class IV, class V or class VI. In one embodiment, the subject harbors at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the first allele and at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the second allele. In a particular embodiment, the subject harbors at least a mutation of class I in the first allele and at least a mutation of class II in the second allele. In a particular embodiment, the subject harbors at least a mutation of class II in the first allele and at least a mutation of class II in the second allele.
  • the subject harbors at least one allelic mutation in the CFTR gene, including but not limited to F508del-CFTR, R117H CFTR, 2184delA CFTR, W1282X CFTR, 2183AA>G CFTR or G551D CFTR.
  • the subject harbors at least a F508del mutation in the CFTR gene.
  • the subject harbors at least a F508del mutation in the first allele and at least a F508del mutation in the second allele.
  • the subject harbors at least a 2184delA mutation in the CFTR gene.
  • the subject harbors at least a W1282X mutation in the CFTR gene.
  • the subject harbors at least a 2184delA mutation in the first allele and at least a W1282X mutation in the second allele.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen means the treatment pattern of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase "induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of particular predetermined criteria [e.g., disease manifestation, etc.]).
  • miRNAs refers to mature microRNA (non-coding small RNAs) molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from longer precursor RNA molecules (“precursor miRNA”: pri-miRNA and pre-miRNA). Pri -miRNAs are transcribed from non-protein-encoding genes or embedded into protein-coding genes (within introns or non-coding exons).
  • the “precursor miRNAs” fold into hairpin structures containing imperfectly base-paired stems and are processed in two steps, catalyzed in animals by two Ribonuclease Ill-type endonucleases called Drosha and Dicer.
  • miR-9 microRNA is a short non-coding RNA gene involved in gene regulation.
  • the mature ⁇ 23nt miRNAs are processed from hairpin precursor sequences by the Dicer enzyme.
  • the dominant mature miRNA sequence is processed from the 5' UTR of the miR-9 precursor, and from the 3' UTR of the mir-9 precursor.
  • the mature products are thought to have regulatory roles through complementarity to mRNA.
  • nucleic acid miR-9 inhibitor i.e., a nucleic acid that inhibits miR- 9 relates to any nucleic acid, for example, an oligonucleotide, that reduces (i.e., inhibits) the biological activity of miR-9.
  • nucleic acid miR-9 inhibitors comprise a nucleic acid sequence that is complementary to at least a portion of the nucleic acid sequence of either miR-9 itself or the target mRNA sequences of miR-9; inhibition of miR-9, therefore, occurs by binding of the inhibitor to miR-9 or to its target mRNA. In both cases, miR-9 is prevented from recognizing and binding its target sequence and thus cannot induce gene silencing.
  • the nucleic acid miR-9 inhibitor of the invention may comprise a structure that is single-stranded, double-stranded, partially double-stranded or hairpin in nature.
  • the nucleic acid miR-9 inhibitor binds to miR-9.
  • the nucleic acid miR-9 inhibitor binds to and sequesters miR-9, preventing it from binding to its target mRNA sequences. Binding occurs via complementary base pairing between at least one nucleotide present in the nucleic acid miR-9 inhibitor and a corresponding nucleotide present in miR-9, such that at least a portion of the nucleic acid miR-9 inhibitor and miR-9 together define a base-paired nucleic acid duplex.
  • Said complementary base pairing can occur over a region of two or more contiguous nucleotides of miR- 9 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides).
  • a base-paired nucleic acid duplex formed when the nucleic acid miR-9 inhibitor binds to miR- 9 may comprise one or more mismatch pairings.
  • two or more regions of complementary base-paired nucleic acid duplex are formed, wherein each region is separated from the next by one or more mismatch pairings.
  • complementary refers to a nucleic acid molecule that forms hydrogen bonds with another nucleic acid molecule with Watson-Crick base pairing.
  • Watson-Crick base pairing refers to the following hydrogen-bonded nucleotide pairings: A:T and C:G (for DNA); and A:U and C:G (for RNA).
  • two or more complementary nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region, with or without an overhang) or have a different number of nucleotides (e.g., one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand).
  • the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence complementary to at least a portion of the miR-9 sequence.
  • the nucleic acid miR-9 inhibitor may comprise a nucleic acid sequence complementary to 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides of miR-9.
  • the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence having at least 70% sequence identity to a complementary (i.e., antisense) sequence of miR-9.
  • a first nucleic acid sequence having at least 70% of identity with a second nucleic acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second nucleic acid sequence.
  • Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • Methods of alignment of sequences for comparison are well- known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc.
  • ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity.
  • these alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance.
  • the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • the BLAST sequence comparison system is available, for instance, from the NCBI website; see also Altschul et al., J. Mol.
  • the nucleic acid miR-9 inhibitor comprises a sequence complementary to miR-9.
  • the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence that differs from the complementary (i.e., antisense) sequence of miR9 at a maximum of 10 (e.g., a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) nucleotide positions.
  • the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence that differs from the complementary (i.e., antisense) sequence of miR9 at a maximum of 5 (e.g., a maximum of 4, 3, 2, or 1) nucleotide positions.
  • said nucleic acid sequence is identical to the complementary (i.e., antisense) sequence of miR9 except at a limited number of nucleotide positions.
  • the nucleic acid miR-9 inhibitor as described above, has a maximum length of 60 (for example, 55, 50, 45, 40, 35, 30, or 25) nucleotides.
  • the nucleic acid miR-9 inhibitor is a miR-9 antagomir.
  • an “antagomir” is a nucleic acid oligomer designed to bind to a specific target microRNA via complementary base pairing (for example, as described above).
  • An antagomir may have a wholly or partially complementary sequence to the microRNA target sequence.
  • Antagomirs may have a single-stranded, double-stranded, partially double-stranded, or hairpin structure.
  • Antagomirs may further comprise chemically modified nucleotides (e.g., as described below). Methods for designing and creating antagomirs are known in the art.
  • the nucleic acid miR-9 inhibitor is a miR-9 microRNA sponge.
  • miR-9 microRNA sponge is a nucleic acid that comprises multiple (e.g., at least 2, 3, 4, 5 or 6) binding sites for a specific target microRNA.
  • a microRNA-sponge can bind and sequester multiple target microRNA molecules.
  • a microRNA sponge may comprise an mRNA expressed from a vector (e.g., a viral vector or plasmid vector). The presence in a microRNA-sponge of multiple binding sites for the target microRNA enables microRNAs to be adsorbed in a manner analogous to a sponge soaking up water.
  • a microRNA-sponge may bind target microRNAs via complementary base pairing (for example, as described above).
  • a microRNA-sponge may comprise multiple (e.g., at least 2, 3, 4, 5 or 6) nucleic acid sequences, each sequence being complementary to at least a portion of the microRNA target sequence.
  • a microRNA- sponge may comprise multiple (e.g., at least 2, 3, 4, 5 or 6) nucleic acid sequences, wherein each sequence is complementary to the microRNA target sequence.
  • the nucleic acid miR-9 inhibitor comprising two or more nucleic acid sequences, wherein each of said two or more nucleic acid sequences has at least 70% sequence identity to the complementary (i.e., antisense) sequence of miR9 is a miR-9 microRNA sponge.
  • the nucleic acid miR-9 inhibitor does not bind directly to miR-9 but instead binds to a miR-9 mRNA target site in the ANO1 nucleic acid sequence. This has the effect of blocking said target site (for example, by steric interference), preventing its recognition and binding by miR-9, and thus inhibiting miR-9 and its actions.
  • the binding of the nucleic acid miR-9 inhibitor of the invention to a miR-9 mRNA target site does not induce gene silencing (e.g., by mRNA degradation or translational repression) of said target mRNA.
  • the miR- 9 mRNA target site is located on an AN01 3 ' UTR.
  • the nucleic acid miR-9 inhibitor is a Target Site Blocker (TSB).
  • TLB Target Site Blocker
  • binding between the nucleic acid miR-9 inhibitor and the miR-9 mRNA target site occurs via complementary base pairing between at least one nucleotide present in the nucleic acid miR-9 inhibitor and a corresponding nucleotide present in the miR- 9 mRNA target site, such that at least a portion of the nucleic acid miR-9 inhibitor and the miR- 9 mRNA target site together define a base-paired nucleic acid duplex.
  • Said complementary base pairing can occur over a region of two or more contiguous nucleotides of the miR-9 mRNA target site (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides).
  • a base-paired nucleic acid duplex formed when the nucleic acid miR-9 inhibitor binds to the miR-9 mRNA target may comprise one or more mismatch pairings.
  • two or more regions of complementary base-paired nucleic acid duplex are formed, wherein each region is separated from the next by one or more mismatch pairings.
  • the mRNA target site is located on the 3' UTR (untranslated region) of the target ANO1 mRNA. In some embodiments, wherein the nucleic acid miR-9 inhibitor competes with miR-9 for binding to a miR-9 mRNA target site.
  • the nucleic acid miR-9 inhibitor is TMEM16a ASO.
  • ANO1 ASO or “TMEM16a ASO” OR “TMEM16a ASO4” refers to the sequence AATCTTTGGTAGTAA (SEQ ID NO: 1).
  • TMEM16a ASO of the present invention competes with miR-9 for binding to a miR-9 mRNA target site.
  • said nucleic acid sequence binds to the miR-9 mRNA target site in TMEM16a via complementary binding at the location targeted by the seed region of miR-9, thus preventing miR-9 from binding.
  • the nucleic acid miR-9 inhibitor is a small interfering RNA (siRNA) targeted against miR-9.
  • siRNA small interfering RNA
  • the term “siRNA” has its general meaning in the art and refers to a double-stranded interfering RNA.
  • siRNA useful in the present methods, comprises short double-stranded RNA from about 17 nucleotides to about 29 nucleotides, preferably from about 19 to about 25 nucleotides in length.
  • the siRNA comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired").
  • the sense strand comprises a nucleic acid sequence substantially identical to a nucleic acid sequence contained within the target miRNA.
  • a nucleic acid sequence in a siRNA that is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence or differs from the target sequence by one or two nucleotides.
  • the sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" area.
  • the siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
  • One or both strands of the siRNA can also comprise a 3 overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand.
  • the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • the nucleic acid miR-9 inhibitor of the invention may be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), or may comprise both RNA and DNA.
  • a nucleic acid miR- 9 inhibitor (as described above) comprises RNA in some embodiments.
  • a nucleic acid miR-9 inhibitor (as described above) comprises DNA.
  • nucleic acid sequences in this document are written in the direction 5 '-3'. As would be understood by a person skilled in the art, nucleic acid sequences written as RNA and containing the nucleotide U may equally be written as DNA by substituting T for U.
  • nucleic acid(s) and/or nucleotide(s) embraces modified nucleic acid(s) and modified nucleotide(s).
  • a nucleic acid or nucleotide may be modified to increase the stability of said nucleic acid or nucleotide, for instance by improving resistance to nuclease degradation.
  • a modified nucleic acid comprises a locked nucleic acid (LNA) nucleotide.
  • LNA locked nucleic acid
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon.
  • the bridge "locks" the ribose in the 3'- endo (North) conformation, which is often found in A-form nucleic acid duplexes.
  • LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide whenever desired.
  • the locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.
  • modified nucleotides include 2'-methoxyethoxy (MOE) nucleotides; 2'- methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides.
  • nucleic acid molecule of the invention may also be conjugated to one or more cholesterol moieties.
  • the nucleic acid miR-9 inhibitor is conjugated to at least one (for example, 1, 2, 3, or 4) cholesterol moiety.
  • a nucleic acid miR-9 inhibitor of the invention may be constructed such that all of its nucleotides are modified nucleotides.
  • the nucleic acid miR-9 inhibitor consists of modified nucleotides.
  • said modified nucleotides consist of LNA nucleotides, 2'-O-methyl modified nucleotides, 2'-0- methoxyethyl modified nucleotides, or 2'- fluoro modified nucleotides.
  • the nucleic acid miR-9 inhibitor is a synthetic 2'-O-methyl RNA oligonucleotide and competes with miR-9 target mRNAs with stronger binding to the miRNA-associated gene silencing complex.
  • Nucleic acid analogs are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of four nucleobases.
  • an analog may have any of these altered.
  • the analog nucleobases confer, among other things, different base pairing and base stacking proprieties. Examples include universal bases, which can pair with all four canon bases, and phosphate-sugar backbone analogs, such as PNA, which affect the chain's properties.
  • Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and LNA, as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the molecule’s backbone.
  • the nucleic acid miR-9 inhibitor is a nucleic acid analog selected from a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), and a morpholino.
  • the nucleic acid miR-9 inhibitor may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20) modified phosphodiester linkage.
  • the modified phosphodiester linkage is a phosphorothioate linkage.
  • all of the phosphodiester linkages are modified phosphodiester linkages.
  • the nucleic acid molecules of the invention may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.
  • a third object of the present invention relates to a vector comprising the nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO: 1)
  • the invention provides a nucleic acid vector comprising a nucleic acid sequence encoding the nucleic acid miR-9 inhibitor as described above.
  • the nucleic acid molecules of the present invention may be delivered into a target cell using a viral vector.
  • the viral vector may be any virus that can serve as a viral vector. Suitable viruses are those which infect the target cells, can be propagated in vitro and can be modified by recombinant nucleotide technology known in the art.
  • the invention provides a viral vector comprising a nucleic acid sequence encoding a nucleic acid miR-9 inhibitor (as described above), for use in the prevention or treatment of cystic fibrosis in a subject.
  • Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and adeno-associated virus (AAV) vectors.
  • a “non-replicating viral vector” is a viral vector that lacks the ability to replicate productively following the infection of a target cell.
  • the ability of a non-replicating viral vector to produce copies of itself following infection of a target cell (such as a human target cell in an individual undergoing vaccination with a non-replicating viral vector) is highly reduced or absent.
  • a viral vector may also be referred to as attenuated or replicationdeficient. The cause can be the loss/deletion of genes essential for replication in the target cell.
  • Non-replicating viral vectors cannot effectively produce copies of itself following the infection of a target cell.
  • Non-replicating viral vectors may therefore advantageously have an improved safety profile as compared to replication-competent viral vectors.
  • a non-replicating viral vector may retain the ability to replicate in cells that are not target cells, allowing viral vector production.
  • a non-replicating viral vector e.g., a non-replicating poxvirus vector
  • a target cell such as a mammalian cell (e.g., a human cell)
  • an avian cell e.g., a chick embryo fibroblast, or CEF, cell.
  • the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MV A) vector, an NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.
  • MV A Modified Vaccinia virus Ankara
  • AVAC canarypox
  • FPV fowlpox
  • MVA and NYVAC are both attenuated derivatives of the vaccinia virus. Compared to the vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.
  • the adenovirus vector is a non-replicating adenovirus vector (wherein non-replicating is defined as above). Adenoviruses can be rendered nonreplicating by deletion of the El or both the El and E3 gene regions.
  • the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.
  • the invention also relates to i) lumacaftor and ivacaftor and ii) a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate, or sequential use in the treatment of cystic fibrosis.
  • the invention also relates to i) tezacaftor and ivacaftor and ii) nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate, or sequential use in the treatment of cystic fibrosis.
  • the invention also relates to i) ivacaftor, tezacaftor and elexacaftor and ii) a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate or sequential use in the treatment of cystic fibrosis.
  • tezacaftor also called “l-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)- N- ⁇ l-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(l-hydroxy-2-methylpropan-2-yl)-lH-indol-5- yl ⁇ cyclopropane- 1 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
  • lumacaftor also called “3- ⁇ 6-[l-(2,2-difhioro-2H-l,3-benzodioxol- 5-yl)cyclopropaneamido]-3-methylpyridin-2-yl ⁇ benzoic acid” has its general meaning in the art and refers to the compound characterized by the formula of:
  • ivacaftor also called “N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo- l,4-dihydroquinoline-3 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
  • the term “elexacaftor” also called “N-(l,3-dimethylpyrazol-4-yl)sulfonyl-6- [3-(3,3,3-trifluoro-2,2-dimethylpropoxy)pyrazol-l-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-l- yl]pyridine-3 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
  • the term “Orkambi” relates to a biotherapy made up of “lumacaftor” and “ivacaftor”. Orkambi is developed by Vertex Pharmaceuticals.
  • Symdeko relates to a biotherapy made up of “tezacaftor” and “ivacaftor”. Symdeko is developed by Vertex Pharmaceuticals.
  • Kaftrio or “Trikfata” relates to a tritherapy made up of “ivacaftor”, “tezacaftor” and “elexacaftor”. Kaftrio/Trikfata is developed by Vertex Pharmaceuticals.
  • the nucleic acid molecule of the present invention is a LNA oligonucleotide.
  • LNA Locked Nucleic Acid
  • LNA oligonucleotide refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogs also referred to as LNA nucleotides and LNA analog nucleotides.
  • LNA oligonucleotides, LNA nucleotides and LNA analog nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO 03/006475; U.S. Patent Nos. 6,043,060, 6268490, 6770748, 6639051, and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840, and 2005/0203042, all of which are incorporated herein by reference.
  • LNA oligonucleotides and LNA analog oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA.
  • Other possible stabilizing modifications include phosphodiester modifications, combinations of phosphodiester and phosphorothioate modifications, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.
  • Chemically stabilized, modified versions of the nucleic acid molecule of the present invention also include “Morpholinos” (phosphorodiamidate morpholino oligomers, PMOs), 2'-O-Met oligomers, tricyclo (tc)-DNAs, U7 short nuclear (sn) RNAs, or tricyclo-DNA-oligoantisense molecules (U.S. Provisional Patent Application Serial No. 61/212,384 For: Tricyclo-DNA Antisense Oligonucleotides, Compositions, and Methods for the Treatment of Disease, filed April 10, 2009, the complete contents of which is hereby incorporated by reference).
  • modified versions of the nucleic acid molecule of the present invention also include 2'-O-methyl modified nucleotides, or 2'-0- methoxyethyl modified nucleotides, or 2'-fluro modified nucleotides.
  • the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third%) drug.
  • the drugs may be administered simultaneously, separately, or sequentially and in any order.
  • the drug is administered to the subject using any suitable method that enables the drug to reach the lungs, pancreas, intestine, skin, and reproductive system.
  • the drug is administered to the subject systemically (i.e., via systemic administration).
  • the drug is administered to the subject such that it enters the circulatory system and is distributed throughout the body.
  • the drug is administered to the subject by local administration, for example, by the local administration to the lungs.
  • the combination of tezacaftor and ivacaftor or the combination of lumacaftor and ivacaftor or the combination of ivacaftor, tezacaftor and elexacaftor are administered to the subject in a pharmaceutical composition.
  • tezacaftor and ivacaftor or lumacaftor and ivacaftor or tezacaftor, ivacaftor and elexacaftor are administered in the form of tablets for oral administration.
  • the nucleic acid molecule of the present invention is delivered by any device adapted to introduce one or more therapeutic compositions into the upper and/or lower respiratory tract, pancreas, intestine, skin, and reproductive system.
  • the devices may be adapted to deliver the therapeutic compositions of the invention in the form of a finely dispersed mist of liquid, foam, or powder.
  • the device may use a piezoelectric effect or ultrasonic vibration to dislodge powder attached on a surface such as a tape in order to generate mist suitable for inhalation.
  • the devices may use any propellant system known to those in the art, including, but not limited to, pumps, liquefied- gas, compressed gas, and the like.
  • Devices of the present invention typically comprise a container with one or more valves through which the flow of the therapeutic composition travels and an actuator for controlling the flow.
  • the devices suitable for administering the constructs of the invention include inhalers and nebulizers.
  • inhalers Various designs of inhalers are available commercially and may be employed to deliver the medicaments of the invention. These include the Accuhaler, Aerohaler, Aerolizer, Airmax, Autohaler, Clickhaler, Diskhaler, Easi-breathe inhaler, Fisonair, Integra, Jet inhaler, Miat-haler, Novolizer inhaler, Pulvinal inhaler, Rotahaler, Spacehaler, Spinhaler, Syncroner inhaler, and Turbohaler devices.
  • the delivery is done by means of a nebulizer or other aerosolisation device, provided the integrity of the nucleic acid miR-9 inhibitor is maintained.
  • the nucleic acid miR-9 inhibitor may be administered to the subject in a single delivery, such as a bolus delivery. Alternatively, the nucleic acid miR-9 inhibitor may be administered to the subject using a continuous delivery technique, such as a timed infusion. The nucleic acid miR- 9 inhibitor may be administered using a repeated delivery regimen, for example, on an hourly, daily or weekly basis. The nucleic acid miR-9 inhibitor dosages may be achieved by single or multiple administrations. By way of example, the nucleic acid miR-9 inhibitor may be administered to the subject in a regimen consisting of a single administration. Alternatively, the nucleic acid miR-9 inhibitor may be administered to the subject in a regimen comprising multiple administrations.
  • an administration regimen may comprise multiple administrations per day, or daily, weekly, bi-weekly, or monthly administrations.
  • An example regimen comprises an initial administration followed by multiple subsequent administrations at weekly or bi-weekly intervals.
  • Another example regimen comprises an initial administration followed by multiple subsequent administrations at monthly or bi-monthly intervals.
  • administration of the nucleic acid miR-9 inhibitor can be guided by monitoring of CF symptoms in the subject.
  • an example regimen comprises an initial administration followed by multiple subsequent administrations carried out on an irregular basis as determined by monitoring CF symptoms in the subject.
  • suitable nucleic acid delivery methods include ionophoresis, microspheres (e.g., bioadhesive microspheres), nanoparticles, dendritic polymers, liposomes, hydrogels, cyclodextrins, and proteinaceous vectors.
  • the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • a fourth object of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO: 1).
  • the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, lumacaftor and ivacaftor.
  • the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, tezacaftor and ivacaftor.
  • the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, tezacaftor, ivacaftor and elexacaftor.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for drugs depend on the disease or condition to be treated and may be determined by the people skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize disease progression.
  • One of the ordinary skills in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example, about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g., be intranasal installation, intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance, administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, at predefined points in time.
  • treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-150 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5,
  • the treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-500 mg/kg
  • the drugs of the present invention are administered to the subject in the form of a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol, and wool fat.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the used herein include intranasal installation, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3 -butanediol.
  • a non-toxic, parenterally acceptable diluent or solvent such as a solution in 1,3 -butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono-or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives
  • injectables are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include, e.g., lactose.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may be added if desired.
  • the compositions of this invention may be administered in the form of suppositories for rectal administration.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.
  • the compositions of this invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.
  • the pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m 2 and 500 mg/m 2 .
  • these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • a pharmaceutical composition of the invention for injection could be prepared to contain sterile buffered water (e.g., 1 ml for subcutaneous injection or intranasal installation or intramuscular), and between about 1 ng to about 100 mg, e.g., about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.
  • sterile buffered water e.g., 1 ml for subcutaneous injection or intranasal installation or intramuscular
  • between about 1 ng to about 100 mg e.g., about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.
  • FIGURES are a diagrammatic representation of FIGURES.
  • TMEM16a ASO4 increases mucociliary clearance in different cftr mutations. Histograms represent the average values ⁇ SDs and were compared using ANOVA coupled with Dunnett' s, Bonferroni's, and Tukey's posthoc test. Cells for patients with F508del/F508del (A), 2184DelA/W1282x (B) and 1717-lG>A/F508del (C) mutations. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • TMEM16a ASO4 treatment increases viability and weight in CF mice.
  • TMEM16a ASO4 and Ctl ASO (10 mg/kg) were subcutaneously injected on days 11 and 18 and every 15 days afterward to 129-cftrtmlEur CF mice.
  • A Kaplan-Meier survival curves of overall survival analysis in CF mice treated with control ASO or TMEM16a ASO4. Data are the mean of more than 100 mice. The dotted lines represent the median survival, the time at which the staircase survival curve crosses 50%.
  • B Body weight curve of CF mice. Each point represents the mean of the group treated with ASO4 TMEM16A (triangular) or the ASO control (dots). P values were determined using the log-rank test. ****p ⁇ 0.0001.
  • FIG. 3 Effects of different ASO TMEM16A sequences on TMEM16a expression and activity.
  • A CF cells (CFBE41O-) were treated by Ctl ASO or the different TMEM16a ASO indicated for 24h.
  • B TMEM16A chloride channel activity assessed by I- quenching of halide-sensitive YFP-H148q/I152L protein on CF cells.
  • Histograms represent average values ⁇ SDs, and the conditions were compared using ANOVA coupled with Dunnett' s post hoc test in comparison to CF Ctl ASO condition.
  • C Mice cell line
  • ASO3 a long version
  • ASO4 a short version
  • Histograms represent average values ⁇ SDs, and the conditions were compared using ANOVA coupled with Dunnett' s post hoc test in comparison Ctl condition.
  • TMEM16a ASO4 increases chloride efflux.
  • A-B The normalized total area under the curves of Ussing-chamber assays of non-CF (NCF) (A) and CF (B) primary human bronchial epithelial (hBEC) cells culture in air-liquid interphase and treated with Ctl ASO or TMEM16a ASO4 for 1 hour each day for 3 days at 50 nM.
  • cell lines were cultured and transfected with ASO (control or TMEM16a ASO4) (1, 2).
  • ASO control or TMEM16a ASO4
  • TMEM16a ASO4 Primary human bronchial epithelial cells
  • hBEC Primary human bronchial epithelial cells
  • Epithelix SARL Epithelix SARL
  • TMEM16a channel activity was assessed by iodine (I-) quenching of halide-sensitive YFP- H148Q/I152L protein (4). Fluorescence was recorded and analyzed using a plate reader as previously described (3).
  • mice (BALB/cAnNRj-Foxnl nu/nu) were treated with fluorescent TMEM16a ASO4 (Eurogentec, Belgium; ex 640 nm/em 680 nm) by different routes of administration (10 mg/kg). Fluorescence was measured at Ih, 4h, Id, 8d, 15d, 21d, and 30 days. Prior to imaging, mice were anesthetized using isoflurane inhalation. Imaging was performed on the IVIS Spectrum imaging system (PerkinElmer, Inc., France) by two-dimensional (2D) epifluorescence imaging at indicated times after probe injection to optimize readouts. 3D images were generated by Ivis software.
  • Toxicity protocol was performed by C-Ris Pharma (Saint-Malo, France) on healthy CD-I mice. Hematology parameters were determined on whole blood. These parameters were determined by using the ProCyte Dx hematology analyzer (IDEXX, France). Biochemical assays were performed using the KBIO04-DUO biochemistry analyzer (Kitvia, France). After sacrifice on day 14, organs were collected and embedded in paraffin before cutting and analyzing.
  • mice homozygous for the F508del mutation and their wild-type littermates were obtained from CDTA-CNRS (Orleans, France).
  • TMEM16a ASO4 subcutaneous injections (10 mg/kg) were given, and their body weights were recorded. Animals had access to food and water ad libitium. All animal studies were approved by the Institutional Animal Care and Use Committee.
  • IMF Ml 6a ASO4 potentiates chloride efflux in CF cells
  • TMEM16a ASO4 robustly potentiated TMEM16a Cl- channel in a concentration manner (Data not shown).
  • concentration ever used at 50 mM is in the middle of the range of activation.
  • modifications in the morphology of the cells were observed, suggesting a toxic dose confirmed by the lactate dehydrogenase method (Data not shown).
  • TMEM16a ASO4 increases mucociliary clearance of CF cells with different mutations hBEC from different donors with different mutations classes were cultured in air-liquid interphase to obtain differentiated cells ( Figure 1A. F508del/F508del (class Il/Class II); Figure IB: 2184delA/W1282X (Class I/Class I) and Figure 1C 1717-lG>A/F508del (Class I/Class II) and. Cells were cultured for 24h in the presence of lumacaftor/ivacaftor (Boston, Massachusetts, USA) or TMEM16a ASO4.
  • TMEM16a ASO4 induced strong and significant activation of mucociliary clearance evaluated by the movement of fluorescent beads.
  • the effects of lumacaftor/ivacaftor (Orkambi®) were only positive and significant on F508del/F508del cells but significantly lower than with TMEM16a ASO4 ( Figure 1A).
  • the effects of TMEM16a ASO4 and lumacaftor/ivacaftor were additive on F508del/F508del cells compared to the treatment with TMEM16a or lumacaftor/ivacaftor alone.
  • Fluorescent TMEM16a ASO4 (10 mg/kg) was administered in nude mice by intravenous, subcutaneous, intraperitoneal injection or intranasal installation to determine the best administration ways and biodistribution. Fluorescence in mice was recorded every week for 1 month (Data not shown). In these pseudocolor images in which white represents the greatest intensity of photon emission, minimal light emission is detected outside the injection site at DO. After 7 days, a good molecule distribution in all mice tissues can be observed with intravenous, subcutaneous, and intranasal administration. However, a strong accumulation in the liver and kidney occurred. With the intraperitoneal injection, the molecule disperses after 14 days to a small extent, while we observed a very good distribution in the mouse tissues for the other routes of administration. From day 21, we observed a decrease in the fluorescence level for all the conditions, as we can observe on day 30. In the following experiments, we chose the subcutaneous injection, a classic way for ASO administration, which was administered every 15 days.
  • TMEM1 6a ASO4 has good specificity and is not toxic
  • TMEM16a ASO4 To characterize TMEM16a ASO4, we have performed an "in vitro pharmaceutical profiling" defined by Bowes et al. (14). This method was described by four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis, and Pfizer) to identify undesirable off- target activity profiles that could hinder or halt candidate drugs’ development. No significant TMEM16a ASO4 impact was identified in the tested targets panel (Data not shown . To confirm TMEM16a ASO4 specificity, we performed experiments on calcium activity, proliferation, inflammation, and infection (Data not shown). A strategy targeting CaCC with Denufosol was proposed to patients in the past. This drug activated calcium mobilization to induce CaCC channels.
  • mice were injected subcutaneously at six doses from 0 to 500 mg/kg (Data not shown). Toxicity was assessed by monitoring general signs of pharmacologic and toxicological (morbidity and mortality, clinical signs, body weight, food, and water consumption) three times a week for 14 days. Before sacrifice, blood and urine were collected, hematology analysis (hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count), blood chemistry analysis (including coagulation parameters), and urinalysis were performed.
  • hematocrit hemoglobin concentration
  • mean corpuscular hemoglobin mean corpuscular volume
  • mean corpuscular hemoglobin concentration mean corpuscular hemoglobin concentration
  • TMEM1 6a ASO4 increases survival and weight gain in CF mice
  • mice usually die of intestine occlusion during or after the weaning period.
  • TMEM16a ASO4 or control ASO 10 mg/kg.
  • TMEM16a ASO To test the most effective TMEM16a ASO, we have generated 2 other long versions ASO1 (TTGGGACCTCGATCTTGG (SEQ ID NO: 5)) and ASO2 (TCTTTGGGACCTCGATCTTG (SEQ ID NO: 6)) of the TMEM16a ASO in addition to the main long TMEM16a ASO (ASO3 (TTTTCTCCGTCTTTGGGACCT (SEQ ID NO:7)) and a short version of TMEM16a ASO (ASO4 (SEQ ID NO: 1).
  • CF cell line CFBE41O-
  • TMEM1 6a ASO4 potentiates chloride efflux in CF cells with different mutations
  • TMEM16a ASO4 increases mucociliary clearance of CF patient cells with different mutations
  • TMEM16A ASO4 induced strong and significant activation of mucociliary clearance evaluated by the movement of fluorescent beads in all the different tested mutations ( Figures 5A and 5B).
  • the effects of Orkambi® were only positively significant on class II F508del/F508del cells, although significantly lower than those treated with TMEM16A ASO4 ( Figure 5A).
  • Trikafta® and ASO4 TMEM16A had a similar effect in class II mutation ( Figure 5A). However, we observed an additive effect when combining the two molecules ( Figure 5A).
  • TMEM16A ASO4 shows a strong and significant activation of mucociliary clearance in class I 2184DelA/W1282x cells compared to cells treated with control, Orkambi® or Trikafta® Figure 5 )
  • the inventors have observed an increase in the survival of CF mice that usually would have died of intestinal obstruction shortly after being weaned.
  • the inventors’ approach offers a novel therapeutic strategy for patients with CF, independently of the CFTR channel, compensating for lung dysfunction by increasing chloride activity and mucociliary clearance, and treating other symptoms such as intestinal obstruction.
  • this therapy could apply to all patients with CF and, in particular, to those who cannot benefit from current curative treatments.
  • MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nature Communications 2017; 8: 710.
  • MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nature Communications 2017; 8: 710. WJ, Qiu J, Sun J, Ma CL, Huang N, Jiang Y, et al. Downregulation of microRNA-9 reduces inflammatory response and fibroblast proliferation in mice with idiopathic pulmonary fibrosis through the ANOl-mediated TGF-beta-Smad3 pathway. J Cell Physiol 2018. YR, Lee ST, Kim SL, Zhu SM, Lee MR, Kim SH, et al. Down-regulation of miR-9 promotes epithelial mesenchymal transition via regulating anoctamin-1 (AN01) in CRC cells. Cancer Genet 2019; 231-232: 22-31. culose SD, Donoghue MT, Rehmet K, de Souza Gomes M, Fort A, Kovvuru P, et al.

Abstract

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the CFTR Cl− channel and affects several organs. However, the most severe consequences are observed in the lung. In order to study the effects of TMEM16a potentiation in vitro and in vivo on CF, the experiments are performed on cell lines and primary cells with different mutation classes, and the CF mouse model is used to study the different administration routes, complete survival data, and study long-term effects. The oligonucleotide (TMEM16a ASO4) potentiates the activity of TMEM16a and increases chloride efflux in CFBE41o- cells and mucociliary clearance of human bronchial epithelial cells (hBEC) from patients with CF with different mutations. The present invention relates to a nucleic acid miR-9 inhibitor and to a method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid miR-9 inhibitor.

Description

NUCLEIC ACID MIR-9 INHIBITOR FOR THE TREATMENT OF CYSTIC
FIBROSIS
FIELD OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for the treatment of cystic fibrosis.
BACKGROUND OF THE INVENTION:
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene (1). More than 2,100 different mutations have been described affecting protein production or activity. The CFTR chloride channel, present in many epithelia, regulates transepithelial salt and water fluxes. In CF, impaired chloride ion efflux coupled with sodium ion hyperabsorption results in epithelial surface dehydration and thick and tenacious mucus obstructing the lungs, pancreatic ducts, the biliary tract, the intestine, and the male reproductive tract. Chronic lung infections are CF’s most common clinical manifestation, leading to pulmonary inflammation, progressive tissue destruction and loss of function, and elevated morbidity and mortality.
Following the cloning of the CFTR gene in 1989 (2), early hope for a therapy to treat patients with CF was founded firmly in the realm of gene therapy (3). For the last 10 years, Vertex Pharmaceuticals’ therapies have revolutionized the clinical approach by providing strategies to address and activate CFTR at the membrane (4). Unfortunately, these approaches are mutationdependent and still exclude more than 15% of patients with nonsense and rare mutations non- responsive to the drugs. Hence the need for alternative therapies that are CFTR independent.
In 2008, TMEM16a (anoctamin-1) was identified by three different groups as the gene encoding the airway Ca2+-activated Cl- channel (CaCC) (5-7). TMEM16a, like CFTR, is located in airway epithelia, participates in the homeostasis of the airway surface fluid, and has been proposed as an alternative strategy to compensate for CFTR deficiency (8). However, in 2013, the inventors demonstrated that this protein is downregulated in CF bronchia (9). Thus, a strategy increasing TMEM16a could bypass CFTR deficiency and be applied to all patients with CF, particularly those who cannot benefit from current curative treatments. In 2017, the inventors first demonstrated the inhibitory role of microRNA-9 (miR-9) in the regulation of TMEM16a (9), which other groups have since confirmed in different pathologies (10-13). Then, they developed a strategy based on antisense oligonucleotide (ASO) to inhibit the fixation of miR-9 on the 3’UTR of TMEM16a by competitive interaction and consequently increase its expression (9).
To limit adverse effects, the design of the ASO is specific to TMEM16a 3’UTR and acts as a corrector to increase the expression at the apical membrane. They report that this strategy could apply to all patients with CF to correct chloride efflux and mucociliary clearance without inducing inflammation or toxicity. Consequently, they demonstrated that this strategy could increase the life expectancy of CF mice. Herein, they support the concept that an increase in TMEM16a chloride activity could represent a therapeutic potential for drug development for all patients with CF.
SUMMARY OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for the treatment of cystic fibrosis. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Patients with cystic fibrosis (CF) bearing mutations non-responsive to current treatments need alternative CFTR independent therapies. The aims here is to study the effects of TMEM16a potentiation in vitro and in vivo and prepare for preclinical studies by assessing the best administration route and the TMEM16a oligonucleotide's toxicity and specificity. The experiments are performed on cell lines and primary cells with different mutation classes. The CF mouse model is used to study the different administration routes, complete survival data, and study long-term effects. The oligonucleotide (TMEM16a ASO4) potentiates the activity of TMEM16a and increases chloride efflux in CFBE41o- cells and mucociliary clearance in human bronchial epithelial cells (hBEC) from patients with CF with different mutations. TMEM16a ASO4 is detectable 30 days after subcutaneous injection in CF mice. TMEM16a ASO4 significantly increases the mice's lifespan. Recovery in CF male mice fertility was noticed. Acute administration of 50 times the effective dose did not show behavioral changes nor macroscopic or pathological changes. TMEM16a ASO4 is very specific and neither induces inflammation nor alters intracellular calcium mobilization or cell proliferation. This strategy could apply to all patients with CF to correct chloride efflux and mucociliary clearance without inducing inflammation or toxicity. Accordingly, the first object of the present invention relates to a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO:1).
Accordingly, a second object of the present invention relates to the method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1).
As used herein, the terms “subject” or “patient” denote a mammal, such as a rodent, a feline, a canine, a pig, a ferret, a rat, mice, a rabbit and a primate. According to the invention, a subject refers to any subject (preferably human) afflicted or at risk of being afflicted with cystic fibrosis. The method of the invention may be performed for any type of cystic fibrosis, such as revised in the World Health Organization Classification of cystic fibrosis and selected from the E84 group: mucoviscidosis, Cystic fibrosis with pulmonary manifestations, Cystic fibrosis with intestinal manifestations and Cystic fibrosis with other manifestations. In some embodiments, the subject of the present is sterile. In some embodiments, the subject, according to the invention, is a human. In some embodiments, the subject, according to the invention, is a girl or a boy. In some embodiments, the subject, according to the invention, is an adult. In some embodiments, the subject, according to the invention, is a child (a human being between the stages of birth and puberty), a teenager (a human being between the stages of puberty to adulthood), or an older person (a human being after puberty). In some embodiments, the subject is less than 15 years old. In some embodiments, the subject is less than 10 years old. In some embodiments, the subject is less than 7 years old. In some embodiments, the subject is less than 5 years old. In some embodiments, the subject is less than 3 years old. In some embodiments, the subject is more than 15 years old. In some embodiments, the subject is more than 20 years old. In some embodiments, the subject is more than 25 years old. In some embodiments, the subject is more than 30 years old. In some embodiments, the subject is more than 35 years old. In some embodiments, the subject is more than 50 years old. In some embodiments, the subject is more than 60 years old. In some embodiments, the subject is more than 65 years old, including the elderly.
As used herein, the term “Cystic fibrosis” (CF) is a genetic disorder that primarily affects the lungs, but also the pancreas, liver, kidneys, reproductive tract, and intestine. Long-term issues include difficulty breathing and coughing up mucus due to 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. Different people may have different degrees of symptoms.
CF is inherited in an autosomal recessive manner. It is caused by mutations in both copies of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Those with a single working copy are carriers and otherwise mostly healthy. CFTR is involved in the production of sweat, digestive fluids, and mucus. When the CFTR is not functional, secretions that are usually thin instead become thick. The condition is diagnosed by a sweat test and genetic testing. Screening of infants at birth takes place in some areas of the world.
As used herein, the term “CFTR protein” refers to the CFTR protein of 1480 amino acids, also called Cystic Fibrosis Transmembrane conductance Regulator. The CFTR protein is a chloride (C1-) channel found in the membranes of secretory tissues such as intestinal and respiratory mucosa. The CFTR protein is represented by the NCBI reference sequence: P13569.3 (SEQ ID NO: 2)
SEQ ID NO: 2:
1 mqrsplekas vvsklffswt rpilrkgyrq rlelsdiyqi psvdsadnls eklerewdre
61 laskknpkli nalrrcffwr fmfygiflyl gevtkavqpl llgriiasyd pdnkeersia
121 iylgiglcll fivrtlllhp aifglhhigm qmriamfsli ykktlklssr vldkisigql
181 vsllsnnlnk fdeglalahf vwiaplqval Imgliwellq asafcglgfl ivlalfqagl
241 grmmmkyrdq ragkiserlv itsemieniq svkaycweea mekmienlrq telkltrkaa
301 yvryfnssaf ffsgffvvfl svlpyalikg iilrkiftti sfcivlrmav trqfpwavqt
361 wydslgaink iqdflqkqey ktleynlttt evvmenvtaf weegfgelfe kakqnnnnrk
421 tsngddslff snfsllgtpv Ikdinfkier gqllavagst gagktsllmv imgelepseg
481 kikhsgrisf csqfswimpg tikeniifgv sydeyryrsv ikacqleedi skfaekdniv
541 Igeggitlsg gqrarislar avykdadlyl Idspfgyldv Itekeifesc vcklmanktr
601 ilvtskmehl kkadkililh egssyfygtf selqnlqpdf ssklmgcdsf dqfsaerms
661 iltetlhrfs legdapvswt etkkqsfkqt gefgekrkns ilnpinsirk fsivqktplq
721 mngieedsde plerrlslvp dseqgeailp risvistgpt Iqarrrqsvl nlmthsvnqg
781 qnihrkttas trkvslapqa nlteldiysr rlsqetglei seeineedlk ecffddmesi
841 pavttwntyl ryitvhksli fvliwclvif laevaaslvv Iwllgntplq dkgnsthsrn
901 nsyaviitst ssyyvfyiyv gvadtllamg ffrglplvht litvskilhh kmlhsvlqap
961 mstlntlkag gilnrfskdi ailddllplt ifdfiqllli vigaiavvav Iqpyifvatv 1021 pvivafimlr ayflqtsqql kqlesegrsp ifthlvtslk glwtlrafgr qpyfetlfhk 1081 alnlhtanwf lylstlrwfq mriemifvif fiavtfisil ttgegegrvg iiltlamnim 1141 stlqwavnss idvdslmrsv srvfkfidmp tegkptkstk pykngqlskv miienshvkk 1201 ddiwpsggqm tvkdltakyt eggnaileni sfsispgqrv gllgrtgsgk stllsaflrl 1261 Integeiqid gvswdsitlq qwrkafgvip qkvfifsgtf rknldpyeqw sdqeiwkvad 1321 evglrsvieq fpgkldfvlv dggcvlshgh kqlmclarsv Iskakillld epsahldpvt 1381 yqiirrtlkq afadctvilc ehrieamlec qqflvieenk vrqydsiqkl Inerslfrqa 1441 ispsdrvklf phmsskcks kpqiaalkee teeevqdtrl
As used herein, the term “CFTR gene” refers to the CFTR gene located on chromosome 7 and may be found in NCBI GenBank locus AC000111 and AC000061, the contents of which are incorporated herein in their entirety by reference. The cDNA for the CFTR gene is found in Audrezet et al., Hum. Mutat. (2004) 23 (4), 343-357. A nucleic acid sequence for human CFTR is represented by SEQ ID NO: 3(NCBI Reference Sequence: NM_000492.3).
SEQ ID NO: 3
1 aattggaagc aaatgacatc acagcaggtc agagaaaaag ggttgagcgg caggcaccca
61 gagtagtagg tctttggcat taggagcttg agcccagacg gccctagcag ggaccccagc
121 gcccgagaga ccatgcagag gtcgcctctg gaaaaggcca gcgttgtctc caaacttttt
181 ttcagctgga ccagaccaat tttgaggaaa ggatacagac agcgcctgga attgtcagac
241 atataccaaa tcccttctgt tgattctgct gacaatctat ctgaaaaatt ggaaagagaa
301 tgggatagag agctggcttc aaagaaaaat cctaaactca ttaatgccct tcggcgatgt
361 tttttctgga gatttatgtt ctatggaatc tttttatatt taggggaagt caccaaagca
421 gtacagcctc tcttactggg aagaatcata gcttcctatg acccggataa caaggaggaa
481 cgctctatcg cgatttatct aggcataggc ttatgccttc tctttattgt gaggacactg
541 ctcctacacc cagccatttt tggccttcat cacattggaa tgcagatgag aatagctatg
601 tttagtttga tttataagaa gactttaaag ctgtcaagcc gtgttctaga taaaataagt
661 attggacaac ttgttagtct cctttccaac aacctgaaca aatttgatga aggacttgca
721 ttggcacatt tcgtgtggat cgctcctttg caagtggcac tcctcatggg gctaatctgg
781 gagttgttac aggcgtctgc cttctgtgga cttggtttcc tgatagtcct tgcccttttt
841 caggctgggc tagggagaat gatgatgaag tacagagatc agagagctgg gaagatcagt
901 gaaagacttg tgattacctc agaaatgatt gaaaatatcc aatctgttaa ggcatactgc
961 tgggaagaag caatggaaaa aatgattgaa aacttaagac aaacagaact gaaactgact
1021 cggaaggcag cctatgtgag atacttcaat agctcagcct tcttcttctc agggttcttt
1081 gtggtgtttt tatctgtgct tccctatgca ctaatcaaag gaatcatcct ccggaaaata 1141 ttcaccacca tctcattctg cattgttctg cgcatggcgg tcactcggca atttccctgg
1201 gctgtacaaa catggtatga ctctcttgga gcaataaaca aaatacagga tttcttacaa
1261 aagcaagaat ataagacatt ggaatataac ttaacgacta cagaagtagt gatggagaat
1321 gtaacagcct tctgggagga gggatttggg gaattatttg agaaagcaaa acaaaacaat
1381 aacaatagaa aaacttctaa tggtgatgac agcctcttct tcagtaattt ctcacttctt
1441 ggtactcctg tcctgaaaga tattaatttc aagatagaaa gaggacagtt gttggcggtt
1501 gctggatcca ctggagcagg caagacttca cttctaatgg tgattatggg agaactggag
1561 ccttcagagg gtaaaattaa gcacagtgga agaatttcat tctgttctca gttttcctgg
1621 attatgcctg gcaccattaa agaaaatatc atctttggtg tttcctatga tgaatataga
1681 tacagaagcg tcatcaaagc atgccaacta gaagaggaca tctccaagtt tgcagagaaa
1741 gacaatatag ttcttggaga aggtggaatc acactgagtg gaggtcaacg agcaagaatt
1801 tctttagcaa gagcagtata caaagatgct gatttgtatt tattagactc tccttttgga
1861 tacctagatg ttttaacaga aaaagaaata tttgaaagct gtgtctgtaa actgatggct
1921 aacaaaacta ggattttggt cacttctaaa atggaacatt taaagaaagc tgacaaaata
1981 ttaattttgc atgaaggtag cagctatttt tatgggacat tttcagaact ccaaaatcta
2041 cagccagact ttagctcaaa actcatggga tgtgattctt tcgaccaatt tagtgcagaa
2101 agaagaaatt caatcctaac tgagacctta caccgtttct cattagaagg agatgctcct
2161 gtctcctgga cagaaacaaa aaaacaatct tttaaacaga ctggagagtt tggggaaaaa
2221 aggaagaatt ctattctcaa tccaatcaac tctatacgaa aattttccat tgtgcaaaag
2281 actcccttac aaatgaatgg catcgaagag gattctgatg agcctttaga gagaaggctg
2341 tccttagtac cagattctga gcagggagag gcgatactgc ctcgcatcag cgtgatcagc
2401 actggcccca cgcttcaggc acgaaggagg cagtctgtcc tgaacctgat gacacactca
2461 gttaaccaag gtcagaacat tcaccgaaag acaacagcat ccacacgaaa agtgtcactg
2521 gcccctcagg caaacttgac tgaactggat atatattcaa gaaggttatc tcaagaaact
2581 ggcttggaaa taagtgaaga aattaacgaa gaagacttaa aggagtgctt ttttgatgat
2641 atggagagca taccagcagt gactacatgg aacacatacc ttcgatatat tactgtccac
2701 aagagcttaa tttttgtgct aatttggtgc ttagtaattt ttctggcaga ggtggctgct
2761 tctttggttg tgctgtggct ccttggaaac actcctcttc aagacaaagg gaatagtact
2821 catagtagaa ataacagcta tgcagtgatt atcaccagca ccagttcgta ttatgtgttt
2881 tacatttacg tgggagtagc cgacactttg cttgctatgg gattcttcag aggtctacca
2941 ctggtgcata ctctaatcac agtgtcgaaa attttacacc acaaaatgtt acattctgtt
3001 cttcaagcac ctatgtcaac cctcaacacg ttgaaagcag gtgggattct taatagattc
3061 tccaaagata tagcaatttt ggatgacctt ctgcctctta ccatatttga cttcatccag
3121 ttgttattaa ttgtgattgg agctatagca gttgtcgcag ttttacaacc ctacatcttt 3181 gttgcaacag tgccagtgat agtggctttt attatgttga gagcatattt cctccaaacc
3241 tcacagcaac tcaaacaact ggaatctgaa ggcaggagtc caattttcac tcatcttgtt
3301 acaagcttaa aaggactatg gacacttcgt gccttcggac ggcagcctta ctttgaaact
3361 ctgttccaca aagctctgaa tttacatact gccaactggt tcttgtacct gtcaacactg
3421 cgctggttcc aaatgagaat agaaatgatt tttgtcatct tcttcattgc tgttaccttc
3481 atttccattt taacaacagg agaaggagaa ggaagagttg gtattatcct gactttagcc
3541 atgaatatca tgagtacatt gcagtgggct gtaaactcca gcatagatgt ggatagcttg
3601 atgcgatctg tgagccgagt ctttaagttc attgacatgc caacagaagg taaacctacc
3661 aagtcaacca aaccatacaa gaatggccaa ctctcgaaag ttatgattat tgagaattca
3721 cacgtgaaga aagatgacat ctggccctca gggggccaaa tgactgtcaa agatctcaca
3781 gcaaaataca cagaaggtgg aaatgccata ttagagaaca tttccttctc aataagtcct
3841 ggccagaggg tgggcctctt gggaagaact ggatcaggga agagtacttt gttatcagct
3901 tttttgagac tactgaacac tgaaggagaa atccagatcg atggtgtgtc ttgggattca
3961 ataactttgc aacagtggag gaaagccttt ggagtgatac cacagaaagt atttattttt
4021 tctggaacat ttagaaaaaa cttggatccc tatgaacagt ggagtgatca agaaatatgg
4081 aaagttgcag atgaggttgg gctcagatct gtgatagaac agtttcctgg gaagcttgac
4141 tttgtccttg tggatggggg ctgtgtccta agccatggcc acaagcagtt gatgtgcttg
4201 gctagatctg ttctcagtaa ggcgaagatc ttgctgcttg atgaacccag tgctcatttg
4261 gatccagtaa cataccaaat aattagaaga actctaaaac aagcatttgc tgattgcaca
4321 gtaattctct gtgaacacag gatagaagca atgctggaat gccaacaatt tttggtcata
4381 gaagagaaca aagtgcggca gtacgattcc atccagaaac tgctgaacga gaggagcctc
4441 ttccggcaag ccatcagccc ctccgacagg gtgaagctct ttccccaccg gaactcaagc
4501 aagtgcaagt ctaagcccca gattgctgct ctgaaagagg agacagaaga agaggtgcaa
4561 gatacaaggc tttagagagc agcataaatg ttgacatggg acatttgctc atggaattgg
4621 agctcgtggg acagtcacct catggaattg gagctcgtgg aacagttacc tctgcctcag
4681 aaaacaagga tgaattaagt ttttttttaa aaaagaaaca tttggtaagg ggaattgagg
4741 acactgatat gggtcttgat aaatggcttc ctggcaatag tcaaattgtg tgaaaggtac
4801 ttcaaatcct tgaagattta ccacttgtgt tttgcaagcc agattttcct gaaaaccctt
4861 gccatgtgct agtaattgga aaggcagctc taaatgtcaa tcagcctagt tgatcagctt
4921 attgtctagt gaaactcgtt aatttgtagt gttggagaag aactgaaatc atacttctta
4981 gggttatgat taagtaatga taactggaaa cttcagcggt ttatataagc ttgtattcct
5041 ttttctctcc tctccccatg atgtttagaa acacaactat attgtttgct aagcattcca
5101 actatctcat ttccaagcaa gtattagaat accacaggaa ccacaagact gcacatcaaa
5161 atatgcccca ttcaacatct agtgagcagt caggaaagag aacttccaga tcctggaaat 5221 cagggttagt attgtccagg tctaccaaaa atctcaatat ttcagataat cacaatacat
5281 cccttacctg ggaaagggct gttataatct ttcacagggg acaggatggt tcccttgatg
5341 aagaagttga tatgcctttt cccaactcca gaaagtgaca agctcacaga cctttgaact 5401 agagtttagc tggaaaagta tgttagtgca aattgtcaca ggacagccct tctttccaca 5461 gaagctccag gtagagggtg tgtaagtaga taggccatgg gcactgtggg tagacacaca 5521 tgaagtccaa gcatttagat gtataggttg atggtggtat gttttcaggc tagatgtatg 5581 tacttcatgc tgtctacact aagagagaat gagagacaca ctgaagaagc accaatcatg 5641 aattagtttt atatgcttct gttttataat tttgtgaagc aaaatttttt ctctaggaaa 5701 tatttatttt aataatgttt caaacatata taacaatgct gtattttaaa agaatgatta 5761 tgaattacat ttgtataaaa taatttttat atttgaaata ttgacttttt atggcactag 5821 tatttctatg aaatattatg ttaaaactgg gacaggggag aacctagggt gatattaacc 5881 aggggccatg aatcaccttt tggtctggag ggaagccttg gggctgatgc agttgttgcc 5941 cacagctgta tgattcccag ccagcacagc ctcttagatg cagttctgaa gaagatggta 6001 ccaccagtct gactgtttcc atcaagggta cactgccttc tcaactccaa actgactctt 6061 aagaagactg cattatattt attactgtaa gaaaatatca cttgtcaata aaatccatac 6121 atttgtgtga aa
As used herein, the terms “ANO1” or “TMEM16a” have their general meaning in the art and refer to the anoctamin-1 protein. AN01 belongs to a protein family composed of 10 members whose primary sequence and predicted structure (eight transmembrane domains) have no similarity with those of other anion channels. An exemplary human nucleic acid sequence is the NCBI Reference Sequence NM_018043.5 (SEQ ID NO:4).
SEQ ID NO:4
1 aaaggcgggc cggctggcgt ccaagttcct gaccaggcgc gggccggccc gcgggaccag
61 cagccgggtg gcggcgcgat cggccccgag aggctcaggc gccccccgca tcgagcgcgc
121 gggccgggcg ggccagggcg gcgggcggag cgggaggcgg ccacgtcccc ggcgggcctg
181 ggcgcgggga ggcccggccc cctgcgagcg cgccgcgaac gctgcggtct ccgcccgcag 241 aggccgccgg ggccgtggat ggggagggcg cgccgcccgg cggtcccagc gcacaggcgg 301 ccacgatgag ggtcaacgag aagtactcga cgctcccggc cgaggaccgc agcgtccaca 361 tcatcaacat ctgcgccatc gaggacatcg gctacctgcc gtccgagggc acgctgctga 421 actccttatc tgtggaccct gatgccgagt gcaagtatgg cctgtacttc agggacggcc 481 ggcgcaaggt ggactacatc ctggtgtacc atcacaagag gccctcgggc aaccggaccc 541 tggtcaggag ggtgcagcac agcgacaccc cctctggggc tcgcagcgtc aagcaggacc 601 accccctgcc gggcaagggg gcgtcgctgg atgcaggctc gggggagccc ccgatggact 661 accacgagga tgacaagcgc ttccgcaggg aggagtacga gggcaacctc ctggaggcgg 721 gcctggagct ggagcgggac gaggacacta aaatccacgg agtcgggttt gtgaaaatcc 781 atgccccctg gaacgtgctg tgcagagagg ccgagtttct gaaactgaag atgccgacga 841 agaagatgta ccacattaat gagacccgtg gcctcctgaa aaaaatcaac tctgtgctcc 901 agaaaatcac agatcccatc cagcccaaag tggctgagca caggccccag accatgaaga 961 gactctccta tcccttctcc cgggagaagc agcatctatt tgacttgtct gataaggatt 1021 cctttttcga cagcaaaacc cggagcacga ttgtctatga gatcttgaag agaacgacgt 1081 gtacaaaggc caagtacagc atgggcatca cgagcctgct ggccaatggt gtgtacgcgg 1141 ctgcataccc actgcacgat ggagactaca acggtgaaaa cgtcgagttc aacgacagaa 1201 aactcctgta cgaagagtgg gcacgctatg gagttttcta taagtaccag cccatcgacc 1261 tggtcaggaa gtattttggg gagaagatcg gcctgtactt cgcctggctg ggcgtgtaca 1321 cccagatgct catccctgcc tccatcgtgg gaatcattgt cttcctgtac ggatgcgcca 1381 ccatggatga aaacatcccc agcatggaga tgtgtgacca gagacacaat atcaccatgt 1441 gcccgctttg cgacaagacc tgcagctact ggaagatgag ctcagcctgc gccacggccc 1501 gcgccagcca cctcttcgac aaccccgcca cggtcttctt ctctgtcttc atggccctct 1561 gggctgccac cttcatggag cactggaagc ggaaacagat gcgactcaac taccgctggg 1621 acctcacggg ctttgaagag gaagaggagg ctgtcaagga tcatcctaga gctgaatacg 1681 aagccagagt cttggagaag tctctgaaga aagagtccag aaacaaagag aagcgccggc 1741 atattccaga ggagtcaaca aacaaatgga agcagagggt taagacagcc atggcggggg 1801 tgaaattgac tgacaaagtg aagctgacat ggagagatcg gttcccagcc tacctcacta 1861 acttggtctc catcatcttc atgattgcag tgacgtttgc catcgtcctc ggcgtcatca 1921 tctacagaat ctccatggcc gccgccttgg ccatgaactc ctccccctcc gtgcggtcca 1981 acatccgggt cacagtcaca gccaccgcag tcatcatcaa cctagtggtc atcatcctcc 2041 tggacgaggt gtatggctgc atagcccgat ggctcaccaa gatcgaggtc ccaaagacgg 2101 agaaaagctt tgaggagagg ctgatcttca aggctttcct gctgaagttt gtgaattcct 2161 acacccccat cttttacgtg gcgttcttca aaggccggtt tgttggacgc ccgggcgact 2221 acgtgtacat tttccgttcc ttccgaatgg aagagtgtgc gccagggggc tgcctgatgg 2281 agctatgcat ccagctcagc atcatcatgc tggggaaaca gctgatccag aacaacctgt 2341 tcgagatcgg catcccgaag atgaagaagc tcatccgcta cctgaagctg aagcagcaga 2401 gcccccctga ccacgaggag tgtgtgaaga ggaaacagcg gtacgaggtg gattacaacc 2461 tggagccctt cgcgggcctc accccagagt acatggaaat gatcatccag tttggcttcg 2521 tcaccctgtt tgtcgcctcc ttccccctgg ccccactgtt tgcgctgctg aacaacatca 2581 tcgagatccg cctggacgcc aaaaagtttg tcactgagct ccgaaggccg gtagctgtca 2641 gagccaaaga catcggaatc tggtacaata tcctcagagg cattgggaag cttgctgtca 2701 tcatcaatgc cttcgtgatc tccttcacgt ctgacttcat cccgcgcctg gtgtacctct
2761 acatgtacag taagaacggg accatgcacg gcttcgtcaa ccacaccctc tcctccttca
2821 acgtcagtga cttccagaac ggcacggccc ccaatgaccc cctggacctg ggctacgagg
2881 tgcagatctg caggtataaa gactaccgag agccgccgtg gtcggaaaac aagtacgaca
2941 tctccaagga cttctgggcc gtcctggcag cccggctggc gtttgtcatc gtcttccaga
3001 acctggtcat gttcatgagc gactttgtgg actgggtcat cccggacatc cccaaggaca
3061 tcagccagca gatccacaag gagaaggtgc tcatggtgga gctgttcatg cgggaggagc
3121 aagacaagca gcagctgctg gaaacctgga tggagaagga gcggcagaag gacgagccgc
3181 cgtgcaacca ccacaacacc aaagcctgcc cagacagcct cggcagccca gcccccagcc
3241 atgcctacca cgggggcgtc ctgtagctat gccagcgggg ctgggcaggc cagccgggca
3301 tcctgaccga tgggcaccct ctcccagggc aggcggcttc ccgctcccac cagggcccgg
3361 tgggtcctgg gttttctgca aacatggagg accactttct gataggacat tttcctttct
3421 tctttctgtt ttctttccct tgtttttgca caaagccatt atgcagggaa tattttttaa
3481 tctgtagtat tcaagatgaa tcaaaatgat ggctggtaat acggcaataa ggtagcaaag
3541 gcaggtgctt tgcagaaaga atgcttggaa acttgagtct ccctagaggt gaaaagtgag
3601 cagaggcccg tagaaaccct cctctgaatc ctcctaattc cttaagatag atgcaaaatg
3661 gtaagccgag gcatcgcgca aaagctggtg cgatgcttca gggaaaatgg aaaacccacg
3721 caagaataat gattgattcc ggttccaaaa ggtgtcacct acctgtttca gaaaagttag
3781 actttccatc gccttttcct tccatcagtt gagtggctga gagagaagtg cctcatccct
3841 gagccacaca gggggcgtgg gagcatccca gttatccctg gaaagctaga aggggacaga
3901 ggtgtccctg attaagcagg aaacagcacc cttggcgtcc ccagcaggct ccccactgtc
3961 agccacacac ctgcccccat cacaccaagc cgacctcaga gttgttcatc ttccttatgg
4021 gacaaaaccg gttgaccaga aaatgggcag agagagatga cctcggaagc atttccacag
4081 atggtgtcag ggtttcaaga agtcttaggg cttccagggg tcccctggaa gctttagaat
4141 atttatgggt ttttttttca aatatcaatt atatggtaga ttgaggattt tttttctgta
4201 gctcaaaggt ggagggagtt tattagttaa ccaaatatcg ttgagaggaa tttaaaatac
4261 tgttactacc aaagattttt attaataaag gcttatattt tggtaacact tctctatatt
4321 tttactcaca ggaatgtcac tgttggacaa ttattttaaa agtgtataaa accaagtctc
4381 ataaatgata tgagtgatct aaatttgcag caatgatact aaacaactct ctgaaatttc
4441 tcaagcacca agagaaacat cattttagca aaggccagga ggaaaaatag aaataaattt
4501 gtcttgaaga tctcattgat gtgatgttac attcccttta atctgccaac tgtggtcaaa
4561 gttcataggt gtcgtacatt tccattattt gctaaaatca tgcaatctga tgcttctctt
4621 ttctcttgta cagtaagtag tttgaagtgg gttttgtata taaatactgt attaaaaatt
4681 aggcaattac caaaaatcct tttatggaaa ccattttttt aaaaagtgaa tgtacacaaa 4741 tccacagagg actgtggctg gacattcatc taaataaatt tgaatatacg acacttttct
4801 cacttgaaaa a
As used herein, the term "gene" has its general meaning in the art and refers to means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
As used herein, the “allele" has its general meaning in the art and refers to an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated, results in functional or dysfunctional (including non-existent) gene products.
As used herein, the term “mutation” has its general meaning in the art and refers to any detectable change in genetic material, e.g., DNA, RNA, cDNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. Mutations include the deletion, insertion, or substitution of one or more nucleotides. The mutation may occur in the coding region of a gene (i.e., in exons), in the introns, or in the regulatory regions (e.g., enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, promoters) of the gene. Generally, a mutation is identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population. Where the mutation is within the gene coding sequence, the mutation may be a “missense” mutation, where it replaces one amino acid with another in the gene product, or a “non-sense” mutation, where it replaces an amino acid codon with a stop codon. A mutation may also occur in a splicing site where it creates or destroys signals for exon-intron splicing, thereby leading to a gene product of altered structure. A mutation in the genetic material may also be “silent”, i.e., the mutation does not result in an alteration of the amino acid sequence of the expression product. In the context of the instant application, mutations identified in the CFTR gene or protein are designated pursuant to the nomenclature of Dunnen and Antonarakis (2000). For instance, ">" indicates a substitution at the DNA level; (underscore) indicates a range of affected residues, separating the first and last residue affected; "del" indicates a deletion, "dup" indicates a duplication; "ins" indicates an insertion, "inv" indicates an inversion and "con" indicates a conversion. More particularly, “X” denotes that an amino acid is changed to a stop codon (X).
As used herein, the term “homozygous” refers to an individual possessing two copies of the same allele. As used herein, the term “homozygous mutant” refers to an individual possessing two copies of the same allele, such allele being characterized as the mutant form of a gene.
As used herein, the term “heterozygous” refers to an individual possessing two different alleles of the same gene, i.e., an individual possessing two different copies of an allele, such alleles are characterized as mutant forms of a gene.
In some embodiments, the subject harbors at least one mutation in the CFTR gene. The CFTR gene mutations were classified into six classes according to their resulting damaging effect on the protein (Elborn JS, 2016):
• Class I mutations are responsible for defects in the early steps of biosynthesis and mainly include premature termination codons or stop codons, often causing mRNA degradation. In class I, some mutations (class la) do not allow mRNA synthesis; they are sometimes called class VII. (De Boeck & Amaral, 2016). Class I is the most challenging CFTR defect to address since no CFTR protein is produced. Nonsense and frameshift mutations account for 8.39% and 16.21% of the total mutations (cftr2.org), respectively. Exemples of class I mutations: W1282X, 1717-1G->A, G542X, R553X, 2183AA>G, 2184delA
• Class II mutations disrupt protein maturation and trafficking to the plasma membrane (PM). Thus, the protein is either absent or present in reduced quantity at the apical membrane. The F508del mutation represents around 70% of all associated alleles; thus, given the recessive inheritance of the disease, approximately 90% of patients with CF carry at least one class II mutation.
• Class III mutations are most often located in the adenosine-5’ -triphosphate (ATP) binding domains and disrupt the regulation of the CFTR channel requiring ATP hydrolysis at nucleotide-binding domains and cAMP-dependent phosphorylation at the cytoplasmic domain for activation. Hence, the abnormal protein can reach the PM but is not activated by ATP or cAMP. These mutations are frequently situated within one of the nucleotide binding domains, (e.g., G551D, R560T).
• Class IV mutations are missense mutations located in the transmembrane channel and affect the conductance of the CFTR channel. Some transmembrane domain segments participate in ionic pore formation. Missense mutations in these regions produce a correctly positioned protein with cAMP-dependent Cl- activity. Nevertheless, these channels’ characteristics differ from those of the endogenous CFTR channel, with decreased ion flux and altered selectivity, making the protein partially functional. Exemples of class IV mutations: R117H, R334W
• Class V mutations correspond to molecular anomalies that affect transcription (quantitative defect). They alter the mRNA stability; thus, the protein is functional, but the density of CFTR protein expressed at the membrane is low. Exemples of class V mutations: 2789+5G->A, A455E
• Class VI mutations include some infrequent mutations resulting in a mature functional protein but with an altered stability. Exemples of class VI mutations: 4326delTC, Glnl412X, 4279insA
(Harriet C., Kristin E. T., Olivier T., et al. Translating the genetics of cystic fibrosis to personalized medicine. Transl Res 2016; 168 40-49 and Elborn JS. Cystic fibrosis. Seminar. 2016).
Examples of CFTR mutations include, but are not limited to 124del23bp CFTR, CFTRdelel CFTR, M1V CFTR, Q2X CFT, S4X CFTR, P5L CFTR, S13F CFTR, L15P CFTR, 182delT CFTR, CFTRdele2 CFTR, CFTRdele2-4 CFTR, 185+1G->T CFTR, CFTRdele2,3 CFTR, W19X CFTR, G27R CFTR, G27X CFTR, Q30X CFTR, R31C CFTR, R31L CFTR, Q39X CFTR, A46D CFTR, 296+lG->A CFTR, 296+lG->T CFTR, CFTRdele3-10,14b-16 CFTR, 296+28A->G CFTR, 296+2T->C CFTR, 296+3insT CFTR, 297-3C->T CFTR, 297-lG->A CFTR, E56K CFTR, W57G CFTR, W57X CFTR, 306insA CFTR, 306delTAGA CFTR, E60X CFTR, P67L CFTR, R74W CFTR, R75X CFTR, R75Q CFTR, 365-366insT CFTR, G85E CFTR, 394delTT CFTR, L88X CFTR, G91R CFTR, CFTRdele4-7 CFTR, CFTRdele4-l l CFTR, CFTR50kbdel CFTR, 4O5+1G->A CFTR, 405+3 A->C CFTR, 406-2A->G CFTR, 406- 1G->A CFTR, E92K CFTR, E92X CFTR, Q98X CFTR, Q98R CFTR, P99L CFTR, L102R CFTR, 442delA CFTR, 444delA CFTR, 457TAT->G CFTR, D110H CFTR, D110E CFTR, R117C CFTR, R117G CFTR, R117H CFTR, R117H;5T CFTR, R117H;7T CFTR, 541delC CFTR, L138ins CFTR, H139R CFTR, 574delA CFTR, I148T CFTR, 602dell4 CFTR, Y161D CFTR , 621+1G->T CFTR, 621+3A->G CFTR, L165S CFTR, R170H CFTR, 663delT CFTR, G178R CFTR, 675del4 CFTR, E193X CFTR, 711+1G->T CFTR, 711+3A->G CFTR, 711+5G->A CFTR, 712-1G->T CFTR, H199Y CFTR, V201M CFTR, P205S CFTR, L206W CFTR, W216X CFTR, Q220X CFTR, L227R CFTR, V232D CFTR, 849delG CFTR, 852del22 CFTR, CFTRdup6b-10 CFTR, M265R CFTR, 935delA CFTR, Y275X CFTR, C276X CFTR, 977insA CFTR, 991del5 CFTR, F311L CFTR, 1078delT CFTR, L320V CFTR, 1119delA CFTR, G330X CFTR, R334W CFTR, R334Q CFTR, R334L CFTR, 1138insG CFTR, I336K CFTR, T338I CFTR, S341P CFTR, 1154insTC CFTR, 1161delC CFTR, R347H CFTR, R347P CFTR, A349V CFTR, R352W CFTR, R352Q CFTR, Q359K/T360K CFTR, 1213delT CFTR, 1248+ 1G->A CFTR, 1249-1G->A CFTR, 1259insA CFTR, 1288insTA CFTR, W401X CFTR, 1341+1G->A CFTR, 5T CFTR, 5T;TG11 CFTR, 5T;TG12 CFTR, 5T;TG13 CFTR, 7T CFTR, 9T CFTR, 1343delG CFTR, Q414X CFTR, 1429del7 CFTR, D443Y CFTR, 1461ins4 CFTR, 1471delA CFTR, L453S CFTR, A455E CFTR, 1497delGG CFTR, V456A CFTR, 1504delG CFTR, 1525-1G->A CFTR, 1525-2A->G CFTR, S466X CFTR, L467P CFTR, M470V CFTR, 1548delG CFTR, E474K CFTR, S489X CFTR, S492F CFTR, 1609delCA CFTR, Q493X CFTR, W496X CFTR, I502T CFTR, I507del CFTR, F508del CFTR, F508C CFTR, D513G CFTR, 1677delTA CFTR, V520F CFTR, C524X CFTR, Q525X CFTR, 1716+1G->A CFTR, CFTRdelel l CFTR, 1717-1G->A CFTR, 1717-8G->A CFTR, G542X CFTR, S549R CFTR, S549N CFTR, G550X CFTR, 1782delA CFTR, G551S CFTR, G551D CFTR, Q552X CFTR, R553X CFTR, 1802delC CFTR, L558S CFTR, A559T CFTR, 1811+1G->C CFTR, R560K CFTR, R560T CFTR, 1811+1G->A CFTR, 1811+1634A->G or 181 l+1.6kbA->G CFTR, 1811+1643G->T CFTR, 1812-1G->A CFTR, R560S CFTR, A561E CFTR, V562I CFTR, Y563N CFTR, Y563D CFTR, 1824delA CFTR, 1833delT CFTR, Y569D CFTR , P574H CFTR , F575Y CFTR, G576A CFTR, D579G CFTR, E585X CFTR, E588V CFTR, 1898+1G- >A CFTR, 1898+1G->C CFTR, 1898+1G->T CFTR, CFTRdelel3,14a CFTR, 1898+3A->G CFTR, 1898+5G->T CFTR, 1924del7 CFTR, H609R CFTR, A613T CFTR, D614G CFTR, G622D CFTR , 2055del9->A CFTR, 2075delA CFTR, 2105-2117dell3insAGAAA CFTR, 2118del4 CFTR, R668C CFTR, 2143delT CFTR, G673X CFTR, 2183AA->G CFTR, 2184insA CFTR, 2184delA CFTR, 2185insC CFTR, Q685X CFTR, R709X CFTR, K710X CFTR, Q715X CFTR, Q720X CFTR, 2307insA CFTR, L732X CFTR, 2347delG CFTR, 2372del8 CFTR, P750L CFTR, V754M CFTR, R764X CFTR, R785X CFTR, R792X CFTR, I807M CFTR, 2556insAT CFTR, 2585delT CFTR, 2594delGT CFTR, E822X CFTR, 2622+lG->A CFTR, E831X CFTR, D836Y CFTR, W846X CFTR Y849X CFTR, R851X CFTR, T854T CFTR, 271 IdelT CFTR, 2721dell 1 CFTR, 2732insA CFTR, CFTRdelel4b-17b CFTR, 2752-26A->G CFTR, W882X CFTR, 2789+2insA CFTR, 2789+5G->A CFTR, 2790- 1G->C CFTR, Q890X CFTR, S912X CFTR, S912L CFTR, 2869insG CFTR, Y913X CFTR, 2896insAG CFTR, L927P CFTR, 2942insT CFTR, 2957delT CFTR, S945L CFTR, 2991del32 CFTR, 3007delG CFTR, 3028delA CFTR, L967S CFTR, G970R CFTR, CFTRdelel6-17b CFTR, G970D CFTR, S977F CFTR, D979V CFTR, 3120G->A CFTR, CFTRdelel7a,17b CFTR, CFTRdelel7a-18 CFTR, 312O+1G->A CFTR, 3121-1G->A CFTR, 3121-2A->G CFTR, 3121-977_3499+248del2515 CFTR, L997F CFTR, 3132delTG CFTR, A1006E CFTR, 3143del9 CFTR, 3171delC CFTR, 3171insC CFTR, Y1014C CFTR, F1016S CFTR, I1027T CFTR, Y1032C CFTR, Q1042X CFTR, 3271delGG CFTR, 3272-26A->G CFTR, F1052V CFTR, T1053I CFTR, H1054D CFTR, G1061R CFTR, L1065P CFTR, R1066C CFTR, R1066H CFTR, G1069R CFTR, R1070W CFTR, R1070Q CFTR, 3349insT CFTR, F1074L CFTR, L1077P CFTR, W1089X CFTR, Y1092X CFTR, W1098X CFTR, W1098C CFTR, F1099L CFTR, Ml 10 IK CFTR, R1102X CFTR, El 104X CFTR, SI 118F CFTR, CFTRdelel8 CFTR, 3500-2A->G CFTR, W1145X CFTR, D1152H CFTR, V1153E CFTR, 3600G->A CFTR, CFTRdelel9 CFTR, CFTRdelel9-21 CFTR, 3600+2insT CFTR, 3600+5G->A CFTR, R1158X CFTR, S1159P CFTR, S1159F CFTR, R1162X CFTR, R1162L CFTR, 3659delC CFTR, 3667ins4 CFTR, S1196X CFTR, 3737delA CFTR, W1204X CFTR, 3791delC CFTR, Y122X CFTR, 3821delT CFTR, I1234V CFTR, S1235R CFTR, 3849G->A CFTR, 3849+4A- >G CFTR, 3849+5G->A CFTR, 3849+40A->G CFTR, 3849+10kbC->T CFTR, 385O-1G->A CFTR, 3850-3T->G CFTR, V1240G CFTR, G1244E CFTR, T1246I CFTR, 3876delA CFTR, 3878delG CFTR, S 125 IN CFTR, L1254X CFTR, S1255P CFTR, S1255X CFTR, 3905insT CFTR, D1270N CFTR, W1282X CFTR, R1283M CFTR, Q1291R CFTR, 4OO5+1G->A CFTR, CFTRdele21 CFTR, 4005+2T->C CFTR, 4010del4 CFTR, 4015delA CFTR, 4016insT CFTR, 4022insT CFTR, 4040delA CFTR, N1303K CFTR, Q1313X CFTR, CFTRdele22-24 CFTR, CFTRdele22,23 CFTR, L1324P CFTR, Q1330X CFTR, L1335P CFTR, 4168delCTAAGCC CFTR, G1349D CFTR, 4209TGTT->AA CFTR, 4218insT CFTR, E1371X CFTR, H1375P CFTR, 4259del5 CFTR, Q1382X CFTR, 4279insA CFTR, 4326delTC CFTR, Q1411X CFTR, Q1412X CFTR, 4374+lG->T CFTR, 4374+lG->A CFTR, 4382delA CFTR, 4428insGA CFTR, A1067T CFTR, E193K CFTR, K1060T CFTR, Glnl412X CFTR (see, e.g., https://www.cftr2.org/mutations_history, for CFTR mutations).
In one embodiment, the subject harbors at least one allelic mutation selected from class I, class II, class III, class IV, class V or class VI. In one embodiment, the subject harbors at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the first allele and at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the second allele. In a particular embodiment, the subject harbors at least a mutation of class I in the first allele and at least a mutation of class II in the second allele. In a particular embodiment, the subject harbors at least a mutation of class II in the first allele and at least a mutation of class II in the second allele.
In a particular embodiment, the subject harbors at least one allelic mutation in the CFTR gene, including but not limited to F508del-CFTR, R117H CFTR, 2184delA CFTR, W1282X CFTR, 2183AA>G CFTR or G551D CFTR.
In one embodiment, the subject harbors at least a F508del mutation in the CFTR gene.
In one embodiment, the subject harbors at least a F508del mutation in the first allele and at least a F508del mutation in the second allele.
In one embodiment, the subject harbors at least a 2184delA mutation in the CFTR gene.
In one embodiment, the subject harbors at least a W1282X mutation in the CFTR gene.
In one embodiment, the subject harbors at least a 2184delA mutation in the first allele and at least a W1282X mutation in the second allele.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of a subject beyond that expected in the absence of such treatment. The term "therapeutic regimen" means the treatment pattern of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of particular predetermined criteria [e.g., disease manifestation, etc.]).
As used herein, the term "miRNAs" refers to mature microRNA (non-coding small RNAs) molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from longer precursor RNA molecules (“precursor miRNA”: pri-miRNA and pre-miRNA). Pri -miRNAs are transcribed from non-protein-encoding genes or embedded into protein-coding genes (within introns or non-coding exons). The “precursor miRNAs” fold into hairpin structures containing imperfectly base-paired stems and are processed in two steps, catalyzed in animals by two Ribonuclease Ill-type endonucleases called Drosha and Dicer. The processed miRNAs (also referred to as “mature miRNA”) are assembled into large ribonucleoprotein complexes (RISCs) that can associate them with their target mRNA to repress translation. All the miRNAs pertaining to the invention are known per se, and their sequences are publicly available from the data base http://www.mirbase.org/cgi-bin/mima_summary. pl?org=hsa. As used herein, the “miR-9” microRNA (homologous to miR-79) is a short non-coding RNA gene involved in gene regulation. The mature ~23nt miRNAs are processed from hairpin precursor sequences by the Dicer enzyme. The dominant mature miRNA sequence is processed from the 5' UTR of the miR-9 precursor, and from the 3' UTR of the mir-9 precursor. The mature products are thought to have regulatory roles through complementarity to mRNA.
As used herein, the term “nucleic acid miR-9 inhibitor” (i.e., a nucleic acid that inhibits miR- 9) relates to any nucleic acid, for example, an oligonucleotide, that reduces (i.e., inhibits) the biological activity of miR-9. In some embodiments, nucleic acid miR-9 inhibitors comprise a nucleic acid sequence that is complementary to at least a portion of the nucleic acid sequence of either miR-9 itself or the target mRNA sequences of miR-9; inhibition of miR-9, therefore, occurs by binding of the inhibitor to miR-9 or to its target mRNA. In both cases, miR-9 is prevented from recognizing and binding its target sequence and thus cannot induce gene silencing. The nucleic acid miR-9 inhibitor of the invention may comprise a structure that is single-stranded, double-stranded, partially double-stranded or hairpin in nature.
In some embodiments, the nucleic acid miR-9 inhibitor binds to miR-9. Thus, in some embodiments, the nucleic acid miR-9 inhibitor binds to and sequesters miR-9, preventing it from binding to its target mRNA sequences. Binding occurs via complementary base pairing between at least one nucleotide present in the nucleic acid miR-9 inhibitor and a corresponding nucleotide present in miR-9, such that at least a portion of the nucleic acid miR-9 inhibitor and miR-9 together define a base-paired nucleic acid duplex. Said complementary base pairing (and thus duplex formation) can occur over a region of two or more contiguous nucleotides of miR- 9 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides). A base-paired nucleic acid duplex formed when the nucleic acid miR-9 inhibitor binds to miR- 9 (as described above) may comprise one or more mismatch pairings. In some embodiments, two or more regions of complementary base-paired nucleic acid duplex (e.g., 3, 4, 5 or 6) are formed, wherein each region is separated from the next by one or more mismatch pairings. As used herein, the term "complementary" refers to a nucleic acid molecule that forms hydrogen bonds with another nucleic acid molecule with Watson-Crick base pairing. Watson-Crick base pairing refers to the following hydrogen-bonded nucleotide pairings: A:T and C:G (for DNA); and A:U and C:G (for RNA). For example, two or more complementary nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region, with or without an overhang) or have a different number of nucleotides (e.g., one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand). Mismatch pairings are formed between any two nucleotide bases that together do not form one of the hydrogen-bonded standard Watson-Crick base pairs of A:U (in RNA), A:T (in DNA) and C:G (in both RNA and DNA). In some embodiments, the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence complementary to at least a portion of the miR-9 sequence. The nucleic acid miR-9 inhibitor may comprise a nucleic acid sequence complementary to 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides of miR-9.
In some embodiments, the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence having at least 70% sequence identity to a complementary (i.e., antisense) sequence of miR-9. According to the invention, a first nucleic acid sequence having at least 70% of identity with a second nucleic acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second nucleic acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well- known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CAB IOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and a per residue gap cost of 1). When aligning short peptides (fewer than 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997. Thus, in some embodiments, the nucleic acid miR-9 inhibitor comprises a sequence complementary to miR-9. In some embodiments, the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence that differs from the complementary (i.e., antisense) sequence of miR9 at a maximum of 10 (e.g., a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) nucleotide positions. In some embodiments, the nucleic acid miR-9 inhibitor comprises a nucleic acid sequence that differs from the complementary (i.e., antisense) sequence of miR9 at a maximum of 5 (e.g., a maximum of 4, 3, 2, or 1) nucleotide positions. Thus, said nucleic acid sequence is identical to the complementary (i.e., antisense) sequence of miR9 except at a limited number of nucleotide positions. In some embodiments, the nucleic acid miR-9 inhibitor, as described above, has a maximum length of 60 (for example, 55, 50, 45, 40, 35, 30, or 25) nucleotides.
In some embodiments, the nucleic acid miR-9 inhibitor is a miR-9 antagomir. As used herein, an “antagomir” is a nucleic acid oligomer designed to bind to a specific target microRNA via complementary base pairing (for example, as described above). An antagomir may have a wholly or partially complementary sequence to the microRNA target sequence. Antagomirs may have a single-stranded, double-stranded, partially double-stranded, or hairpin structure. Antagomirs may further comprise chemically modified nucleotides (e.g., as described below). Methods for designing and creating antagomirs are known in the art.
In some embodiments, the nucleic acid miR-9 inhibitor is a miR-9 microRNA sponge. As used herein, the term “microRNA-sponge” is a nucleic acid that comprises multiple (e.g., at least 2, 3, 4, 5 or 6) binding sites for a specific target microRNA. Thus, a microRNA-sponge can bind and sequester multiple target microRNA molecules. A microRNA sponge may comprise an mRNA expressed from a vector (e.g., a viral vector or plasmid vector). The presence in a microRNA-sponge of multiple binding sites for the target microRNA enables microRNAs to be adsorbed in a manner analogous to a sponge soaking up water. A microRNA-sponge may bind target microRNAs via complementary base pairing (for example, as described above). Thus, a microRNA-sponge may comprise multiple (e.g., at least 2, 3, 4, 5 or 6) nucleic acid sequences, each sequence being complementary to at least a portion of the microRNA target sequence. A microRNA- sponge may comprise multiple (e.g., at least 2, 3, 4, 5 or 6) nucleic acid sequences, wherein each sequence is complementary to the microRNA target sequence. Methods for designing and creating microRNA-sponges are known in the art. In some embodiments, the nucleic acid miR-9 inhibitor comprising two or more nucleic acid sequences, wherein each of said two or more nucleic acid sequences has at least 70% sequence identity to the complementary (i.e., antisense) sequence of miR9 is a miR-9 microRNA sponge.
Thus, in some embodiments, the nucleic acid miR-9 inhibitor does not bind directly to miR-9 but instead binds to a miR-9 mRNA target site in the ANO1 nucleic acid sequence. This has the effect of blocking said target site (for example, by steric interference), preventing its recognition and binding by miR-9, and thus inhibiting miR-9 and its actions. In contrast to the binding of miR-9 to an mRNA target site, the binding of the nucleic acid miR-9 inhibitor of the invention to a miR-9 mRNA target site does not induce gene silencing (e.g., by mRNA degradation or translational repression) of said target mRNA. In some embodiments, the miR- 9 mRNA target site is located on an AN01 3 ' UTR. In some embodiments, the nucleic acid miR-9 inhibitor is a Target Site Blocker (TSB). The binding of the nucleic acid miR-9 inhibitor to a miR-9 mRNA target site may occur via complementary base pairing, as described above. Thus, in some embodiments, binding between the nucleic acid miR-9 inhibitor and the miR-9 mRNA target site occurs via complementary base pairing between at least one nucleotide present in the nucleic acid miR-9 inhibitor and a corresponding nucleotide present in the miR- 9 mRNA target site, such that at least a portion of the nucleic acid miR-9 inhibitor and the miR- 9 mRNA target site together define a base-paired nucleic acid duplex. Said complementary base pairing (and thus duplex formation) can occur over a region of two or more contiguous nucleotides of the miR-9 mRNA target site (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides). A base-paired nucleic acid duplex formed when the nucleic acid miR-9 inhibitor binds to the miR-9 mRNA target (as described above) may comprise one or more mismatch pairings. In some embodiments, two or more regions of complementary base-paired nucleic acid duplex (e.g., 3, 4, 5 or 6) are formed, wherein each region is separated from the next by one or more mismatch pairings. In some embodiments, the mRNA target site is located on the 3' UTR (untranslated region) of the target ANO1 mRNA. In some embodiments, wherein the nucleic acid miR-9 inhibitor competes with miR-9 for binding to a miR-9 mRNA target site.
In some embodiments, the nucleic acid miR-9 inhibitor is TMEM16a ASO. As used herein, the term “ANO1 ASO” or “TMEM16a ASO” OR “TMEM16a ASO4” refers to the sequence AATCTTTGGTAGTAA (SEQ ID NO: 1). In some embodiments, wherein the TMEM16a ASO of the present invention competes with miR-9 for binding to a miR-9 mRNA target site.
Thus, said nucleic acid sequence binds to the miR-9 mRNA target site in TMEM16a via complementary binding at the location targeted by the seed region of miR-9, thus preventing miR-9 from binding.
In some embodiments, the nucleic acid miR-9 inhibitor is a small interfering RNA (siRNA) targeted against miR-9. As used herein, the term “siRNA” has its general meaning in the art and refers to a double-stranded interfering RNA. siRNA, useful in the present methods, comprises short double-stranded RNA from about 17 nucleotides to about 29 nucleotides, preferably from about 19 to about 25 nucleotides in length. The siRNA comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired"). The sense strand comprises a nucleic acid sequence substantially identical to a nucleic acid sequence contained within the target miRNA. As used herein, a nucleic acid sequence in a siRNA that is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence or differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" area. The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. One or both strands of the siRNA can also comprise a 3 overhang. As used herein, a "3' overhang" refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand. Thus, in one embodiment, the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In a preferred embodiment, the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
The nucleic acid miR-9 inhibitor of the invention may be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), or may comprise both RNA and DNA. Thus, a nucleic acid miR- 9 inhibitor (as described above) comprises RNA in some embodiments. In some embodiments, a nucleic acid miR-9 inhibitor (as described above) comprises DNA. Unless specifically indicated otherwise, nucleic acid sequences in this document are written in the direction 5 '-3'. As would be understood by a person skilled in the art, nucleic acid sequences written as RNA and containing the nucleotide U may equally be written as DNA by substituting T for U. Reference to nucleic acid(s) and/or nucleotide(s) embraces modified nucleic acid(s) and modified nucleotide(s). For example, a nucleic acid or nucleotide may be modified to increase the stability of said nucleic acid or nucleotide, for instance by improving resistance to nuclease degradation. In some embodiments, a modified nucleic acid comprises a locked nucleic acid (LNA) nucleotide. In more detail, the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'- endo (North) conformation, which is often found in A-form nucleic acid duplexes. LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide whenever desired. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides. Other examples of modified nucleotides include 2'-methoxyethoxy (MOE) nucleotides; 2'- methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides. 2'-deoxy-2'-chloro nucleotides, 2'- azido nucleotides, and 2'-0-methyl nucleotides. A nucleic acid molecule of the invention may also be conjugated to one or more cholesterol moieties. Thus, in some embodiments, the nucleic acid miR-9 inhibitor is conjugated to at least one (for example, 1, 2, 3, or 4) cholesterol moiety. A nucleic acid miR-9 inhibitor of the invention may be constructed such that all of its nucleotides are modified nucleotides. Thus, in some embodiments, the nucleic acid miR-9 inhibitor consists of modified nucleotides. In some embodiments, said modified nucleotides consist of LNA nucleotides, 2'-O-methyl modified nucleotides, 2'-0- methoxyethyl modified nucleotides, or 2'- fluoro modified nucleotides. In some embodiments, the nucleic acid miR-9 inhibitor is a synthetic 2'-O-methyl RNA oligonucleotide and competes with miR-9 target mRNAs with stronger binding to the miRNA-associated gene silencing complex. Nucleic acid analogs are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analog may have any of these altered. Typically, the analog nucleobases confer, among other things, different base pairing and base stacking proprieties. Examples include universal bases, which can pair with all four canon bases, and phosphate-sugar backbone analogs, such as PNA, which affect the chain's properties. Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and LNA, as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the molecule’s backbone. Thus, in some embodiments, the nucleic acid miR-9 inhibitor (as described above) is a nucleic acid analog selected from a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), and a morpholino. The nucleic acid miR-9 inhibitor (as described above) may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20) modified phosphodiester linkage. In some embodiments, the modified phosphodiester linkage is a phosphorothioate linkage. In some embodiments, all of the phosphodiester linkages are modified phosphodiester linkages. The nucleic acid molecules of the invention may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.
Accordingly, a third object of the present invention relates to a vector comprising the nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO: 1)
In one aspect, the invention provides a nucleic acid vector comprising a nucleic acid sequence encoding the nucleic acid miR-9 inhibitor as described above. For example, the nucleic acid molecules of the present invention may be delivered into a target cell using a viral vector. The viral vector may be any virus that can serve as a viral vector. Suitable viruses are those which infect the target cells, can be propagated in vitro and can be modified by recombinant nucleotide technology known in the art. Thus, in one aspect, the invention provides a viral vector comprising a nucleic acid sequence encoding a nucleic acid miR-9 inhibitor (as described above), for use in the prevention or treatment of cystic fibrosis in a subject. Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and adeno-associated virus (AAV) vectors. As used herein, a “non-replicating viral vector” is a viral vector that lacks the ability to replicate productively following the infection of a target cell. Thus, the ability of a non-replicating viral vector to produce copies of itself following infection of a target cell (such as a human target cell in an individual undergoing vaccination with a non-replicating viral vector) is highly reduced or absent. Such a viral vector may also be referred to as attenuated or replicationdeficient. The cause can be the loss/deletion of genes essential for replication in the target cell. Thus, a non-replicating viral vector cannot effectively produce copies of itself following the infection of a target cell. Non-replicating viral vectors may therefore advantageously have an improved safety profile as compared to replication-competent viral vectors. A non-replicating viral vector may retain the ability to replicate in cells that are not target cells, allowing viral vector production. By way of example, a non-replicating viral vector (e.g., a non-replicating poxvirus vector) may lack the ability to productively replicate in a target cell such as a mammalian cell (e.g., a human cell), but retain the ability to replicate (and hence allow vector production) in an avian cell (e.g., a chick embryo fibroblast, or CEF, cell). In some embodiments, the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MV A) vector, an NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. MVA and NYVAC are both attenuated derivatives of the vaccinia virus. Compared to the vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames. In some embodiments, the adenovirus vector is a non-replicating adenovirus vector (wherein non-replicating is defined as above). Adenoviruses can be rendered nonreplicating by deletion of the El or both the El and E3 gene regions. In some embodiments, the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.
In a particular embodiment, the invention also relates to i) lumacaftor and ivacaftor and ii) a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate, or sequential use in the treatment of cystic fibrosis.
In a particular embodiment, the invention also relates to i) tezacaftor and ivacaftor and ii) nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate, or sequential use in the treatment of cystic fibrosis.
In a particular embodiment, the invention also relates to i) ivacaftor, tezacaftor and elexacaftor and ii) a nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO: 1) for simultaneous, separate or sequential use in the treatment of cystic fibrosis.
As used herein the term “tezacaftor” also called “l-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)- N-{ l-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(l-hydroxy-2-methylpropan-2-yl)-lH-indol-5- yl} cyclopropane- 1 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0001
As used herein, the term “lumacaftor” also called “3-{6-[l-(2,2-difhioro-2H-l,3-benzodioxol- 5-yl)cyclopropaneamido]-3-methylpyridin-2-yl}benzoic acid” has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0002
As used herein, the term “ivacaftor” also called “N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo- l,4-dihydroquinoline-3 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0003
As used herein, the term “elexacaftor” also called “N-(l,3-dimethylpyrazol-4-yl)sulfonyl-6- [3-(3,3,3-trifluoro-2,2-dimethylpropoxy)pyrazol-l-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-l- yl]pyridine-3 -carboxamide” has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0004
As used herein, the term “Orkambi” relates to a biotherapy made up of “lumacaftor” and “ivacaftor”. Orkambi is developed by Vertex Pharmaceuticals.
As used herein, the term “Symdeko” relates to a biotherapy made up of “tezacaftor” and “ivacaftor”. Symdeko is developed by Vertex Pharmaceuticals.
As used herein, the term “Kaftrio” or “Trikfata” relates to a tritherapy made up of “ivacaftor”, “tezacaftor” and “elexacaftor”. Kaftrio/Trikfata is developed by Vertex Pharmaceuticals.
In some embodiments, the one skilled in the art can easily provide some modifications that will improve the clinical efficacy of the nucleic acid molecule of the present invention. In a particular embodiment, the nucleic acid molecule of the present invention, according to the invention, is a LNA oligonucleotide. As used herein, the term "LNA" (Locked Nucleic Acid) (or "LNA oligonucleotide") refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogs also referred to as LNA nucleotides and LNA analog nucleotides. LNA oligonucleotides, LNA nucleotides and LNA analog nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO 03/006475; U.S. Patent Nos. 6,043,060, 6268490, 6770748, 6639051, and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840, and 2005/0203042, all of which are incorporated herein by reference. LNA oligonucleotides and LNA analog oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA. Other possible stabilizing modifications include phosphodiester modifications, combinations of phosphodiester and phosphorothioate modifications, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. Chemically stabilized, modified versions of the nucleic acid molecule of the present invention also include “Morpholinos” (phosphorodiamidate morpholino oligomers, PMOs), 2'-O-Met oligomers, tricyclo (tc)-DNAs, U7 short nuclear (sn) RNAs, or tricyclo-DNA-oligoantisense molecules (U.S. Provisional Patent Application Serial No. 61/212,384 For: Tricyclo-DNA Antisense Oligonucleotides, Compositions, and Methods for the Treatment of Disease, filed April 10, 2009, the complete contents of which is hereby incorporated by reference). Chemically stabilized, modified versions of the nucleic acid molecule of the present invention also include 2'-O-methyl modified nucleotides, or 2'-0- methoxyethyl modified nucleotides, or 2'-fluro modified nucleotides.
As used herein, the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third...) drug. The drugs may be administered simultaneously, separately, or sequentially and in any order. According to the invention, the drug is administered to the subject using any suitable method that enables the drug to reach the lungs, pancreas, intestine, skin, and reproductive system. In some embodiments, the drug is administered to the subject systemically (i.e., via systemic administration). Thus, in some embodiments, the drug is administered to the subject such that it enters the circulatory system and is distributed throughout the body. In some embodiments, the drug is administered to the subject by local administration, for example, by the local administration to the lungs. In some embodiments, the combination of tezacaftor and ivacaftor or the combination of lumacaftor and ivacaftor or the combination of ivacaftor, tezacaftor and elexacaftor are administered to the subject in a pharmaceutical composition. In some embodiments, tezacaftor and ivacaftor or lumacaftor and ivacaftor or tezacaftor, ivacaftor and elexacaftor are administered in the form of tablets for oral administration.
In some embodiments, the nucleic acid molecule of the present invention is delivered by any device adapted to introduce one or more therapeutic compositions into the upper and/or lower respiratory tract, pancreas, intestine, skin, and reproductive system. The devices may be adapted to deliver the therapeutic compositions of the invention in the form of a finely dispersed mist of liquid, foam, or powder. The device may use a piezoelectric effect or ultrasonic vibration to dislodge powder attached on a surface such as a tape in order to generate mist suitable for inhalation. The devices may use any propellant system known to those in the art, including, but not limited to, pumps, liquefied- gas, compressed gas, and the like. Devices of the present invention typically comprise a container with one or more valves through which the flow of the therapeutic composition travels and an actuator for controlling the flow. The devices suitable for administering the constructs of the invention include inhalers and nebulizers. Various designs of inhalers are available commercially and may be employed to deliver the medicaments of the invention. These include the Accuhaler, Aerohaler, Aerolizer, Airmax, Autohaler, Clickhaler, Diskhaler, Easi-breathe inhaler, Fisonair, Integra, Jet inhaler, Miat-haler, Novolizer inhaler, Pulvinal inhaler, Rotahaler, Spacehaler, Spinhaler, Syncroner inhaler, and Turbohaler devices. Thus, in some embodiments, the delivery is done by means of a nebulizer or other aerosolisation device, provided the integrity of the nucleic acid miR-9 inhibitor is maintained.
The nucleic acid miR-9 inhibitor may be administered to the subject in a single delivery, such as a bolus delivery. Alternatively, the nucleic acid miR-9 inhibitor may be administered to the subject using a continuous delivery technique, such as a timed infusion. The nucleic acid miR- 9 inhibitor may be administered using a repeated delivery regimen, for example, on an hourly, daily or weekly basis. The nucleic acid miR-9 inhibitor dosages may be achieved by single or multiple administrations. By way of example, the nucleic acid miR-9 inhibitor may be administered to the subject in a regimen consisting of a single administration. Alternatively, the nucleic acid miR-9 inhibitor may be administered to the subject in a regimen comprising multiple administrations. For example, an administration regimen may comprise multiple administrations per day, or daily, weekly, bi-weekly, or monthly administrations. An example regimen comprises an initial administration followed by multiple subsequent administrations at weekly or bi-weekly intervals. Another example regimen comprises an initial administration followed by multiple subsequent administrations at monthly or bi-monthly intervals. Alternatively, administration of the nucleic acid miR-9 inhibitor can be guided by monitoring of CF symptoms in the subject. Thus, an example regimen comprises an initial administration followed by multiple subsequent administrations carried out on an irregular basis as determined by monitoring CF symptoms in the subject. Methods for delivering nucleic acids are known in the art and will be familiar to a skilled person. By way of example, suitable nucleic acid delivery methods include ionophoresis, microspheres (e.g., bioadhesive microspheres), nanoparticles, dendritic polymers, liposomes, hydrogels, cyclodextrins, and proteinaceous vectors.
As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.
As used herein, the term “administration simultaneously” refers to the administration of 1, 2 or 3 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 3 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 3 active ingredients at different times, the administration route being identical or different. Accordingly, a fourth object of the present invention relates to a pharmaceutical composition comprising the nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO: 1).
In some embodiment, the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, lumacaftor and ivacaftor.
In some embodiment, the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, tezacaftor and ivacaftor.
In some embodiment, the pharmaceutical composition of the present invention comprises a combination of the nucleic acid miR-9 inhibitor of the present invention, tezacaftor, ivacaftor and elexacaftor.
A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drugs depend on the disease or condition to be treated and may be determined by the people skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the drug employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize disease progression. One of the ordinary skills in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example, about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g., be intranasal installation, intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance, administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-150 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. The treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-500 mg/kg
Typically, the drugs of the present invention are administered to the subject in the form of a pharmaceutical composition that comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol, and wool fat. For use in administration to a subject, the composition will be formulated for the administration to the subject. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein include intranasal installation, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may be added if desired. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m2 and 500 mg/m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., subcutaneous injection or intranasal installation or intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g., 1 ml for subcutaneous injection or intranasal installation or intramuscular), and between about 1 ng to about 100 mg, e.g., about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. TMEM16a ASO4 increases mucociliary clearance in different cftr mutations. Histograms represent the average values ± SDs and were compared using ANOVA coupled with Dunnett' s, Bonferroni's, and Tukey's posthoc test. Cells for patients with F508del/F508del (A), 2184DelA/W1282x (B) and 1717-lG>A/F508del (C) mutations. **p<0.01; ***p<0.001; ****p<0.0001.
Figure 2. TMEM16a ASO4 treatment increases viability and weight in CF mice. TMEM16a ASO4 and Ctl ASO (10 mg/kg) were subcutaneously injected on days 11 and 18 and every 15 days afterward to 129-cftrtmlEur CF mice. (A). Kaplan-Meier survival curves of overall survival analysis in CF mice treated with control ASO or TMEM16a ASO4. Data are the mean of more than 100 mice. The dotted lines represent the median survival, the time at which the staircase survival curve crosses 50%. (B). Body weight curve of CF mice. Each point represents the mean of the group treated with ASO4 TMEM16A (triangular) or the ASO control (dots). P values were determined using the log-rank test. ****p<0.0001.
Figure 3. Effects of different ASO TMEM16A sequences on TMEM16a expression and activity. (A). CF cells (CFBE41O-) were treated by Ctl ASO or the different TMEM16a ASO indicated for 24h. A. TMEM16A protein expression was analyzed and quantified by western blotting using anti-TMEM16A antibody. P-actin was used for normalization, (n = 4, in triplicates). Histograms represent average values ± SD. (B). TMEM16A chloride channel activity assessed by I- quenching of halide-sensitive YFP-H148q/I152L protein on CF cells. Histograms represent average values ± SDs, and the conditions were compared using ANOVA coupled with Dunnett' s post hoc test in comparison to CF Ctl ASO condition. (C). Mice cell line (MLE15) was treated by Ctl ASO, a long version (ASO3), or a short version (ASO4) of TMEM16a ASO for 24h. Histograms represent average values ± SDs, and the conditions were compared using ANOVA coupled with Dunnett' s post hoc test in comparison Ctl condition.
***p<0.001. Figure 4. TMEM16a ASO4 increases chloride efflux. (A-B). The normalized total area under the curves of Ussing-chamber assays of non-CF (NCF) (A) and CF (B) primary human bronchial epithelial (hBEC) cells culture in air-liquid interphase and treated with Ctl ASO or TMEM16a ASO4 for 1 hour each day for 3 days at 50 nM. (C). The normalized total area under the curves of Ussing-chamber assays of non-CF hBEC when stimulated with UTP with or without Ani9 pretreatment (1 pM) (n=3). Data are represented as mean values ± SD. ****p<0.0001, ANOVA followed by Dunnett' s, Bonferroni's, and Tukey's posthoc test.
Figure. 5. TMEM16a ASO4 increases mucociliary clearance in different cells in various CFTR mutations. Bead mean speed measured in hBEC with (A) F508del/F508del and (B) 2184DelA/W1282x mutations. After the indicated treatments (n = 5 per condition, 75 beads tracked per sample). Data are represented as mean values ± SD and compared to ASO4 TMEM16a condition. ****p<0.0001, ANOVA followed by Dunnett's, Bonferroni's, and Tukey's posthoc test.
EXAMPLE:
Material & Methods
Cell cultures & cell transfections
As previously described, cell lines were cultured and transfected with ASO (control or TMEM16a ASO4) (1, 2). Primary human bronchial epithelial cells (hBEC) provided by Epithelix SARL (Geneva, Switzerland) were cultured and transfected with ASO (3).
TMEM1 6a chloride channel activity assay
TMEM16a channel activity was assessed by iodine (I-) quenching of halide-sensitive YFP- H148Q/I152L protein (4). Fluorescence was recorded and analyzed using a plate reader as previously described (3).
Mucociliary clearance assay
One day after transfection of primary hBEC cell with TMEM16a ASO4 or control ASO, FluoSpheres Carboxylate-Modified Microspheres, 1.0 pm, yellow-green fluorescent (505/515) (Thermo Fisher Scientific) diluted in culture medium (1/50) were added to the apical face of the cultures. The movement of the beads was recorded under an Axiovert 200 microscope (Zeiss) and analyzed as previously described (3). In Vivo Fluorescence Assessment
Nude mice (BALB/cAnNRj-Foxnl nu/nu) were treated with fluorescent TMEM16a ASO4 (Eurogentec, Belgium; ex 640 nm/em 680 nm) by different routes of administration (10 mg/kg). Fluorescence was measured at Ih, 4h, Id, 8d, 15d, 21d, and 30 days. Prior to imaging, mice were anesthetized using isoflurane inhalation. Imaging was performed on the IVIS Spectrum imaging system (PerkinElmer, Inc., France) by two-dimensional (2D) epifluorescence imaging at indicated times after probe injection to optimize readouts. 3D images were generated by Ivis software.
In Vivo toxicity evaluation
Toxicity protocol was performed by C-Ris Pharma (Saint-Malo, France) on healthy CD-I mice. Hematology parameters were determined on whole blood. These parameters were determined by using the ProCyte Dx hematology analyzer (IDEXX, France). Biochemical assays were performed using the KBIO04-DUO biochemistry analyzer (Kitvia, France). After sacrifice on day 14, organs were collected and embedded in paraffin before cutting and analyzing.
Survival and fertility studies
129-Cftr tmlEur CF model mice homozygous for the F508del mutation and their wild-type littermates were obtained from CDTA-CNRS (Orleans, France). TMEM16a ASO4 subcutaneous injections (10 mg/kg) were given, and their body weights were recorded. Animals had access to food and water ad libitium. All animal studies were approved by the Institutional Animal Care and Use Committee.
Statistical analysis
Statistical analyses were done using GraphPad Prism 7.05 software (GraphPad Software Inc, San Diego, USA) as indicated in the respective figure legends.
Results
IMF Ml 6a ASO4 potentiates chloride efflux in CF cells
To complete previously published results (9), the effects of TMEM16a ASO4 on Cl- efflux were assayed at different concentrations (from 10 nM to 200 nM during 24h) on the CFBE41o- cell line (Data not shown). TMEM16a ASO4 robustly potentiated TMEM16a Cl- channel in a concentration manner (Data not shown). The concentration ever used at 50 mM is in the middle of the range of activation. At 200 nM, modifications in the morphology of the cells were observed, suggesting a toxic dose confirmed by the lactate dehydrogenase method (Data not shown).
The effects of Cl- efflux were analyzed by YFP probe in different F508del cells from lungs (KM4, CUFI, IB3-1, human primary glandular cells (HBG)) or pancreas (CFPAC). The effects are significant in all cells tested, with differences in the activation level (Data not shown). The induction of chloride efflux was between 20 to 25% in KM4, CUFI, and IB3-1. In the most responsive cells (HBG and CFPAC), the induction is close to 50%.
TMEM16a ASO4 increases mucociliary clearance of CF cells with different mutations hBEC from different donors with different mutations classes were cultured in air-liquid interphase to obtain differentiated cells (Figure 1A. F508del/F508del (class Il/Class II); Figure IB: 2184delA/W1282X (Class I/Class I) and Figure 1C 1717-lG>A/F508del (Class I/Class II) and. Cells were cultured for 24h in the presence of lumacaftor/ivacaftor (Boston, Massachusetts, USA) or TMEM16a ASO4. In all cases, TMEM16a ASO4 induced strong and significant activation of mucociliary clearance evaluated by the movement of fluorescent beads. The effects of lumacaftor/ivacaftor (Orkambi®) were only positive and significant on F508del/F508del cells but significantly lower than with TMEM16a ASO4 (Figure 1A). Interestingly, the effects of TMEM16a ASO4 and lumacaftor/ivacaftor were additive on F508del/F508del cells compared to the treatment with TMEM16a or lumacaftor/ivacaftor alone. Very interesting results were observed on class I mutations cells (Figure IB), with a significant increase of chloride efflux of cells treated with TMEM16a ASO4 compared to cells treated with lumacaftor/ivacaftor. Surprisingly, we have found that the combination is not efficient with certain mutations. Indeed, the impact of lumacaftor/ivacaftor on Cl- efflux with or without TMEM16a ASO4 was negative on mucociliary clearance with 1717-lG>A/F508del cells (Figure 1C). The addition of lumacaftor/ivacaftor does not seem to have any effect on cells already treated with TMEM16a ASO4.
Subcutaneous administration allowed a good distribution in all organs in mice
Fluorescent TMEM16a ASO4 (10 mg/kg) was administered in nude mice by intravenous, subcutaneous, intraperitoneal injection or intranasal installation to determine the best administration ways and biodistribution. Fluorescence in mice was recorded every week for 1 month (Data not shown). In these pseudocolor images in which white represents the greatest intensity of photon emission, minimal light emission is detected outside the injection site at DO. After 7 days, a good molecule distribution in all mice tissues can be observed with intravenous, subcutaneous, and intranasal administration. However, a strong accumulation in the liver and kidney occurred. With the intraperitoneal injection, the molecule disperses after 14 days to a small extent, while we observed a very good distribution in the mouse tissues for the other routes of administration. From day 21, we observed a decrease in the fluorescence level for all the conditions, as we can observe on day 30. In the following experiments, we chose the subcutaneous injection, a classic way for ASO administration, which was administered every 15 days.
TMEM1 6a ASO4 has good specificity and is not toxic
To characterize TMEM16a ASO4, we have performed an "in vitro pharmaceutical profiling" defined by Bowes et al. (14). This method was described by four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis, and Pfizer) to identify undesirable off- target activity profiles that could hinder or halt candidate drugs’ development. No significant TMEM16a ASO4 impact was identified in the tested targets panel (Data not shown . To confirm TMEM16a ASO4 specificity, we performed experiments on calcium activity, proliferation, inflammation, and infection (Data not shown). A strategy targeting CaCC with Denufosol was proposed to patients in the past. This drug activated calcium mobilization to induce CaCC channels. In our case, we demonstrate that calcium mobilization was not affected in all treated cells. Because overexpression of TMEM16a is associated with different cancer (15), we have decided to study this parameter. After TMEM16a ASO4 treatment and consequently mild induction of TMEM16a activation, we have not observed any modification of the proliferation. Finally, as inflammation is overexpressed and is considered a critical dysregulated parameter in CF cells, we have analyzed the impact of TMEM16a ASO4 on 24 cytokines. No significant induction of cytokines was observed in all tested CF cells (Data not shown). In the same way, infection or bacterial proliferation were not affected by TMEM16a ASO4 (Data not shown).
Finally, we have performed a toxicity analysis of TMEM16a ASO4 on CF mice. Mice were injected subcutaneously at six doses from 0 to 500 mg/kg (Data not shown). Toxicity was assessed by monitoring general signs of pharmacologic and toxicological (morbidity and mortality, clinical signs, body weight, food, and water consumption) three times a week for 14 days. Before sacrifice, blood and urine were collected, hematology analysis (hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count), blood chemistry analysis (including coagulation parameters), and urinalysis were performed. In blood samples (Data not shown), the different parameters representing liver (ALAT and ASAT), kidney (urea), or pancreas (amylase) were similar in all tested doses. Similar results were obtained on the other parameters (data not shown). No significant difference was observed in the behavior of the mice, on macroscopic autopsy, or in histology (Data not shown).
TMEM1 6a ASO4 increases survival and weight gain in CF mice
CF mice usually die of intestine occlusion during or after the weaning period. We treated the mice on days 11 and 18 after birth and repeatedly every 15 days after weaning by subcutaneous injections of TMEM16a ASO4 or control ASO (10 mg/kg). We observed a significant increase in the mice's survival that went up to 196 days vs. 36 days in control mice (p<0.0001) (Figure 2A). These data are consistent with the chronically treated mice's weight gain being higher than control mice (Figure 2B).
Recovery of fertility in treated male mice
The literature clearly shows that homozygous male mice (F508del/F508del) are sterile (21, 22). This is explained by the atresia of the vas deferens, which makes them non-functional (Data not shown). In our study, homozygous mice (F508del/F508del) chronically treated from day 11 after birth (Data not shown) were put in the same cage. Mice could reproduce, and the pups were viable (Data not shown).
Screening of different TMEM16a ASO on TMEM16a expression and activity
To test the most effective TMEM16a ASO, we have generated 2 other long versions ASO1 (TTGGGACCTCGATCTTGG (SEQ ID NO: 5)) and ASO2 (TCTTTGGGACCTCGATCTTG (SEQ ID NO: 6)) of the TMEM16a ASO in addition to the main long TMEM16a ASO (ASO3 (TTTTCTCCGTCTTTGGGACCT (SEQ ID NO:7)) and a short version of TMEM16a ASO (ASO4 (SEQ ID NO: 1). CF cell line (CFBE41O-) were transfected during 24 hours as published (Sonneville et al., 2017). Results were compared to non-CF and CF cells treated with control ASO. We have observed a restoration of TMEM16a expression (Figure 3 A) and chloride activity (Figure 3B) with the long and the short version (ASO3 (SEQ ID NO: 7) and ASO4 (SEQ ID NO: 1)), which was similar to the non-CF cells (16HBE14o-). ASO1 (SEQ ID NO:5) and ASO2 (SEQ ID NO:6) had no effects on TMEM16a expression or activity.
In vitro effect of the short and long version of TMEM16a AS04 on chloride efflux Sequence comparison between humans and mice showed that one nucleotide could interfere with the binding of the TMEM16a ASO to the miR-9 site. We compared the effects of the two oligonucleotides (the long TMEM16a ASO3 (SEQ ID NO: 7) and the short TMEM16a ASO4 (SEQ ID NO: 1)) on chloride activity in a mouse cell line (MLE15). The results demonstrate a significant increase in chloride activity on MLE15 with the short sequence (ASO4 (SEQ ID NO: 1)) (Figure 3C) compared to the control ASO or long version (ASO3 (SEQ ID NO:7)), which did not demonstrate any effect on the mouse cell line. Thus, since the short ASO sequence (ASO4 (SEQ ID NO: 1)) could work in human and mouse models, we used the short ASO sequence (ASO4 (SEQ ID NO: 1)) for the rest of the in vivo and in vitro studies.
TMEM1 6a ASO4 potentiates chloride efflux in CF cells with different mutations
We measured by Ussing chamber the transepithelial Cl- current, driven by cAMP for CFTR or Ca2+ mediated signaling pathways for TMEM16a activity. After adding, to the apical compartment of primary human bronchial epithelial cells (hBEC), amiloride to block ENaC- mediated Na+ absorption and forskolin to activate CFTR efflux by an agonist of cAMP- dependant protein kinase, we observed a strong CFTR-dependent Cl- efflux in non-CF hBEC (Figure 4A~). This activity was abolished when we added the specific CFTR inhibitor Inh-172 (Figure 4 A). In CF hBEC, no cAMP-dependent Cl- activity was observed as expected (Figure 41 f
After adding UTP, Ca2+-mediated Cl- efflux was significantly increased in ASO4 TMEM16a treated CF similarly to non-CF cells and compared to control (Figures 4A and 4B). To verify the specificity of TMEM16a ASO4 and ensure the CaCC's molecular identity, we pretreated the cells with Ani9, the most potent and selective inhibitor of TMEM16a. The TMEM16A- mediated Cl- efflux was strongly inhibited (Figure 4C), which confirms the ASO's targeted channel specificity.
TMEM16a ASO4 increases mucociliary clearance of CF patient cells with different mutations
TMEM16A ASO4 induced strong and significant activation of mucociliary clearance evaluated by the movement of fluorescent beads in all the different tested mutations (Figures 5A and 5B). The effects of Orkambi® were only positively significant on class II F508del/F508del cells, although significantly lower than those treated with TMEM16A ASO4 (Figure 5A). Trikafta® and ASO4 TMEM16A had a similar effect in class II mutation (Figure 5A). However, we observed an additive effect when combining the two molecules (Figure 5A). TMEM16A ASO4 shows a strong and significant activation of mucociliary clearance in class I 2184DelA/W1282x cells compared to cells treated with control, Orkambi® or Trikafta® Figure 5 ) We observe an additive effect when combining the Trikafta® and TMEM16A ASO4 (Figure 5B).
Conclusions
The present results validate the concept of using an ASO targeting an alternative chloride channel with TMEM16a in the context of CF. The inventors have observed an increase in the survival of CF mice that usually would have died of intestinal obstruction shortly after being weaned. Furthermore, the inventors’ approach offers a novel therapeutic strategy for patients with CF, independently of the CFTR channel, compensating for lung dysfunction by increasing chloride activity and mucociliary clearance, and treating other symptoms such as intestinal obstruction. Thus, this therapy could apply to all patients with CF and, in particular, to those who cannot benefit from current curative treatments.
In order to determine the most effective way of administration, the inventor have injected fluorescent TMEM16a ASO4 in mice by intravenous, subcutaneous, intraperitoneal injection, and intranasal instillation. As we have observed, airway administration could be interesting in our study because activity could be significantly more effective in the airway than in intraperitoneal or intravenous administration. These data indicate a critical role of ASO4 in a murine CF model and suggest that local delivery of antisense oligonucleotides may be a novel approach for treating CF lung disorders. Additionally, our results show that the molecule's half- life in animals is quite long since we can still see the ASO4 fluorescence 30 days after administration. More, an administration every 15 days is sufficient to significantly increase the survival of the animals. These data are consistent with the literature. For example, a 20-MOE ASO4 administered subcutaneously weekly at 300 mg per week has been shown to reduce Factor XI levels by 80% and reduce deep vein thromboses in patients undergoing total knee replacement by 7-fold compared to enoxaparin (5). In this condition, an administration twice- monthly of TMEMA16a ASO4 has been demonstrated to increase the survival of mice from an average survival of 36 days without treatment to 294 days with chronic treatment.
Finally, one result that was not expected was the results the inventors obtained on the improvement of fertility in male CF mice by restoring vas deferent abnormalities, also described on CF male patients (6). Of course, these results will have to be pursued to understand better the role that TMEM16a may play in vas deferens as little data is available (7, 8). Nevertheless, these results show that targeting TMEM16a can be an alternative strategy for different symptoms. Furthermore, because TMEM16a expression and function are independent of CFTR function, this therapeutic approach is predicted to be suitable for all patients with CF. These results show that strategies targeting TMEM16a can be considered a strategy applied to all patients and could be used alone or in combination with other CFTR-targeted strategies for eligible patients.
REFERENCES:
1. Sonneville F, Ruffin M, Coraux C, Rousselet N, Le Rouzic P, Blouquit-Laye S, et al.
TARGETING MIR-9 AND AN01 : NEW THERAPEUTIC STRATEGY IN CYSTIC FIBROSIS. Pediatric Pulmonology 2017; 52: S291-S291.
2. Tabary O, Escotte S, Couetil JP, Hubert D, Dusser D, Puchelle E, et al. Relationship between
IkappaBalpha deficiency, NFkappaB activity and interleukin-8 production in CF human airway epithelial cells. Pflugers Arch 2001; 443 Suppl 1 : S40-44.
3. Sonneville F, Ruffin M, Coraux C, Rousselet N, Rouzic PL, Blouquit-Laye S, et al.
MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nature Communications 2017; 8: 710.
4. Galietta LJ, Haggie PM, Verkman AS. Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett 2001; 499: 220-224.
5. Yu RZ, Kim TW, Hong A, Watanabe TA, Gaus HJ, Geary RS. Cross-species pharmacokinetic comparison from mouse to man of a second-generation antisense oligonucleotide, ISIS 301012, targeting human apolipoprotein B-100. Drug Metab Dispos 2007; 35: 460-468.
6. Elborn JS. Cystic fibrosis. Lancet 2016; 388: 2519-2531.
7. Beauvillard D, Perrin A, Drapier H, Ravel C, Freour T, Ferec C, et al. [Congenital bilateral absence of vas deferens: From diagnosis to assisted reproductive techniques - the experience of three centers], Gynecol Obstet Fertil 2015; 43: 367-374.
8. Chan HC, Ruan YC, He Q, Chen MH, Chen H, Xu WM, et al. The cystic fibrosis transmembrane conductance regulator in reproductive health and disease. J Physiol 2009; 587: 2187-2195. eville F, Ruffin M, Coraux C, Rousselet N, Rouzic PL, Blouquit-Laye S, et al.
MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nature Communications 2017; 8: 710. WJ, Qiu J, Sun J, Ma CL, Huang N, Jiang Y, et al. Downregulation of microRNA-9 reduces inflammatory response and fibroblast proliferation in mice with idiopathic pulmonary fibrosis through the ANOl-mediated TGF-beta-Smad3 pathway. J Cell Physiol 2018. YR, Lee ST, Kim SL, Zhu SM, Lee MR, Kim SH, et al. Down-regulation of miR-9 promotes epithelial mesenchymal transition via regulating anoctamin-1 (AN01) in CRC cells. Cancer Genet 2019; 231-232: 22-31. cuklu SD, Donoghue MT, Rehmet K, de Souza Gomes M, Fort A, Kovvuru P, et al.
MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells. J Biol Chem 2012; 287: 29516-29528.n J, Chen W, Zhao L, Zang X, Liu Y. A negative Smad2/miR-9/AN01 regulatory loop is responsible for LPS-induced sepsis. Biomed Pharmacother 2019; 116: 109016. es J, Brown AJ, Hamon J, Jarolimek W, Sridhar A, Waldron G, et al. Reducing safety- related drug attrition: the use of in vitro pharmacological profiling. Nat Rev Drug Discov 2012; 11 : 909-922. ttes D, Jan LY. The multifaceted role of TMEM16A in cancer. Cell Calcium 2019; 82:
102050.

Claims

CLAIMS:
1. A nucleic acid miR-9 inhibitor comprising the nucleic acid sequence of AATCTTTGGTAGTAA (SEQ ID NO: 1).
2. A vector comprising the nucleic acid sequence of claim 1.
3. A pharmaceutical composition comprising the nucleic acid sequence of claim 1.
4. A method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of nucleic acid miR-9 inhibitor comprising the nucleic acid sequence AATCTTTGGTAGTAA (SEQ ID NO:1).
5. The method of claim 4 wherein the subject harbors at least one allelic mutation selected from class I, class II, class III, class IV, class V or class VI.
6. The method of claim 4 wherein the subject harbors at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the first allele and at least one mutation selected from class I, class II, class III, class IV, class V or class VI in the second allele.
7. The method of claim 6 wherein the subject harbors at least a mutation of class I in the first allele and at least a mutation of class II in the second allele.
8. The method of claim 5 wherein the subject harbors at least one allelic mutation selected from F508del-CFTR, R117H CFTR, 2184delA CFTR, W1282X CFTR, 2183 AA>G CFTR or G551D CFTR.
9. The method of claim 4 wherein the nucleic acid miR-9 inhibitor does not bind directly to miR-9 but instead binds to a miR-9 mRNA target site in the AN01 nucleic acid sequence.
10. The method of claim 5 wherein the nucleic acid miR-9 inhibitor is a Target Site Blocker (TSB). The method of claim 4 wherein the nucleic acid miR-9 inhibitor is delivered using a viral vector The method of claim 9 wherein the vector is an AAV vector. The method of claim 4 wherein the nucleic acid miR-9 inhibitor is administered to the subject using any suitable method that enables the nucleic acid miR-9 inhibitor to reach the lungs. The method of claim 4 wherein the nucleic acid miR-9 inhibitor is administered in combination with lumacaftor and ivacaftor. The method of claim 4 wherein the nucleic acid miR-9 inhibitor is administered in combination with tezacaftor and ivacaftor. The method of claim 4 wherein the nucleic acid miR-9 inhibitor is administered in combination with tezacaftor, ivacaftor and elexacaftor. The pharmaceutical composition of claim 3 which comprises lumacaftor and ivacaftor. The pharmaceutical composition of claim 3 which comprises tezacaftor and ivacaftor. The pharmaceutical composition of claim 3 which comprises tezacaftor, ivacaftor and elexacaftor.
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