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WO2025237990A1 - Oligonucléotides antisens et leur utilisation pour le traitement de la fibrose pulmonaire - Google Patents

Oligonucléotides antisens et leur utilisation pour le traitement de la fibrose pulmonaire

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Publication number
WO2025237990A1
WO2025237990A1 PCT/EP2025/063054 EP2025063054W WO2025237990A1 WO 2025237990 A1 WO2025237990 A1 WO 2025237990A1 EP 2025063054 W EP2025063054 W EP 2025063054W WO 2025237990 A1 WO2025237990 A1 WO 2025237990A1
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WIPO (PCT)
Prior art keywords
hspb5
inhibitor
aav
seq
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/063054
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English (en)
Inventor
Carmen GARRIDO-FLEURY
Philippe Barthelemy
Palma Rocchi
Françoise GOIRAND
Olivier Burgy
Philippe Bonniaud
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Bourgogne Europe
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Bordeaux
Universite de Bordeaux
Institut Jean Paoli and Irene Calmettes
Original Assignee
Universite Bourgogne Europe
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Bordeaux
Universite de Bordeaux
Institut Jean Paoli and Irene Calmettes
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Application filed by Universite Bourgogne Europe, Aix Marseille Universite, Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Centre Hospitalier Universitaire de Bordeaux, Universite de Bordeaux, Institut Jean Paoli and Irene Calmettes filed Critical Universite Bourgogne Europe
Publication of WO2025237990A1 publication Critical patent/WO2025237990A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • 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
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • the present invention relates to antisense oligonucleotides efficiency in reducing the expression of HSPB5 (heat shock protein aB-crystallin) in Fibrotic interstitial lung diseases (Fibrotic ILDs) in particular Progressive pulmonary fibrosis (PPF) such as idiopathic pulmonary fibrosis (IPF) of a subject.
  • HSPB5 heat shock protein aB-crystallin
  • PPF Progressive pulmonary fibrosis
  • IPPF idiopathic pulmonary fibrosis
  • ILDs connective tissues diseases including rheumatoid arthritis-ILD (RA-ILD) and systemic sclerosis associated ILD (SSc-ILD).
  • RA-ILD rheumatoid arthritis-ILD
  • SSc-ILD systemic sclerosis associated ILD
  • PPF progressive pulmonary fibrosis
  • TGF- P1 is a key profibrotic growth factor overexpressed during PPF which promotes fibroblast transition into pathological myofibroblasts (7, 8).
  • TGF-pi classically signals via the Smads proteins, a crucial pathway in fibrogenesis (9).
  • HSPs Heat shock proteins
  • HSPB5 also termed aB-crystallin
  • BLM bleomycin
  • Inventors team has demonstrated that HSPB5 knock-out mice are resistant to BLM-induced fibrosis.
  • HSPB5 hampers SMAD4 ubiquitination and, consequently, SMAD4 accumulates in the nucleus and promotes TGF-pi signaling and fibrogenesis (11).
  • HSPB5 appears as a relevant target for pulmonary fibrosis.
  • ASO Antisense Oligonucleotides
  • ASO targeting HSPB5 can efficiently limit pulmonary fibrosis development and may be a novel therapeutic strategy to hamper lung fibrosis progression.
  • a first object of the invention concerns an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 in a subject in need thereof and targets the gene or the mRNA of HSPB5.
  • a second object of the present invention concerns the inhibitor and/or an antisense oligonucleotide according to the invention for use in the treatment of Fibrotic interstitial lung diseases (Fibrotic ILDs) in a subject in need thereof.
  • Fibrotic ILDs Fibrotic interstitial lung diseases
  • Fibrotic ILDs is progressive pulmonary fibrosis (PPF)
  • HSPB5 heat shock protein aB- crystallin
  • TGF Transforming-growth-factor
  • ASO22 was selected by its capacity to inhibit TGF-pi -induced expression ofHSPB5 and additional key markers of fibrosis such as PAI-1, collagen, fibronectin and a-SMA in fibroblastic human CCD-19Lu cells as well as PAI-1 and a-SMA in pulmonary epithelial A549 cells.
  • Intra-tracheal or intravenous administration of ASO22 in bleomycin-induced pulmonary fibrotic mice decreased HSPB5 expression and reduced fibrosis as measured by the decrease in pulmonary remodeling, collagen accumulation and Acta2 and Col lai expression.
  • the present invention relates to antisense oligonucleotides efficiency in reducing the expression of HSPB5 in a subject suffering of fibrotic interstitial lung diseases (ILDs), in particular progressive pulmonary fibrosis (PPF) such as idiopathic pulmonary fibrosis (IPF).
  • ILDs fibrotic interstitial lung diseases
  • PPF progressive pulmonary fibrosis
  • IPPF idiopathic pulmonary fibrosis
  • a first aspect of the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets the gene or the mRNA of HSPB5.
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets the nucleic acids sequence SEQ ID NO: 27.
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets a region comprising at least between 15 nucleic acids to 25 nucleic acids of SEQ ID NO: 27.
  • the inhibitor targets at least, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleic acids of SEQ ID NO: 27.
  • the invention also relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets at least the region comprising or consisting of the nucleic acids 256-783 of SEQ ID NO: 27.
  • the invention also relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets the region selected from the list comprising or consisting of the nucleic acids 356-375 of SEQ ID NO: 27, the nucleic acids 536-555 of SEQ ID NO: 27, the nucleic acids 676-695 of SEQ ID NO: 27, the nucleic acids 736-755 of SEQ ID NO:27 and the nucleic acids 756-775 of SEQ ID NO: 27.
  • the invention also relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets at least the region comprising or consisting of the nucleic acids 676-695 of SEQ ID NO: 27.
  • HSPB5 for “Probable ATP-dependent RNA helicase DDX5” and also knows as “heat shock protein oB-crystallin” or “Alpha-crystallin B chain” (CRY AB) is member of the small heat shock protein (sHSP also known as the HSP20) family and can be induced by heat shock, ischemia, and oxidation.
  • sHSP small heat shock protein
  • HSP20 Alpha-crystallin B chain
  • HSPB5 is a protein that in humans is encoded by the CRYAB gene (gene ID 1410).
  • HSPB5 functions as molecular chaperone that primarily binds misfolded proteins to prevent protein aggregation, as well as inhibit apoptosis and contribute to intracellular architecture (Yamamoto S, et al.
  • the naturally occurring human HSPB5 gene has a nucleotide sequence as shown in NCBI accession number Entrez Gene ID 1410 or Ensembl accession number ENSG00000109846. According to the NCBI database, HSPB5 has 6 major protein- coding mRNA sequences, which are NM_001885.3, NM_001289807.1, NM_001289808.2, NM 001330379.1, NM_001368245.1; NM_001368246.1 corresponding to the mRNA variant 1, variant 2, variant 3, variant 4, variant 5 and variant 6, respectively.
  • the variant 1, variant 2, variant 3, variant 5 concern HSPB5 isoform 1 protein and variant 4, variant 6 concern HSPB5 isoform 2 protein
  • the CDSs are strictly identical among the 4 HSPB5 transcript variants (variant 1, variant 2, variant 3, variant 5 encode the same isoform: HPB5 isoform 1).
  • the two other variants (variant 4 and 6 which encode for HPSBP5 isoform 2) have a distinct N terminus and are shorter length as the main isoform.
  • the reference sequence used to design ASOs was from the 1st transcript variant of HSPB5 i.e. NM_001885 and restricted to the CDS, which is defined as CCDS83 1.1 (between nucleotides 256 and 783 of SEQ ID NO: 27).
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets any variants 1, 2, 3 4, 5 and/or 6 ofHSPB5.
  • said inhibitor targets the nucleic acids sequence SEQ ID NO: 27 (mRNA of Homo sapiens HSPB5 (variant 1)).
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets the nucleic acids sequence SEQ ID NO: 27.
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor reduces the expression and/or activity of HSPB5 and targets at least between 15 nucleic acids to 25 nucleic acids of SEQ ID NO: 27.
  • nucleotide sequence of mRNA (CDS in bold) of Homo sapiens HSPB5 is defined by the sequence SEQ ID NO: 27:
  • inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit or reduce the expression and/or activity of HSPB5.
  • the inhibitor of the invention refers to a natural or synthetic compound that has a biological effect to reduce and/or inhibit the expression of HSPB5 gene.
  • inhibiting expression of a gene typically results in a decrease or even abolition of the gene product (ARN and/or protein, e.g. HSPB5 protein) in target cells or tissues, although various levels of inhibition may be achieved. Inhibiting or decreasing expression is typically referred to as knockdown.
  • the inhibitor of activity of HSPB5 refers to a natural or synthetic compound that has a biological effect to reduce and/or inhibit the activity of HSPB5.
  • the inhibitor of HSPB5 of the invention is a siRNA, a shRNA, an antisense oligonucleotide, miRNA or a ribozyme.
  • the inhibitor of HSPB5 according to the invention is a siRNA.
  • Small inhibitory RNAs also referred to as short interfering RNAs (siRNAs) can also function as HSPB5 expression inhibitors for use in the present invention.
  • HSPB5 activity can be reduced by treating the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that HSPB5 expression is specifically inhibited (i.e. RNA interference or RNAi) by degradation of mRNAs in a sequence specific manner.
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • the invention relates to an inhibitor of HSPB5 wherein said inhibitor is siRNA.
  • the siRNA according to the invention targets the region selected from the list consisting of the nucleic acids 356-375, 536-555, 676-695, 736-755, 756- 775 of SEQ ID NO: 27.
  • the inhibitor of HSPB5 according to the invention is a shRNA.
  • Short hairpin RNAs can also function as HSPB5 expression inhibitors for use in the present invention.
  • a short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.
  • RISC RNA-induced silencing complex
  • the inhibitor of HSPB5 according to the invention is a miRNA.
  • miRNAs can also function as HSPB5 expression inhibitors for use in the present invention.
  • miRNA has its general meaning in the art and refers to microRNA molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported, and suppress translation of targeted mRNAs. miRNAs are each processed from a longer precursor RNA molecule (“precursor miRNA”). Precursor miRNAs are transcribed from non-protein-encoding genes.
  • the precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer.
  • the processed miRNA is typically a portion of the stem.
  • the processed miRNA (also referred to as “mature miRNA”) becomes part of a large complex to downregulate, e.g. decrease translation, of a particular target gene.
  • miRNAs may be employed to knockdown HSPB5.
  • the miRNAs may be complementary to different target transcripts or different binding sites of a target transcript.
  • Polycistronic transcripts may also be utilized to enhance the efficiency of target gene knockdown
  • multiple genes encoding the same miRNAs or different miRNAs may be regulated together in a single transcript, or as separate transcripts in a single vector cassette.
  • the vector is a viral vector, including but not limited to recombinant adeno-associated viral (rAAV) vectors, lentiviral vectors, retroviral vectors and retrotransposon-based vector systems.
  • rAAV recombinant adeno-associated viral
  • the inhibitor of HSPB5 is an antisense nucleic acid.
  • linhibitor of HSPB5 expression of the invention is based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including antisense RNA molecules and antisense DNA molecules, would act to directly block the translation of HSPB5 mRNA by binding thereto and thus preventing protein translation or by increasing mRNA degradation, thus decreasing the level of HSPB5 proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding HSPB5 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732, each of which is incorporated by reference herein in its entirety).
  • the antisense RNA that is complementary to the sense target sequence is encoded by a DNA sequence for the production of any of the foregoing inhibitors (e.g., antisense, siRNAs, shRNAs or miRNAs).
  • the DNA encoding double stranded RNA of interest is incorporated into a gene cassette, e.g. an expression cassette in which transcription of the DNA is controlled by a promoter.
  • the inhibitor of HSPB5 gene expression is an antisense oligonucleotide.
  • the inhibitor of HSPB5 gene expression is an isolated, synthetic or recombinant antisense oligonucleotide targeting the HSPB5 mRNA transcript.
  • the oligonucleotide of the invention can be of any suitable type.
  • the oligonucleotide is a RNA oligonucleotide. In some embodiments, the oligonucleotide is a DNA oligonucleotide.
  • the invention also relates to an antisense oligonucleotide which reduces the expression and/or activity of HSPB5 and targets the gene or the mRNA of HSPB5.
  • the antisense oligonucleotide of the invention reduces the expression and/or activity of HSPB5 and targets the nucleotides sequence SEQ ID NO: 27. In a further embodiment, the antisense oligonucleotide of the invention reduces the expression and/or activity of HSPB5 and targets at least between 15 nucleic acids to 25 nucleic acids of SEQ ID NO: 27.
  • the antisense oligonucleotide targets at least, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleic acids of SEQ ID NO: 27.
  • the antisense oligonucleotide of the invention reduces the expression and/or activity of HSPB5 and targets at least the region comprising or consisting of the nucleic acids 256-783 of SEQ ID NO: 27.
  • the antisense oligonucleotide of the invention reduces the expression and/or activity of HSPB5 and targets at least the region selected from the list consisting of the nucleic acids 356-375 of SEQ ID NO: 27, the nucleic acids 536-555of SEQ ID NO: 27, the nucleic acids 676-695 of SEQ ID NO: 27, the nucleic acids 736-755 of SEQ ID NO: 27, the nucleic acids 756-775 of SEQ ID NO: 27.
  • the antisense oligonucleotide of the invention reduces the expression and/or activity of HSPB5 and targets at least the region comprising or consisting of the nucleic acids 676-695 of SEQ ID NO: 27.
  • the antisense oligonucleotide of the present invention has a length of at least 15 nucleic acids.
  • the antisense oligonucleotide of the present invention has a length from 15 to 25 nucleic acids.
  • the antisense oligonucleotide of the present invention has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleic acids.
  • the antisense oligonucleotide of the present invention has a length of 20 nucleic acids.
  • the antisense oligonucleotide is selected from the group consisting of but not limited to: SEQ ID NO: 1 to SEQ ID NO:26 (see Table 1).
  • the antisense oligonucleotide is selected from the group consisting of but not limited to: SEQ ID NO: 1 to SEQ ID NO:26 (see Table 1). In a particular embodiment, the antisense oligonucleotide is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 25 and SEQ ID NO:26.
  • the antisense oligonucleotide is set forth as SEQ ID NO:22.
  • the term “acid nucleic” or “nucleotide” is defined as a modified or naturally occurring deoxyribonucleotide or ribonucleotide. Nucleotides typically include purines and pyrimidines, which include thymidine (T), cytidine (C), guanosine (G), adenosine (A) and uridine (U).
  • oligonucleotide refers to an oligomer of the nucleotides defined above.
  • oligonucleotide refers to a nucleic acid sequence, 3 '-5' or 5'-3' oriented, which may be single- or double-stranded.
  • the oligonucleotide used in the context of the invention may in particular be DNA or RNA.
  • oligonucleotide analog refers to an oligonucleotide having (i) a modified backbone structure, e.g.
  • Oligonucleotide analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequencespecific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide ⁇ e.g., single-stranded RNA or single-stranded DNA).
  • analogs are those having a substantially uncharged, phosphorus containing backbone.
  • a substantially uncharged, phosphorus containing backbone in an oligonucleotide analog is one in which a majority of the subunit linkages, e.g., between 50-100%, typically at least 60% to 100% or 75% or 80% of its linkages, are uncharged, and contain a single phosphorous atom.
  • oligonucleotide also refers to an oligonucleotide sequence that is inverted relative to its normal orientation for transcription and so correspond to a RNA or DNA sequence that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule through Watson-Crick base pairing).
  • an antisense strand can be constructed in a number of different ways, provided that it is capable of interfering with the expression of a target gene.
  • the antisense strand can be constructed by reverse-complementing the coding region (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement, (e.g., RNAs encoded by the antisense and sense gene may be complementary).
  • the oligonucleotide need not have the same intron or exon pattern as the target gene, and noncoding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments such as antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the oligonucleotide has the same exon pattern as the target gene such as siRNA and antisense oligonucleotide (ASO).
  • target or “targeting” refers to an oligonucleotide able to specifically bind to a CRY AB gene or a HSPB5 mRNA (any variants of the mRNA) encoding a HSPB5 gene product.
  • it refers to an oligonucleotide able to inhibit said gene or said mRNA by the methods known to the skilled in the art (e.g. antisense, RNA interference).
  • the antisense oligonucleotide of the present invention targets an mRNA and/or DNA encoding HSPB5 gene product, and is capable of reducing the amount of HSPB5 expression and/or activity in cells.
  • the antisense oligonucleotide comprises a sequence that is at least partially complementary, particularly perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions.
  • a sequence that is “perfectly complementary to” a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule.
  • a sequence is “partially complementary to” a second sequence if there are one or more mismatches.
  • the antisense oligonucleotide of the present invention that targets a cDNA or mRNA encoding HSPB5 gene can be designed by using the sequence of said mRNA as a basis, e.g. using bioinformatic tools.
  • the antisense oligonucleotide according to the invention is capable of reducing the expression and/or activity of HSPB5 in lung cells.
  • Methods for determining whether an oligonucleotide is capable of reducing the expression and/or activity of HSPB5 in cells are known to those skilled in the art.
  • the oligonucleotide is targeted to a translation initiation site (AUG codon), sequences in the coding region e.g. one or more exons), 5 ’-untranslated region or 3 ’-untranslated region of an mRNA.
  • the aim is to interfere with functions of the messenger RNA include all vital functions including translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA.
  • the overall effect of such interference with the RNA function is to cause interference with protein expression.
  • the oligonucleotide of the present invention is further modified, particularly chemically modified, in order to increase the stability and/or therapeutic efficiency in vivo.
  • the one skilled in the art can easily provide some modifications that will improve the efficacy of the oligonucleotide such as stabilizing modifications (C. Frank Bennett and Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annu. Rev. Pharmacol. Toxicol. 2010.50:259-293; Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016 Aug 19;44(14):6518-48).
  • the oligonucleotide used in the context of the invention may comprise modified nucleotides.
  • Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the oligonucleotide.
  • chemical modifications include backbone modifications, heterocycle modifications, sugar modifications, and conjugation strategies.
  • the oligonucleotide is be selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, small regulatory RNAs (sRNAs), U7- orUl- mediated ASOs or conjugate products thereof such as peptide-conjugated or nanoparticle- complexed ASOs, chemically modified oligonucleotide by backbone modifications such as morpholinos, phosphorodi ami date morpholino oligomers (Phosphorodi ami date morpholinos, PMO), peptide nucleic acid (PNA), phosphorothioate (PS) oligonucleotides, stereochemically pure phosphorothioate (PS) oligonucleotides, phosphoramidates modified oligonucleotides, thiophosphoramidate-modified oligonucleotides, and methylphosphonate modified oligonucleotides; chemically modified
  • the oligonucleotide may be stabilized.
  • a “stabilized” oligonucleotide refers to an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure.
  • oligonucleotide stabilization can be accomplished via phosphate backbone modifications, phosphodiester modifications, phosphorothioate (PS) backbone modifications, combinations of phosphodiester and phosphorothioate modifications, thiophosphoramidate modifications, 2' modifications (2'- O-Me, 2'-O-(2-methoxyethyl) (MOE) modifications and 2'-fluoro modifications), methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.
  • PS phosphorothioate
  • MOE 2-methoxyethyl
  • the antisense oligonucleotide is lipid-conjugated, known as LASO.
  • LASO lipid-conjugated
  • the antisense oligonucleotide of the present invention is modified by substitution at the 3’ or the 5’ end by a moiety comprising at least three saturated or unsaturated, particularly saturated, linear or branched, particularly linear, hydrocarbon chains comprising from 2 to 30 carbon atoms, particularly from 5 to 20 carbon atoms, more particularly from 10 to 18 carbon atoms as described in WO2014/195432.
  • the antisense oligonucleotide of the present invention is modified by substitution at the 3 ’ or the 5 ’ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, particularly saturated, linear or branched, particularly linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, particularly from 6 to 20 carbon atoms, in particular 10 to 19 carbon atoms, and even more particularly from 12 to 18 carbon atoms as described in WO2014/195430.
  • the oligonucleotide may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom), which have increased resistance to nuclease digestion.
  • 2’ -methoxy ethyl (MOE) modification (such as the modified backbone commercialized by IONIS Pharmaceuticals) is also effective.
  • the oligonucleotide of the present invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-methyl group (2'-O-Me) substitution, 2-methoxyethyl group (2'-0-M0E) substitution, fluoro group (2'- fluoro) substitution, chloro group (2'-Cl) substitution, bromo group (2'-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2'-CF3) substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-O-alkyl) substitution, S-alkyl group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl group (2'-O- alkenyl) substitution, S-alkenyl group (2'-S-
  • the oligonucleotide of the present invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3'-endo configuration.
  • LNA locked nucleic acid
  • These molecules are extremely stable in biological medium, able to activate RNase H such as when LNA are located to extremities (Gapmer) and form tight hybrids with complementary RNA and DNA.
  • the oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2’-0Me analogs, 2'-O-Met, 2'-O-(2-methoxyethyl) (MOE) oligomers, 2’-phosphorothioate analogs, 2’-fluoro analogs, 2’-Cl analogs, 2’-Br analogs, 2’-CN analogs, 2’-CF3 analogs, 2’-OCF3 analogs, 2’- OCN analogs, 2’-O-alkyl analogs, 2’-S-alkyl analogs, 2’-N-alkyl analogs, 2’-O-alkenyl analogs, 2’-S-alkenyl analogs, 2’-N-alkenyl analogs, 2’-SOCH3 analogs, 2’-SO2CH3 analogs, 2’- ONO2 analogs, 2’-NO2 analogs, 2’-NO2 analogs
  • the oligonucleotide according to the invention is a LNA oligonucleotide.
  • LNA Locked Nucleic Acid
  • LNA oligonucleotide refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA analogue nucleotides.
  • LNA oligonucleotides, LNA nucleotides and LNA analogue nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos.
  • LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA.
  • oligonucleotide sequences coupled to small nuclear RNA molecules such as U1 or U7 in combination with a viral transfer method based on, but not limited to, lentivirus or adeno-associated virus (Denti, MA, et al, 2008; Goyenvalle, A, et al, 2004).
  • oligonucleotides of the present invention are peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • the deoxyribose backbone of oligonucleotides is replaced with a backbone more akin to a peptide than a sugar.
  • Each subunit, or monomer has a naturally occurring or non-naturally occurring base attached to this backbone.
  • One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. Because of the radical deviation from the deoxyribose backbone, these compounds were named peptide nucleic acids (PNAs) (Dueholm et al., New J. Chem., 1997, 21, 19-31).
  • PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes.
  • the resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA, DNA/RNA or RNA/RNA duplexes as determined by Tm's.
  • This high thermal stability might be attributed to the lack of charge repulsion due to the neutral backbone in PNA.
  • the neutral backbone of the PNA also results in the Tm's of PNAZDNA(RNA) duplex being practically independent of the salt concentration.
  • the PNA/DNA(RNA) duplex interaction offers a further advantage over DNA/DNA, DNA/RNA or RNA/RNA duplex interactions which are highly dependent on ionic strength.
  • Homopyrimidine PNAs have been shown to bind complementary DNA or RNA in an anti-parallel orientation forming (PNA)2/DNA(RNA) triplexes of high thermal stability (see, e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677). In addition to increased affinity, PNA has also been shown to bind to DNA or RNA with increased specificity.
  • the binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations.
  • the orientation is said to be anti-parallel when the DNA or RNA strand in a 5' to 3' orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5' end of the DNA or RNA and amino end of the PNA is directed towards the 3' end of the DNA or RNA.
  • PNA peptide nucleic acid
  • PNA have shown strong binding affinity and specificity to complementary DNA (Egholm, M., et al., Chem. Soc., Chem. Commun., 1993, 800; Egholm, M., et.al., Nature, 1993, 365, 566; and Nielsen, P., et.al. Nucl. Acids Res., 1993, 21, 197). Furthermore, PNA's show nuclease resistance and stability in cell-extracts (Demidov, V. V., et al., Biochem. Pharmacol., 1994, 48, 1309-1313). Modifications of PNA include extended backbones (Hyrup, B., et.al. Chem. Soc., Chem.
  • the oligonucleotides of the present invention are obtained by conventional methods well known to those skilled in the art.
  • the oligonucleotide of the invention can be synthesized de novo using any of a number of procedures well known in the art.
  • the b-cyanoethyl phosphorami dite method (Beaucage et al., 1981); nucleoside H-phosphonate method (Garegg et al., 1986; Froehler et al., 1986, Garegg et al., 1986, Gaffney et al., 1988).
  • These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market.
  • nucleic acids may be referred to as synthetic nucleic acids.
  • oligonucleotide can be produced on a large scale in plasmids (see Sambrook, et al., 1989). Oligonucleotide can be prepared from existing nucleic acid sequences using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Oligonucleotide prepared in this manner may be referred to as isolated nucleic acids.
  • oligonucleotides such as chemical modification of the oligonucleotides, lipid- and polymer-based nanoparticles or nanocarriers, ligand- oligonucleotide conjugates by linking oligonucleotides to targeting agents such as carbohydrates, peptides, antibodies, aptamers, lipids or small molecules and small molecules that improve oligonucleotide delivery such as described in Juliano RL.
  • targeting agents such as carbohydrates, peptides, antibodies, aptamers, lipids or small molecules and small molecules that improve oligonucleotide delivery such as described in Juliano RL.
  • the delivery of therapeutic oligonucleotides Nucleic Acids Res. 2016 Aug 19;44(14):6518-48.
  • Lipophilic conjugates and lipid conjugates include fatty acid-oligonucleotide conjugates; sterololigonucleotide conjugates and vitamin-oligonucleotide conjugates.
  • the oligonucleotide of the present invention is conjugated to a second molecule.
  • said second molecule is selected from the group consisting of aptamers, antibodies or polypeptides.
  • the oligonucleotide of the present invention may be conjugated to a cell penetrating peptide.
  • Cell penetrating peptides are well known in the art and include for example the TAT peptide (Bechara C, Sagan S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013 Jun 19;587(12): 1693-702).
  • the oligonucleotide of the present invention is associated with a carrier or vehicle, e g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art.
  • a carrier or vehicle e g., liposomes or micelles
  • Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the oligonucleotide, or improve the oligonucleotide's pharmacokinetic or therapeutic properties.
  • the oligonucleotide of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the oligonucleotide depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature.
  • the diameters of the liposomes generally range from about 15 nm to about 5 microns.
  • the use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane.
  • liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active.
  • a liposome delivery vehicle originally designed as a research tool, such as Lipofectin can deliver intact nucleic acid molecules to cells.
  • Specific advantages of using liposomes include the following: they are nontoxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues.
  • cost-effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product has demonstrated the viability of this technology as an acceptable drug delivery system.
  • the oligonucleotide of the present invention is complexed with a complexing agent to increase cellular uptake of oligonucleotides.
  • a complexing agent includes cationic lipids.
  • Cationic lipids can be used to deliver oligonucleotides to cells.
  • the term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells.
  • cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof.
  • Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Particularly, straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., C1-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • counterions anions
  • cationic lipids examples include: polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, Lipofectamine, DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif ).
  • Cationic liposomes may comprise the following: N-[l-(2,3- dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoloxy)- propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3p-[N-(N ' ,N ' dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-
  • DOTMA N-[l-(2,3- dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride
  • DOTAP N-[l-(2,3-dioleoloxy)- propyl]-N,N,N-trimethylammonium methylsulfate
  • DC-Chol 3p-[N-(N ' ,N ' di
  • DOSPA l,2-dimyristyloxypropyl-3-dimethy-l-hydroxyethyl ammonium bromide
  • DDAB dimethyldioctadecylammonium bromide
  • DOTMA cationic lipid N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • DOTMA cationic lipid N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • Oligonucleotides can also be complexed with, e.g., poly(L-lysine) or avidin and lipids may, or may not, be included in this mixture (e.g., steryl-poly(L-lysine).
  • Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci.
  • lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods.
  • other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.
  • the antisense oligonucleotide according to the invention comprises a sequence consisting of any sequences of SEQ ID NO:1 to SEQ ID NO:26.
  • the antisense oligonucleotide according to the invention consists of a sequence consisting of any sequences of SEQ ID NO: 1 to SEQ ID NO:26.
  • the antisense oligonucleotide is selected from the group consisting of SEQ ID NO:6, SEQ ID NO: 15, SEQ ID NO 22, SEQ ID NO 25 and SEQ ID NO:26.
  • the antisense oligonucleotide is set forth as SEQ ID NO:22.
  • the inhibitor and/or the antisense oligonucleotide according to the invention is capable of reducing the amount of HSPB5 in lung cells.
  • the invention relates to an ASO having at least 70% of identity with an ASO of SEQ ID NO: 1 to SEQ ID NO:26.
  • the percentage of identity can be 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; or 99%.
  • Nucleic acid sequence identity is particularly determined using a suitable sequence alignment algorithm and default parameters, such as BLAST N (Karlin and Altschul, Proc. Natl Acad. Sci. USA 87(6):2264-2268 (1990)).
  • the present invention relates to a vector for delivery of a heterologous nucleic acid, wherein the nucleic acid encodes an inhibitor according to the invention.
  • the invention relates to a vector for delivery of a heterologous nucleic acid, wherein the nucleic acid encodes for an inhibitor according to the invention that specifically binds to HSPB5 mRNA and inhibits expression of HSPB5 in a cell.
  • the vector according to invention, wherein the inhibitor is a siRNA or an antisense oligonucleotide as described above.
  • the acid nucleic acid (e.g. antisense nucleic acid) of the invention may be delivered in vivo alone (naked ASO/LASO) or in association with a vector.
  • a "vector” is any vehicle capable of facilitating the transfer of the oligonucleotide of the invention to the cells.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, naked plasmids, non-viral delivery systems (cationic transfection agents, liposomes, lipid nanoparticles, and the like), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the oligonucleotide sequences.
  • Viral vectors include, but are not limited to nucleic acid sequences from the following viruses: RNA viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus (AAV); SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus.
  • retrovirus as for example moloney murine leukemia virus and lentiviral derived vectors
  • harvey murine sarcoma virus murine mammary tumor virus
  • rous sarcoma virus adenovirus, adeno-associated virus (AAV)
  • AAV adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses
  • an object of the invention relates to a vector comprising an oligonucleotide sequence that encodes a portion or fragment of HSPB5, or variants thereof.
  • the vector of the invention comprises any variant of the oligonucleotide sequence that encodes a portion or fragment of HSPB5.
  • the vector of the invention comprises any variant of the oligonucleotide sequence that encodes any variant of HSPB5.
  • the invention relates to a vector comprising an antisense oligonucleotide sequence that encodes a portion or fragment of HSPB5, or variants thereof.
  • the invention relates to a vector comprising a shRNA sequence that encodes a portion or fragment of the HSPB5, or variants thereof.
  • the invention relates to a vector comprising a miRNA sequence that encodes a portion or fragment of HSPB5, or variants thereof.
  • the vector according to the invention comprises an antisense oligonucleotide which targets the region selected from the list consisting of the nucleic acids 356-375; 536-555, 676-695, 736-755 and 756-775 of SEQ ID NO: 27.
  • the invention relates to a vector comprising or consisting of any sequences from SEQ ID NO: 1 to SEQ ID NO:26. - l' l -
  • the invention relates to a vector comprising or consisting of any sequences selected from the group consisting of SEQ IDNO:6, SEQ IDNO: 15, SEQ IDNO:22, SEQ ID NO:25 and SEQ ID NO 26.
  • the invention in another embodiment, relates to a vector comprising an oligonucleotide sequence that encodes a portion or fragment of HSPB5, or variants thereof and a CAG promoter.
  • the invention in another embodiment, relates to a vector comprising a miRNA sequence that encodes a portion or fragment of HSPB5, or variants thereof and a CAG promoter or a PolII promoter.
  • the invention in another embodiment, relates to a vector comprising a shRNA sequence that encodes a portion or fragment of HSPB5, or variants thereof and a U6 promoter.
  • variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc.
  • the term variant also includes genes sequences of the invention from other sources or organisms. Variants are preferably substantially homologous to sequences according to the invention, i.e., exhibit a nucleotide sequence identity of typically at least about 75%, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95% with sequences of the invention. Variants of the genes of the invention also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions.
  • Typical stringent hybridisation conditions include temperatures above 30° C, preferably above 35°C, more preferably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM.
  • Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc.
  • the vector use according to the invention is a non-viral vector or a viral vector.
  • the non-viral vector is a plasmid comprising a nucleic acid sequence that encodes HSPB5.
  • the vector may a viral vector.
  • Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology.
  • viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
  • transgene refers to the antisense oligonucleotide of the invention.
  • gene transfer or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e. g. episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • transferred replicons e. g. episomes
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94/19478.
  • the viral vector may be an adenoviral, a retroviral, a lentiviral, a herpesvirus or an adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • adeno-associated viral (AAV) vectors are employed.
  • the invention in another embodiment, relates to an adeno-associated virus (AAV) vector comprising an oligonucleotide sequence that targets a portion or fragment HSPB5, or variants thereof.
  • AAV adeno-associated virus
  • the AAV vector is AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10 or any other serotypes of AAV that can infect human, rodents, monkeys or other species.
  • an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, etc.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g. the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
  • an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus.
  • ITRs do not need to be the wild-type polynucleotide sequences, and may be altered, e.g, by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the nucleic acid sequences of the invention) and a transcriptional termination region.
  • the viral vectors utilized in the compositions and methods of the invention are recombinant adeno-associated virus (rAAV).
  • the rAAV may be of any serotype, modification, or derivative, known in the art, or any combination thereof (e.g., a population of rAAV that comprises two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9) known in the art.
  • the rAAV are rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVIO, rAAV-11, rAAV-12, rAAV-13, rAAV-14, rAAV-15, rAAV-16, rAAV.rh8, rAAV.rhlO, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-l, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, rAAV.HSCl, rAAV.HSC2, rAAV
  • the rAAV used in the compositions and methods of the invention comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-
  • the rAAV comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., vpl, vp2 and/or vp3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-
  • the AAV that is used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015: 1056-1068, which is incorporated by reference in its entirety.
  • the AAV that is used in the methods described herein comprises one of the following amino acid insertions: LGETTRP (SEQ ID NO: 14) or LALGETTRP (SEQ ID NO: 15), as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is any AAV disclosed in United States Patent No. 9,585,971, such as AAV-PHP.B.
  • the AAV that is used in the methods described herein is any AAV disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is any AAV disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • the AAV that is used in the methods described herein is any AAV disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • the AAV that is used in the methods described herein is any AAV disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
  • the AAV that is used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos.
  • the rAAV have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), W0 2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S.
  • Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; International Patent Application Nos.
  • the rAAV comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes.
  • the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV 1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HS
  • a single-stranded AAV can be used.
  • a self-complementary vector e.g., scAAV
  • scAAV single-stranded AAV
  • the recombinant AAV vector used for delivering the transgene have a tropism for cells in the DRG.
  • Such vectors can include non-replicating “rAAV”, particularly those bearing an AAV8 or AAVrhlO capsid are preferred.
  • the viral vectors provided herein are AAV9 or AAVrhlO based viral vectors.
  • the AAV8 or AAVrhlO based viral vectors provided herein retain tropism for DRG.
  • AAV variant capsids can be used, including but not limited to those described by Wilson in US Patent No.
  • the present invention relates to a recombinant adeno-associated virus (rAAV) comprising (i) an expression cassette containing a transgene under the control of regulatory elements and flanked by ITRs, and (ii) an AAV capsid, wherein the transgene encodes an inhibitory RNA that specifically binds HSPB5 mRNA and inhibits expression of HSPB5 in a cell.
  • rAAV recombinant adeno-associated virus
  • AAV vectors comprising an artificial genome comprising (i) an expression cassette containing the transgene under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has the amino acid sequence of the AAV capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV capsid protein while retaining the biological function of the AAV capsid.
  • AAVrhlO vectors comprising an artificial genome comprising (i) an expression cassette containing the transgene under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has the amino acid sequence of the AAVrhlO capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAVrhlO capsid protein while retaining the biological function of the AAVrhlOcapsid.
  • the encoded AAVrhlO capsid has the sequence of SEQ ID NO: 81 set forth in U.S. Patent No.
  • 9,790,427 which is incorporated by reference herein in its entirety, with 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 or 30 amino acid substitutions and retaining the biological function of the AAVrhlO capsid.
  • the control elements are selected to be functional in a mammalian cell.
  • the resulting construct which contains the operatively linked components is flanked by (5’ and 3’) functional AAV ITR sequences.
  • AAV ITRs adeno-associated virus inverted terminal repeats
  • AAV ITRs the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus.
  • AAV ITRs, together with the AAV rep coding region provide for the efficient excision and rescue from, and integration of a polynucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
  • the polynucleotide sequences of AAV ITR regions are known.
  • an "AAV ITR” does not necessarily comprise the wild-type polynucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, etc. Furthermore, 5' and 3' ITRs which flank a selected polynucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.
  • AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV 5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, etc.
  • 5' and 3' ITRs which flank a selected polynucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
  • Particular embodiments are vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian DRG.
  • a review and comparison of transduction efficiencies of different serotypes is provided in this patent application.
  • AAV2, AAV5, AAV8, AAV9 and rh.10 based vectors direct long-term expression of transgenes in DRG.
  • the selected polynucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
  • control elements can comprise control sequences normally associated with the selected gene.
  • the vector of the present invention comprises an expression cassette.
  • expression cassette refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the present invention.
  • nucleic acid molecule encodes a heterologous gene and may also include suitable regulatory elements.
  • the heterologous gene refers to a transgene that encodes an RNA of interest.
  • Each expression cassette may comprise at least a promoter sequence operably linked to a sequence encoding the RNA of interest.
  • Each expression cassette may consist of additional regulatory elements, spacers, introns, UTRs, polyadenylation site, and the like.
  • the expression cassette is polycistronic with respect to the transgenes encoding e.g. two or more miRNAs.
  • the expression cassette comprises a promoter, a nucleic acid encoding one or more RNA molecules of interest, and a polyA.
  • the expression cassette comprises 5’ - promoter sequence, a sequence encoding a first RNA of interest, a sequence encoding a second RNA of interest, and a polyadenylation sequence- 3’.
  • an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck posttranscriptional response element (WPRE), and/or other elements known to affect expression levels of the encoding sequence.
  • an expression cassette comprises the nucleic acid molecule of the present invention operatively linked to a promoter sequence.
  • operatively linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter).
  • Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation.
  • promoter sequence refers to a polynucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3’- direction) coding sequence.
  • Transcription promoters can include “inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • the promoter is a heterologous promoter.
  • heterologous promoter refers to a promoter that is not found to be operatively linked to a given encoding sequence in nature.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phophoglycerate kinase (PKG) promoter, CAG (composite of the (CMV) cytomegalovirus enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron), U6 promoter, neuronal promoters (Human synapsin 1 (hSyn) promoter, NeuN promoters, CamKII promoter, promoter of Dopamine- 1 receptor and Dopamine-2 receptor), the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a CMV promoter such as the CMV immediate early promoter region (CMV-IE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • heterologous promoters and other control elements such as DRG-specific and inducible promoters, enhancers and the like, will be of particular use.
  • An “enhancer” is a polynucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • the promoter is derived in its entirety from a native gene.
  • the promoter is composed of different elements derived from different naturally occurring promoters.
  • the promoter comprises a synthetic polynucleotide sequence.
  • promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor.
  • Ubiquitous, cell-type- specific, tissue-specific, developmental stage-specific, and conditional promoters for example, drug-responsive promoters (e.g. tetracycline-responsive promoters) are well known to those of skill in the art.
  • Pol I promoters control transcription of large ribosomal RNAs
  • Pol II promoters control the transcription of mRNAs (that are translated into protein) and small nuclear RNAs (snRNAs); and Pol III promoters uniquely transcribe small non-coding RNAs.
  • Pol III promoters are useful for synthesizing small interfering RNAs (shRNAs) from DNA templates in vivo.
  • Pol II promoters are preferred but can only be used for transcription of miRNAs. When a Pol II promoter is used, however, it may be preferred to omit translation initiation signals so that the RNAs function as antisense, siRNA, shRNA or miRNAs and are not translated into peptides in vivo.
  • the AAV expression vector which harbors the DNA molecule of interest flanked by AAV ITRs can be constructed by directly inserting the selected sequence (s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • ORFs major AAV open reading frames
  • Such constructs can be designed using techniques well known in the art. See, e.g., U. S. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993).
  • AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3'of a selected nucleic acid construct that is present in another vector using standard ligation techniques.
  • AAV vectors which contain ITRs have been described in, e.g., U. S. Patent No. 5,139,941.
  • AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.
  • chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5'and 3'of one or more selected nucleic acid sequences.
  • Preferred codons for expression of the chimeric gene sequence in mammalian DRG cells can be used, and in certain embodiments codon optimization of the transgene is performed by well-known methods.
  • the complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods.
  • an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
  • transfection techniques include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.
  • a particular viral vector comprises, in addition to a nucleic acid sequence of the invention, the backbone of AAV vector plasmid with ITR derived from AAV-2, the promoter, such as the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus/p- actin hybrid promoter (CAG) consisting of the enhancer from the CMV immediate gene, the promoter, splice donor and intron from the chicken P-actin gene, the splice acceptor from rabbit P-globin, or any neuronal promoter such as the promoter of Dopamine- 1 receptor or Dopamine- 2 receptor, or the synapsin promoter, with or without the wild-type or mutant form of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a rabbit betaglobin polyA sequence.
  • the viral vector may comprise in addition, a nucleic acid sequence encoding an antibiotic resistance gene such as the genes of resistance ampicillin (AmpR), kanamycin
  • retroviral vectors are employed.
  • Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell-lines.
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replicationdefective.
  • a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components.
  • Retroviral vectors are able to infect a broad variety of cell types.
  • lentiviral vectors are employed.
  • the invention relates to a lentivirus vector comprising an oligonucleotide sequence that targets a portion or fragment of HSPB5, or variants thereof.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
  • Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S.
  • the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell.
  • the gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
  • This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell.
  • Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.
  • the env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
  • the nucleic acid molecule or the vector of the present invention include “control sequences”, which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • the invention relates to the inhibitor and/or the antisense oligonucleotide as described above for use in the treatment of pulmonary fibrosis in a subject in need thereof.
  • the invention relates to a method for treating pulmonary fibrosis in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor and/or the antisense oligonucleotide as described above.
  • Fibrotic interstitial lung diseases or “Fibrotic ILD” or “Pulmonary Fibrosis” refers to a group of diseases affecting the pulmonary interstitium (the tissue and space between the air sacs of the lungs and lung capillaries). Fibrotic ILD refers to more than 200 chronic lung disorders. With fibrotic ILD, the tissue between the air sacs of the lungs (the interstitium) and the lung capillaries is affected by the accumulation of extracellular matrix rich in fibrillary collagens (fibrosis). It concerns alveolar epithelium, pulmonary capillary endothelium, basement membrane, perivascular and perilymphatic tissues.
  • Fibrotic interstitial lung diseases Fibrotic ILDs
  • progressive pulmonary fibrosis PPF
  • the term " progressive pulmonary fibrosis” among Fibrotic ILDs refers to a disease associated with worsening respiratory symptoms, a decline in lung function, a decreased quality of life, and a risk of early death, independent of the classification of the ILD.
  • progressive pulmonary fibrosis is selected from the list consisting of Idiopathic pulmonary fibrosis (IPF), idiopathic non-specific interstitial pneumonia (NSIP), fibrotic ILD associated with inflammatory rheumatic disease such as rheumatoid arthritis, fibrotic ILD associated with autoimmune connective tissue disease such as systemic sclerosis, Sjogren’s syndrome, undifferentiated connective tissue disease, chronic hypersensitivity pneumonitis, drug induced pulmonary fibrosis, and lung fibrosis associated with unresolving acute respiratory distress syndrome (ARDS) including SARS-COV2 induced ARDS, sarcoidosis or others progressive pulmonary fibrosis.
  • Fibrotic ILD is progressive pulmonary fibrosis (PPF) such as Idiopathic pulmonary fibrosis (IPF).
  • Idiopathic pulmonary fibrosis or “IPF” means an interstitial lung disease for which no obvious cause can be identified (idiopathic), and which is associated with typical findings both radiographic (basal and pleural based fibrosis with honeycombing) and pathologic (usual interstitial pneumonia pattern comprising temporally and spatially heterogeneous fibrosis, histopathologic honeycombing and fibroblastic foci).
  • radiographic basic and pleural based fibrosis with honeycombing
  • pathologic usual interstitial pneumonia pattern comprising temporally and spatially heterogeneous fibrosis, histopathologic honeycombing and fibroblastic foci.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment 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 patient 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 patient during treatment of an illness, e.g., to keep the patient 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 a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human, a mouse or a rat. As used herein, the term “subject” encompasses “patient”.
  • the subject suffers or is susceptible to suffer from Fibrotic interstitial lung diseases (Fibrotic ILDs) and particularly progressive pulmonary fibrosis (PPF) diseases such as Idiopathic pulmonary fibrosis (IPF).
  • Fibrotic ILDs Fibrotic interstitial lung diseases
  • PPF progressive pulmonary fibrosis
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of HSPB5 such as an ASO of the invention) into the subject, such as by, intravenous, intramuscular, enteral, subcutaneous, parenteral, systemic, local, spinal, nasal, topical or epidermal administration (e.g., by injection or infusion).
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • the administration is performed by a patch, a paste, an ointment, a suspension, a solution or a cream, a gel or a spray.
  • the administration of the inhibitor and/or antisense oligonucleotide is performed by an intrathecal, subcutaneous, topical or intravenous administration.
  • an antisense oligonucleotide according to the invention and a ii) classical treatment for simultaneous, separate or sequential use in the treatment of n as a combined preparation.
  • the term “classical treatment” refers to any compound, natural or synthetic.
  • the classical treatment is selected from the group consisting of but not limited to: aspirin, paracetamol, Nonsteroidal anti-inflammatory drugs (NSAIDs); codeine, cryotherapy, virtual therapy, cannabis, morphine and its derivatives, opium and its derivatives.
  • NSAIDs Nonsteroidal anti-inflammatory drugs
  • a “therapeutically effective amount” is intended for a minimal amount of active agent (e.g. ASO according to the invention) which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount” to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the invention relates to a pharmaceutical composition which comprises the inhibitor and/or the antisense oligonucleotide according to the invention.
  • the invention relates to the pharmaceutical composition according to the invention for use in the treatment of pulmonary fibrosis.
  • the inhibitor and/or the antisense oligonucleotide as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, glycerol, glycerol, glycerol, adiluent, encapsulating material or formulation auxiliary of any type.
  • the pharmaceutical composition according to the invention is administered by an intratracheal, subcutaneous, aerosol, nasal, topical or intravenous or oral (per os, enteral) administration.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatine.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation may be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the present invention provides a topical formulation comprising antisense oligonucleotides.
  • the present invention provides a topical formulation comprising antisense oligonucleotides.
  • Dosage forms for the topical or transdermal administration of the inhibitors of the present invention include, but are not limited to, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • a topical formulation comprises antisense oligonucleotides comprised in micelles, liposomes, or non-lipid based microspheres.
  • such a topical formulation may comprise a permeability enhancing agent such as but not limited to dimethyl sulfoxide, hydrocarbons (for example, alkanes and alkenes), alcohols (for example, glycols and glycerols), acids (for example, fatty acids), amines, amides, esters (for example, isopropyl myristate), surfactants (for example, anionic, cationic, or non- ionic surfactants), terpenes, and lipids (for example, phospholipids).
  • a permeability enhancing agent such as but not limited to dimethyl sulfoxide, hydrocarbons (for example, alkanes and alkenes), alcohols (for example, glycols and glycerols), acids (for example, fatty acids), amines, amides, esters (for example, isopropyl myristate), surfactants (for example, anionic, cationic, or non- ionic surfactants
  • the formulation is a patch, a paste, an ointment, a suspension, a solution or a cream, a gel or a spray. In a particular embodiment, the formulation is a cream.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.
  • anti- pulmonary fibrosis agents may be added to the pharmaceutical composition as described below.
  • Anti-pulmonary fibrosis agents may be pirfenidone and nintedanib (4).
  • Additional anti-pulmonary fibrosis agents may be selected from, but are not limited to, modulators targeting cytokines, chemokines, growth factors, tyrosine kinases, hormones, soluble receptors, decoy receptors, corticosteroids, monoclonal or polyclonal antibodies, mono- specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • the further therapeutic active agent can be an antifibrosis agent.
  • Suitable antifibrosis agents include, but are not limited to treprostinil, an inhaled prostaglandin mimetic, ii) phosphodiesterase 4B (PDE4B) oral inhibitor BI- 1015550, iii) a LPA1 antagonist, BMS-986278. (These drugs are evaluated in phase 3 studies) iv) ., anti-CTGF antibodies.
  • the other therapeutic active agent can be an opioid or nonopioid analgesic agent (to treat the pulmonary fibrosis symptoms).
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 HSPB5 expression is associated with fibrosis development in pulmonary fibrosis patients.
  • Figure 3 Effects of ASO22 on HSPB5 and mesenchymal markers expression of pulmonary cells.
  • A) Protein expression tested by western blot in CCD-19Lu (data representative of n 6 western blots) and B) the associated densitometric analysis of collagen expression (relative to TGF-pi condition).
  • D) Protein expression tested by western blot in A549 (data representative of n 5 western blots).
  • mRNA level tested by RT-qPCR (n 8-9) of HSPB5, ACTA2 and SERPINEJ in E) CCD-19Lu and F) A549.
  • FIG. 5 Intravenous ASO22 hampers BLM-induced fibrosis development.
  • A Representative images of lung sections of mice, 21 days after i.t. instillation of bleomycin or NaCl, with or without ASO22 or control scramble. (Hematoxylin-Eosin staining). The mice were treated every other day (D) from D9 to D21 by intravenous injections of either control scramble (Scr 6, 12, 18 mg/kg) or ASO22 (6, 12, 18 mg/kg).
  • B Collagen quantification using Sircol Assay on the left lung.
  • FIG. 6 Intra-tracheal injection of ASO22 hampers BLM- induced fibrosis development.
  • A Representative images of lung sections of mice, 21 days after i t. instillation of bleomycin, and ASO22 or control scramble treatment (Hematoxylin-Eosin Staining). The mice were treated at day (D)3, D6, D15 and D18 by intra-tracheal injection of either NaCl, control scramble (Scr) or ASO22 at 12 mg/kg.
  • Collagen quantification using B) Sircol Assay on the left-lung and (C) Picosirius red staining of lung sections of mice.
  • FIG. 7 Cy5-ASO22 accumulates in the lung after intravenous ou intra-tracheal injection.
  • ASOs antisense oligonucleotides targeting the entire HSPB5 mRNA
  • a specific R software package was developed by Pascal Finetti and Palma Rocchi (PDA16130, 2017).
  • This program uses the sequence of the transcripts of the gene of interest in FASTA format. Firstly, the gene coding part of interest is selected and segmented into consecutive sequences of 20 bases. Subsequently, to define potential ASOs sequences, this software identifies the complementary sequences of the resulting sequences and reverses them to 5'-3' direction. In a second step, the ASOs are selected according to their percentage of GC (40-60%) and their specificity to the gene of interest sequence transcripts.
  • Oligonucleotide molecules were synthesized on an automated AKTA OP10 (GE Healthcare) at 40 pmol scale on primer support (loading 350 pmol/g, GE Healthcare, crosslinked polystyrene). Conventional -cyanoethyl phosphorami di te chemistry was used. Phosphorothioate linkage was introduced during the synthesis cycle with Sulfurizing Reagent II (3-((N,N-dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-5-thione from Glen Research). The deprotection was realized in an ammoniac solution during 4 hours at 55 °C.
  • the quantification was obtained using a micro-volume spectrophotometer (mySPEC, VWR, USA) at 260 nm. (data not shown)
  • human lung epithelial A549 cells ATCC, CCL-185
  • human pulmonary fibroblastic CCD-19Lu cells ATCC, CCL210
  • ASOs at 100 nM were pre-incubated for 25 min with 3,75 pL TransIT-X2® Dynamic Delivery System (Mirus Bio LLC, USA) in 250 JJ.1 of Gibco Opti-MEM, a serum-reduced medium (Life Technologies SAS, Courtaboeuf, France) before being added drop by drop to the cells in their complete growth medium containing 10% foetal bovine serum (FBS).
  • FBS foetal bovine serum
  • the medium containing ASOs and Transit- X2 was replaced with the 0% FBS DMEM (A549) or MEM (CCD-19Lu) medium overnight before being stimulated 48h with recombinant human TGF-pi (rTGF- 1 R&D systems, Minneapolis, MN) in complete medium at 10 ng/mL.
  • mice When indicated animals were treated with ASO22 at 6, 12 or 18 mg/kg. At day 21, mice were euthanized by abdominal aortic bleeding during deep anesthesia. Lungs were collected for histological, transcriptomics and collagen quantification analyses. Blood was collected and plasma separated by centrifugation for quantification of liver and renal markers.
  • Histomorphometric assay The amount of collagen in paraffin-embedded tissue sections was quantified by Picrosirius Red staining as previously described (34).
  • RNA from lung tissue was extracted with Trizol (Invitrogen). Shortly, 300 ng of total RNA were transcribed into cDNA by M-MLV reverse transcriptase with random primers in the presence of RNaseOUT RNAse inhibitor (Invitrogen). cDNAs were quantified by realtime PCR with a SYBR Green Real-time PCR kit (Applied Biosystems) on a Viaa7 detection system (Applied Biosystems, France). Relative mRNA levels were determined with the AACt method. Oligonucleotides used for RT-qPCR are described in supplemental methods.
  • ALT Alanine Aminotransferase
  • AST Aspartate Aminotransferase
  • Urea Urea
  • HSPB5 is associated with fibrosis development in patients with idiopathic pulmonary fibrosis
  • a total of 26 ASOs targeting HSPB5 were designed as described in material and methods. Their efficiency to inhibit HSPB5 protein and mRNA expression induced by TGF-pi was screened in CCD-19Lu cells. At the mRNA level, three ASO (ASO5, 22 and 25) showed a significant inhibitory effect on HSPB5 expression ( Figure 2). At the protein level, four ASOs reproducibly inhibited HSPB5 expression (data not shown) among which one (ASO6) was specific to human HSPB5 while three (ASO15, 22 and 26) were characterized by their crossreactivity with both human and murine HSPB5. Since among them ASO22 was the most efficient to inhibit human and mouse HSPB5 both at the mRNA and protein levels, this ASO was used in the following experiments in vitro and in vivo.
  • ASO22 inhibits TGF-PI downstream signaling in pulmonary human cells.
  • ASO22 In lung fibroblast CCD-19Lu cells, ASO22 (100 ng/mL) inhibited the TGF-pi -induced expression of HSPB5, as well as PAI-1, collagen, fibronectin and a-SMA ( Figure 3A, B). The effects of ASO22 on a-SMA expression were confirmed by immunofluorescence ( Figure 3C). Similarly, ASO22 decreased HSPB5, ACTA2 and SERPINE1 gene expression upon TGF-pi stimulation ( Figure 3D).
  • ASO22 attenuates TGF-PI -induced cells motility
  • ASQ22 limits bleomycin-induced fibrosis.
  • ASO22 or a control scramble ASO were administered intravenously (i.v.) in a dosedependent manner (6-12-18 mg/kg) in our model of bleomycin-induced lung fibrosis every other day from D9 to D21 after BLM or NaCl intra-tracheal (i.t.) administration (Figure 5).
  • ASO22 does not induce liver or kidney toxicity in mice
  • ASO22 or control scramble ASO (6, 12 or 18 mg/kg) in healthy mice by following the weight change, plasmatic transaminases as hepatic function markers and plasmatic urea as a renal marker.
  • ASO22 or control scramble ASO administration did not induce any significant modification of the weight gain profile compared to the control animals treated with NaCl (data not shown).
  • control scramble ASO and ASO22 treatment did not induce either significant changes in hepatic alanine aminotransferase (ALT) aspartate aminotransferase (AST) or urea levels (data not shown).
  • Intra-tracheal ASO22 hampers the development of BLM-induced fibrosis.
  • TGF-P 1 signaling is a seminal pathway involved in a large panel of pathologies from fibrotic disorders to cancer (15, 16). Consequently, the inhibition of TGF-pi signaling has been extensively investigated leading to the development of many inhibitors targeting the cytokine itself or its receptors. However, clinical studies in cancer have demonstrated that these molecules had mitigated efficacy and sometimes an unacceptable toxicity (17), due to the pleiotropic properties of TGF-pi. Therefore, research is now focused on upstream or downstream events and regulatory mechanisms of TGF-pi signaling (17).
  • HSPB5 a small HSP that regulates TGF-pi signaling through the promotion of SMAD4 nuclear location, was overexpressed in myofibroblasts and hyperplastic alveolar epithelial cells in IPF patients (11). Furthermore, the lack of HSPB5 in mice significantly reduced pulmonary fibrosis development in different animal models (11). In the present study, we observed using the LTRC cohort including 255 IPF patients that HSPB5 expression was associated with fibrosis development, further substantiating the clinical relevance of targeting HSPB5.
  • HSPB5 can be targeted both in vitro and in vivo by silencing oligonucleotides.
  • Our screening based on the inhibition of HSPB5 expression at both RNA and protein levels allowed us to identify ASO22. It reproductively inhibited HSPB5 expression in human cell lines of alveolar epithelial cells and human pulmonary fibroblasts, as well as their differentiation into myofibroblasts induced by TGF- 1.
  • ASO22 was also efficient to limit pulmonary fibrosis progression in the murine bleomycin model, when applied both systemically (intravenous) or locally (intra-tracheal administration). ASO22 significantly reduced collagen accumulation and lung architecture disorganization, and this was associated with a reduction of HSPB5 expression in lung tissues. These results are consistent with those of Tanguy et al. (23) that demonstrated that the chemical molecule NCI-41356, reported among other things to inhibit HSPB5, was able to limit TGF- pi signaling and the lung fibrosis in a BLM mice model, thus supporting the concept of HSPB5 as an antifibrotic therapeutic target. In what concerns the use of a RNA silencing strategy for a more specific targeting, Wettstein et al.
  • ASOs are usually described as having limited biodistribution following systemic administration (25) and one of the greatest challenges in ASO therapies is the delivery of the drug to the site of action.
  • One possible explanation of the efficacy of ASO22 observed after its systemic administration may be due because we added a phosphorothioate backbone, which gives nuclease resistance and favors binding to proteins in the plasma compared to classic ASO (26). Such interactions, for example with the albumin, can decrease its clearance and thus promote ASO half-life in the circulation.
  • TGF-beta orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1. Nature. 2020;577(7791):566-71.
  • Spigel DR et al. A Randomized, Double-Blinded, Phase II Trial of Carboplatin and Pemetrexed with or without Apatorsen (OGX-427) in Patients with Previously Untreated Stage IV Non-Squamous-Non-Small-Cell Lung Cancer: The SPRUCE Trial. Oncologist. 2019;24(12):el409-el6. 14. Ko AH, et al. A Randomized, Double - Blinded, Phase II Trial of Gemcitabine and Nab - Paclitaxel Plus Apatorsen or Placebo in Patients with Metastatic Pancreatic Cancer: The RAINIER Trial. The Oncologist. 2017;22(12): 1427-el29.
  • Huynh LK, et al. A Perspective on the Development of TGF-beta Inhibitors for Cancer Treatment. Biomolecules. 2019;9(l 1).
  • Baylot V, et al. OGX-427 inhibits tumor progression and enhances gemcitabine chemotherapy in pancreatic cancer. Cell Death Dis. 2011;2(10):e221.
  • Emblom-Callahan MC et al. Genomic phenotype of non-cultured pulmonary fibroblasts in idiopathic pulmonary fibrosis. Genomics. 2010;96(3): 134-45.

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Abstract

La présente invention concerne le traitement de maladies pulmonaires interstitielles avec fibrose (MPI avec fibrose) et, en particulier, la fibrose pulmonaire progressive (FPP) telle que la fibrose pulmonaire idiopathique (FPI). En particulier, les inventeurs développent des oligonucléotides antisens ciblant HSPB5 et étudient leurs effets sur le développement de la fibrose pulmonaire expérimentale. Des oligonucléotides antisens spécifiques (ASO) ont été conçus et criblés in vitro, sur la base de leur capacité à inhiber à la fois l'expression de HSPB5 humaine et murine. L'un d'entre eux, ASO22, a été sélectionné par sa capacité à inhiber l'expression induite par TGF-β1 de HSPB5 et des marqueurs clés supplémentaires de fibrose tels que PAI-1, le collagène, la fibronectine et α-SMA dans des cellules CCD-19Lu humaines fibroblastiques ainsi que PAI-1 et α-SMA dans des cellules A549 épithéliales pulmonaires. L'administration intra-trachéale ou intraveineuse d'ASO22 dans des souris à fibrose pulmonaire induite par la bléomycine a diminué l'expression de HSPB5 et une fibrose réduite telle que mesurée par la diminution de remodelage pulmonaire, d'accumulation de collagène et d'expression d'Acta2 et de Col1a1. Ensemble, ces résultats suggèrent un réel intérêt d'utiliser une stratégie oligonucléotidique antisens ciblant HSPB5 pour le traitement de la fibrose pulmonaire.
PCT/EP2025/063054 2024-05-14 2025-05-13 Oligonucléotides antisens et leur utilisation pour le traitement de la fibrose pulmonaire Pending WO2025237990A1 (fr)

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