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WO2025003222A1 - Oligonucléotides pour le traitement de maladies neuromusculaires - Google Patents

Oligonucléotides pour le traitement de maladies neuromusculaires Download PDF

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Publication number
WO2025003222A1
WO2025003222A1 PCT/EP2024/067949 EP2024067949W WO2025003222A1 WO 2025003222 A1 WO2025003222 A1 WO 2025003222A1 EP 2024067949 W EP2024067949 W EP 2024067949W WO 2025003222 A1 WO2025003222 A1 WO 2025003222A1
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oligonucleotide
nucleotides
ribonucleotides
deoxyribonucleotides
sequence
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Inventor
Cecilia JIMENEZ MALLEBRERA
Arístides LÓPEZ MÁRQUEZ
Haiyan Zhou
Francesco Muntoni
Sara AGUTI
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Hospital Sant Joan de Deu
UCL Business Ltd
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Hospital Sant Joan de Deu
UCL Business Ltd
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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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present disclosure pertains to the field of medical treatment, in particular it refers to the treatment of neuromuscular diseases by gene therapy.
  • Neuromuscular diseases are a heterogeneous group of rare disorders with prevalence between 1 and 10 per 100.000 in the population. There are several genes involved in these diseases, a fact that complicates the diagnosis. In addition, as there is a high variability in the phenotypes, the research and healthcare of the patients are complex.
  • Congenital muscular dystrophies are a group of neuromuscular diseases caused by genetic alterations that occur at birth or in infancy. Among the different types of congenital muscular dystrophies, the collagen Vl-rel ated dystrophies (COL6-RDs) are one of the most common disorders, with prevalence below 1 per 100.000.
  • Collagen type VI is a microfibrillar component of the extracellular matrix, synthesized in the interstitial fibroblasts (Zou, Zhang, Sabatelli, Chu, & Bbnnemann, 2008) and found in different tissues such as muscle, skin, tendon, cartilage, internal organs, and blood vessels. Collagen VI is involved in the structural and mechanical stabilization of the tissues, as well as in the interactions between the cells and the extracellular matrix; it links different components of the basement membranes and participates in cell adhesion and proliferation; and it stimulates the DNA synthesis of mesenchymal cells and the migration of the neural crest cells.
  • Collagen Vl-related dystrophies are congenital muscular dystrophies caused by mutations in the COL6A1, COL6A2 and COL6A3 genes. These patients present very variable phenotypes. The most severe is the Ullrich congenital muscular dystrophy and the milder is Bethlem myopathy.
  • intermediate clinical phenotypes In between, there are intermediate clinical phenotypes.
  • the clinical presentations of patients with intermediate clinical phenotypes are a mix between features of Ullrich Congenital Muscular Dystrophy and Bethlem myopathy, such as significant weakness in early childhood, distal laxity of the most distal interphalangeal joins, or contractures of the long finger flexors. Patients achieve ambulation, but they may lose it in the late teenage or early adult years. A typical feature of these patients is also progressive respiratory impairment.
  • Curative treatments for the COL6-RDs are still in development and for the moment most of the patients only receive treatments to support the orthopedic and respiratory symptoms.
  • Pharmacological treatments were first investigated, based on the cascade of events known to degenerate the myofibers in the collagen VI deficient skeletal muscle.
  • treatments based on genetic therapeutic approaches such as therapies focused on silencing gene expression are being investigated.
  • the mutation (CO/_6A/)het.c.877G ⁇ A, p.Gly293Arg causes a COL6-RD with an intermediate phenotype. It is found in the position 19 of the exon 10 and it has a dominant negative effect, because it is a missense mutation that causes the substitution of a glycine from the triple helix domain by an arginine. Presently there is no treatment that can modify the course of the COL6-RD caused by this mutation, therefore there is a need in the art to provide therapeutical approaches for this disease.
  • a first aspect of the invention is an oligonucleotide that downregulates the expression of an allele carrying a mutation at position 877 in the COL6A1 coding sequence (CDS), wherein G is mutated to A compared to wild-type COL6A1..
  • the invention also provides, as a second aspect, a composition comprising a delivery agent and an oligonucleotide as defined in the first aspect, wherein the oligonucleotide is associated to the delivery agent.
  • the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotides as defined in the first aspect, and/or the composition as defined in the second aspect, and a pharmaceutically acceptable excipient or carrier.
  • a fourth aspect of the invention is an oligonucleotide as defined in the first aspect, and/or the composition as defined in the second aspect, and/or the pharmaceutical composition as defined in the third aspect, for use as a medicament. In other words, they are for use in therapy.
  • the fourth aspect can also be formulated as the use of the oligonucleotide as defined in the first aspect, and/or the composition as defined in the second aspect, an/or the pharmaceutical composition as defined in the third aspect, for the manufacture of a medicament.
  • This aspect can also be formulated as a method for the prevention and/or treatment of a disease, the method comprising administering a therapeutically effective amount of the oligonucleotide as defined in the first aspect, and/or the composition as defined in the second aspect, and/ or the pharmaceutical composition as defined in the third aspect, in a subject in need thereof.
  • a fifth aspect provided by the invention is an oligonucleotide as defined in the first aspect, and/or the composition as defined in the second aspect, and/or the composition as defined in the third aspect, for use in the treatment of a condition associated to the mutation (CO/_6A/)c.877G>A.
  • the condition is a neuromuscular disease, more in particular it is a muscular dystrophy.
  • the fifth aspect can also be formulated as the use of the oligonucleotide as defined in the first aspect, and/or the composition as defined in the second aspect, an/or the pharmaceutical composition as defined in the third aspect, for the treatment of a condition associated to the mutation (CO/_6A/)c.877G>A.
  • the condition is a neuromuscular disease, more in particular it is a muscular dystrophy.
  • This aspect can also be formulated as a method for the prevention and/or treatment of a condition associated to the mutation (CO/_6A/)c.877G>A.
  • Figure 1 shows chromatogram of the sequences of exon 10 of COL6A1 showing the heterozygous c.877G>A mutation (in the position of the circled nucleotide (G)).
  • Figure 2 shows: (A) ddPCR showing the abundance (%) of wild-type (in white) and mutant transcripts (in black) in the healthy control fibroblasts and patient's primary fibroblasts, the y-axis represents the abundance in percentage (%); (B) ddPCR for total COL6A1 expression (copies of the transcript/pL) of the healthy control, and patient's primary fibroblasts, the y-axis represents the concentration (copies/ pL). Data are represented as mean ⁇ SD. The x-axis is ordered showing the healthy control's data in the left, and the patient's data in the right.
  • Figure 4 shows immunofluorescence of patient's fibroblasts non-treated and treated with the tested oligonucleotide at 150 nM:
  • A HyVolution analysis of the patient's non treated fibroblasts (images above) and after the treatment with the tested oligonucleotide at 150 nM (images below). Scale bar: 10 pm.;
  • B 3D reconstructions of the patient's non treated fibroblasts (image above) and after the treatment with the tested oligonucleotide at 150 nM (image below). Scale bar: 5pm.
  • Figure 5 shows the percentage of mutant and wild type transcripts derived from the allele specific transcriptional analysis using ddPCR of the patient fibroblasts (P) treated with AON 1 at concentrations of 25 nM, 50 nM, 100 nM, 150 nM and 200 nM. Results are compared with untreated (NT) and Scrambled (Negative Control). Data are presented as the mean ⁇ SD of the abundance in percentage of each allele. Mutant (black), wild type (white). Y axis represents Abundance (A) in percentage (%)
  • FIG. 7 Concentration of copies of the mutant allele for the patient fibroblasts (P) treated with AON 4 at concentrations of 25 nM, 50 nM, 100 nM, 150 nM and 200 nM. Results are compared with P untreated (NT) and P Scrambled (Negative Control). Y axis represents Concentration (copies/piL): “C” stands for Concentration; “c.” stands for copies.
  • Figure 8. shows in the x-axis and ordered from left to right, allele specific transcription analysis using ddPCR of the patients' primary fibroblasts non-treated (NT), AON 2, AON 5, AON 6 and AON 7 at a concentration of 150 nM. Ratio between the WT and MUT allele was calculated and normalized to the non-treated, represented as mean ⁇ SD, and were statistically analysed using one-way ANOVA and Dunnett's multiple comparison test (**p ⁇ 0,01; *** p ⁇ 0,001; **** p ⁇ 0,0001). The y-axis represents the relative expression, in other words: the radio between mutated allele (MUT) and wild type allele (WT).
  • the first aspect provided by the invention is an oligonucleotide that downregulates the expression an allele carrying a mutation (COL6A1)c.877G>/ ⁇ .
  • the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele, at the site of the mutation. More in particular, which either does not downregulate the expression of a COL6A1 wild-type allele or does downregulate the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  • the oligonucleotide of the first aspect downregulates the expression of an allele carrying a mutation at position 877 in the COL6A1 coding sequence (CDS), wherein G is mutated to A compared to wild-type COL6A1, wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele, at the site of the mutation, and which either does not downregulate the expression of a COL6A1 wild-type allele or does downregulate the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  • CDS COL6A1 coding sequence
  • wild-type allele refers in the present disclosure to both a COL6A1 allele with the reference sequence (NM_001848 version NM_001848.3 of August 31, 2019).
  • the oligonucleotide of the first aspect would also not downregulate the expression of a COL6A1 allele different than the COL6A1 wild type allele and not carrying the mutation (CO/_6A1)c.877G>A mutation or would downregulate this allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  • the term “COL6A1” refers both to the collagen type VI alpha 1 chain gene, with genomic NCBI reference NG_008674 version NG_008674.1 of May 4, 2006, and its mRNA transcript with NCBI reference NM_001848 version NM_001848.3 of August 31, 2019.
  • the COL6A1 encodes for one of the three o-chains that compound the collagen VI in humans and it is found in chromosome 21 .
  • the encoded peptide is the alpha 1 subunit of type VI collagen, with protein NCBI reference NP_001839.2 (of February 11, 2006) or UniProt KB accession number P12109, version 3 of February 6 th , 2007, and version 222 of the database release.
  • the encoded peptide is a short o-chain domain flanked by N-terminal and C-terminal globular domains of the type "factor von Willebrand A”. It presents a beaded filament structure.
  • the central domain is a triple helix (TH) formed by conserved Gly-X-Y repeats. These Glycine residues are essential for the correct assembly of the collagen VI chains.
  • the union of the three o-chains (o1, o2 and o3) through the Glycine residues of the triple helix domain forms heterotrimeric o1 [VI]o2[VI]o3[VI] monomers which join in antiparallel dimers and two of them associate, forming a tetramer. After the assembly, tetramers are secreted to the extracellular space and the higher-order structure is formed due to associations between them, giving rise to the functional collagen VI.
  • COL6A )c.877 G> A refers, in the standard nomenclature format established by the Human Genome Variation Society (HGVS) (Ogino, S., et al. (2007)), to the mutation in the position 877 of the COL6A1 CDS in which a guanine (G) nucleotide present in the reference nucleotide sequence (NCBI Reference Sequence: NM_001848, version NM_001848.3 of August 31, 2019) is substituted by an adenine (A).
  • NCBI Reference Sequence: NM_001848, version NM_001848.3 of August 31, 2019 is substituted by an adenine (A).
  • NCBI Reference Sequence NM_001848, version NM_001848.3 of August 31, 2019
  • NCBI Reference Sequence NM_001848, version NM_001848.3 of August 31, 2019
  • A adenine
  • (CO _6A1)c.877G>A refers to the mutation in which the guanine (G) in the position 877 in the COL6A1 CDS reference nucleotide sequence (SEQ ID NO: 24) is substituted by an adenine (A).
  • the nucleotide in position 877 of the COL6A1 CDS corresponds to the nucleotide in position 958 of the full sequence of the COL6A1 mRNA transcript (with NCBI reference NM_001848.3), which is also be a reference.
  • the (CO/_6A1)c.877G>A mutated gene comprise a CDS that consists of the sequence SEQ ID NO: 25.
  • the mRNA transcript of the (CO/_6A1)c.877G>A mutated gene comprises the CDS with sequence SEQ ID NO: 25.
  • This mutation causes the substitution of a glycine from the triple helix domain by an arginine (p.Gly293Arg), thus affecting the correct assembly of the collagen VI chains.
  • the mutation is one of the two identified in the single nucleotide polymorphism with reference rs398123643 (build 156, released on September 21, 2022) in the position ch r21 :45989626 (GRCh38.p14) of the Homo sapiens.
  • expression of the encoded protein(s) is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%.
  • expression of the encoded protein(s) is reduced by from around 10% to around 60%, from around 20% to around 50%, or from around 30 to around 40%.
  • the oligonucleotide of the invention downregulates the expression by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least
  • the oligonucleotide downregulates the expression by at least 20%. More in particular by at least 25%.
  • the oligonucleotide of the invention downregulates the expression by at least 30% of an allele carrying a mutation (COL6A1)c.877G>A, wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele, at the site of the mutation, and which either does not downregulate the expression of a COL6A1 wild-type allele or does downregulate the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  • Reduction of the expression of the encoded protein(s) can be measured by any suitable technique known in the art. For example, reverse transcription, Sanger sequencing and/or quantitative real-time PCR are frequently used techniques. The examples below describe a method to determine the expression of the genes in greater detail.
  • oligonucleotide refers to an oligomer of nucleotide.
  • Nucleotides consist of a naturally occurring nitrogenous base or nucleobase (purines (adenine and guanine) and pyrimidines (cytosine, uracil, and thymine)) which is covalently bond to the T position of a 5-carbon sugar (deoxyribose or ribose) which is in turn covalently bond at its 5' position to a phosphate.
  • Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides, which are respectively oligomers of "deoxyribonucleotides” or “ribonucleotides”.
  • An oligonucleotide formed by deoxyribonucleotides may be referred as "DNA oligonucleotide”, “DNA molecules” or simply as “DNA”; and an oligonucleotide formed by ribonucleotides may be referred as "RNA oligonucleotide”, “RNA molecules” or simply "RNA”.
  • a deoxyribooligonucleotide consists of a deoxyribonucleotide (containing adenine or guanine as purines, or cytosine or thymine as pyrimidines) repeating structure wherein the phosphate of a deoxyribonucleotide covalently bonds to the 3' carbon of the deoxyribose of another deoxyribonucleotide, form an alternating, unbranched polymer.
  • a ribooligonucleotide (containing adenine or guanine as purines, or cytosine or uracil as pyrimidines) consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • oligonucleotide contains a sugar-phosphate backbone or phosphate backbone.
  • a "thymine nucleotide”, an “uracil nucleotide”, a “guanine nucleotide”, a “cytosine nucleotide” or an “adenine nucleotide” refer to the nucleotide that contains the correspondent nitrogenous base, in particular the nucleotides that contain these bases and that are part of the DNA or RNA.
  • oligonucleotide sequences are represented by strand in the 5' to 3' direction from left to right, and the first nucleotide presented in the sequence is residue position number 1 .
  • the term "oligonucleotide” also includes oligomers comprising monomers other than deoxy adenosine 3'- monophosphate, deoxyguanosine 3'-monophosphate, deoxycytidine 3'-monophosphate, deoxythymidine 3'- monophosphate, adenosine 3'-monophosphate, guanosine 3'-monophosphate, cytidine 3'-monophosphate, or uridine 3'-monophosphate, but are functionally and structurally similar thereto.
  • oligonucleotide analogues such as, for example, enhanced binding ability, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.
  • oligonucleotide analogue is indeed composed by nucleotide analogues (or modified nucleotides, or RNA/DNA-like nucleotides), which may have one or more of the three parts of it (phosphate, pentose sugar or nucleobase) altered compared to the above-mentioned "standard” nucleotides.
  • nucleotide analogues or modified nucleotides, or RNA/DNA-like nucleotides
  • the backbone of an oligonucleotide can be modified.
  • a common modification is the substitution of the phosphate backbone for a phosphoroti ated backbone.
  • the oligonucleotide contains a phosphorothioated backbone (i.e. modifying the phosphodiester linkage to phosphorothioate of a sugarphosphate backbone).
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or all of the oligonucleotide bases have a phosphorothioated backbone.
  • from 1 to 31, 2 to 30, 3 to 29, 4 to 28, 5 to 27, 6 to 26, 7 to 25, 8 to 24, 9 to 23, 10 to 22, 11 to 21, 12 to 20, 13 to 19, 14 to 18, or 15 to 17 bases have a phosphorothioated backbone.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides have a phosphorotioated backbone.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 deoxyribonucleotides have a phosphorothioated backbone.
  • More in particular all the deoxyribonucleotides have a phosphororthioated backbone.
  • Oligonucleotides that contain phosphorothioated backbones provide an increased resistance to nucleases compared to unmodified oligonucleotides (containing 100% phosphodiester backbone).
  • the oligonucleotide comprises modifications to help enhance their properties.
  • the oligonucleotide may be modified by the substitution of at least one nucleotide with at least one modified nucleotide, ideally so that the in vivo and in vitro stability of the oligonucleotide is enhanced as compared to a corresponding unmodified oligonucleotide.
  • the oligonucleotide comprises 2'-deoxy guanosine, 2'-deoxy adenosine, 2'-0-methylguanosine, 2'-0-methyl (e.g., 2'-O-methylcytidine, 2'-0-methylpseudouridine, 2'-0-methyluridine, 2'-0-methyladenosine (2prime-O- methyladenosine as referred in the sequence listing), 2'-0-methylguanosine) ribonucleotide, 2'- amino, 2'-thio and 2'-fluoro modified ribonucleotide, 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'- fluoro-guanosine, 2'-fluoro- adenosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino- adenosine, 2'--amino
  • the oligonucleotide includes derivatization of the 5 position, for instance being selected from 5-(2-amino) propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine; derivatization of the 6 position, for instance 6-(2-amino)propyl uridine; derivatization of the 8-position for adenosine and/or guanosines, for instance 8- bromo guanosine, 8-chloro guanosine, or 8-fluoroguanosine.
  • the oligonucleotide comprises nucleotide analogs such as deaza nucleotides, e.g., 7-deaza- adenosine; 0- and N-modified (for instance alkylated, such as N6-methyl adenosine) nucleotides; and other heterocyclically modified nucleotide analogs.
  • nucleotide analogs such as deaza nucleotides, e.g., 7-deaza- adenosine; 0- and N-modified (for instance alkylated, such as N6-methyl adenosine) nucleotides; and other heterocyclically modified nucleotide analogs.
  • the oligonucleotide comprises a modified sugar portion.
  • modifications to the sugar portion of the nucleotides which may be employed include the 2' OH-group being replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, H2, NHR, NR2, COOR, or OR, wherein R is substituted or unsubstituted 01-06 alkyl, alkenyl, alkynyl, aryl and so on.
  • the phosphate group of the nucleotide may also be modified, such as by substituting one or more of the oxygens of the phosphate group with sulfur (for instance by employing phosphorothioates).
  • Modifications may decrease the rate of hydrolysis of polynucleotides comprising the modified bases, for example by inhibiting degradation by exonucleases.
  • the oligonucleotide is resistant to ribonucleases.
  • Oligonucleotide which may be employed includes those with modifications to promote such resistance, for instance an oligonucleotide of the invention may have particularly been modified with a 2'- O-methyl group (e.g., 2'-0-methylcytidine, 2'-0- methylpseudouridine, 2'-0- methylguanosine, 2'-0-methyluridine, 2'-0-methyladenosine, 2'-0-methyl).
  • the oligonucleotide contains a modification to increase resistance to ribonucleases and a phosphorothioate backbone.
  • the oligonucleotide contains peptide nucleic acid (PNA), Morpholino nucleic acid, glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acids (HNA).
  • PNA peptide nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • HNA hexitol nucleic acids
  • the oligonucleotide contains locked nucleic acids (LNA) (an oligonucleotide comprising at least one 2'-C,4'-C-oxy- methylene-linked bicyclic ribonucleotide monomer), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1- methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
  • LNA
  • the oligonucleotide includes modifications to the phosphate backbone such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates.
  • the oligonucleotide contains a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2- propenyl, and isopropyl).
  • the oligonucleotide of the invention is complementary to (and thus hybridize with) a region of the RNA transcript from the genes, in particular to the mRNA.
  • the targeted mRNA transcript is the NM_001848.3 (NCBI reference) with the (CO/_6A1)c.877G>A mutation.
  • the targeted mRNA transcript comprises a CDS which is of SEQ ID NO: 25.
  • the oligonucleotide is complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides of the target sequence within the transcript; in particular it is complementary to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; more particularly it is complementary to at least 13, 14, 15, 16, 17, 18 or 19 nucleotides; and even more particularly it is complementary to at least 17 nucleotides of the target transcript.
  • the oligonucleotide is complementary to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides of the target sequence within the transcript; in particular it is complementary to 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; more particularly it is complementary to 13, 14, 15, 16, 17, 18 or 19 nucleotides; and even more particularly it is complementary to 17 nucleotides of the target transcript.
  • the oligonucleotide is complementary to from 9 to 31, 10 to 30, 11 to 29, 12 to 28, 13 to 27, 14 to 26, 15 to 25, 16 to 24, or from 17 to 23 nucleotides of the target sequence within the transcript, more in particular from 13 to 31 ; in particular it is complementary to from 13 to 25, 14 to 24, 15 to 23, 16 to 22 or 17 to 21 nucleotides; more particularly it is complementary to from 13 to 19, 14 to 18, or 15 to 17 nucleotides of the target transcript.
  • the target sequence comprises the sequence delimited by the nucleotides in position 862 to 892 of the SEQ ID NO:25; more in particular in position 865 to 889 of the SEQ ID NO:25; more in particular in position 868 to 886 of the SEQ ID NO:25; more in particular in position 869 to 885 of the SEQ ID NO:25.
  • the target sequence consists of the sequence delimited by the nucleotides in position 862 to 892 of the SEQ ID NO:25; more in particular in position 865 to 889 of the SEQ ID NO:25; more in particular in position 868 to 886 of the SEQ ID NO:25; more in particular in position 869 to 885 of the SEQ ID NO:25.
  • the oligonucleotide is from 9 to 31, 10 to 30, 11 to 29, 12 to 28, 13 to 27, 14 to 26, 15 to 25, 16 to 24, 17 to 23, 18 to 22, or 19 to 21 nucleotides in length, particularly from 13 to 31, more in particular from 13 to 19 nucleotides in length, more particularly it is from 15 to 19, or from 16 to 18 nucleotides in length.
  • the oligonucleotide is 17 to 25 nucleotides in length. In another particular embodiment, the oligonucleotide is 17 to 20 nucleotides in length.
  • the region of the oligonucleotide capable of hybridizing to the target transcript has a length as described herein, or at least that length, but there are also additional nucleotides at the 5' and/or 3' ends of the oligonucleotide (overhangs), though in other instances no overhangs are present and the whole length of the oligonucleotide hybridizes with the target.
  • oligonucleotide sequences which are 100% complementary to a portion of the target RNA may particularly be employed. In some instances, though, sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence may be present.
  • oligonucleotide sequences with insertions, deletions, and single point mutations relative to the target sequence may also be effective for reducing the target gene expression.
  • the oligonucleotide is at least around 80%, around 81%, around 82%, around 83%, around 84%, around 85%, around 86%, around 87%, around 88%, around 89%, around 90%, around 91%, around 92%, around 93%, around 94%, around 95%, around 96%, around 97%, around 98%, around 99% or even around 100% complementary to the target sequence within the mRNA transcript.
  • the oligonucleotide is at least around 94%, around 95%, around 96%, around 97%, around 98%, around 99% complementary to the target sequence. In more particular embodiments, the oligonucleotide is around 100% complementary to the target sequence.
  • the oligonucleotide is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides in length, particularly at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In a more particular embodiment, the oligonucleotide is at least 13 nucleotides in length. In a more particular embodiment, the oligonucleotide is at least 17 nucleotides in length.
  • the oligonucleotide is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides in length, particularly 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, more particularly 13, 14, 15, 16, 17, 18 or 19, more in particular 16, 17 or 18 nucleotides in length. More in particular the oligonucleotide is 17 nucleotides in length.
  • hybridizes are terms of art that refer to the pairing of nucleobases in complementary strands of oligonucleotides (e.g., an antisense oligomer and a selected/target sequence in a pre-mRNA molecule). While embodiments of this disclosure are not limited to a particular pairing mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases or nitrogenous bases.
  • the natural nucleobase adenine is complementary to the natural nucleobases thymidine and uracil, which pair through the formation of hydrogen bonds.
  • the natural base guanine is complementary to the natural nucleobases cytosine and 5-methyl cytosine.
  • An oligonucleotide complementary to a certain target sequence is understood as an oligonucleotide having a sequence that will bind to the target sequence. Complementarity can be 100%, when all of the nucleotides bind to the target sequence, or less that 100%.
  • the above embodiments can be alternatively expressed as an oligonucleotide that comprises a sequence that has identity with respect to the reverse complimentary of a target sequence within a transcript mRNA.
  • Identity is of 100% or, alternatively, at least around 80%, around 81%, around 82%, around 83%, around 84%, around 85%, around 86%, around 87%, around 88%, around 89%, around 90%, around 91%, around 92%, around 93%, around 94%, around 95%, around 96%, around 97%, around 98%, or around 99% with respect to the reverse complimentary of the target sequence within the transcript mRNA.
  • Sequence identity including determination of sequence complementarity for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the field. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity i.e., a local alignment.
  • a preferred, non- limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the skilled person may determine whether an oligonucleotide downregulates the expression of an allele carrying the mutation at position 877 in the COL6A1 coding sequence (CDS), and does not downregulate the expression of a COL6A1 wild-type allele (or downregulates the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation), by methods well known in the state of the art. For example, this may be done or this is achieved by Droplet Digital PCT (ddPCR) technique, as shown in the examples.
  • ddPCR Droplet Digital PCT
  • oligonucleotides described herein can be single-stranded or double-stranded.
  • the oligonucleotide of the first aspect is double stranded.
  • double-stranded RNA include, e.g., siRNA, short hairpin RNA (shRNA) and other RNAi agents such as pre-miRNA.
  • the oligonucleotide is a small interfering RNA (siRNA).
  • siRNA acts by activating the RNAi-induced suppression complex. Once the siRNA molecules according to the present disclosure enter into the cell it gets incorporated into other proteins to form the RISC complex, and once the siRNA is part of said complex, the siRNA is unwond to form single stranded siRNA (the strand that is thermodynamically less stable due to its base pairing at the 5' end is chosen to remain part of the RISC complex).
  • siRNAs according to the present invention indeed downregulates the expression of an allele carrying a mutation (COL6A1)c.877G>A by hybridizing to its mRNA. Therefore, it is herein considered that the siRNAs of the present invention downregulate the expression of the mutant allele by hybridization to the target mRNA.
  • the siRNA sequences may have overhangs or blunt ends. In a particular embodiment, the siRNA sequences have overhangs. Suitable siRNA sequences can be identified using any means known in the art.
  • the oligonucleotide as defined in the first aspect is single-stranded.
  • Single-stranded oligonucleotides include, e.g., ribozymes, mature miRNA, guide RNA, and triplex-forming oligonucleotides and antisense oligonucleotides (AONs or ASOs) such as gapmers.
  • the oligonucleotide is a miRNA.
  • miRNAs are involved in RNA silencing and post- transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then gene silence said mRNA molecules by one or more of the processes of cleavage of mRNA strand into two pieces, destabilization of mRNA by shortening its poly(A) tail, or translation of mRNA into proteins.
  • the sequence of the miRNA comprises a portion that corresponds with that of a portion of the mRNA transcript. In particular, the portion will usually be 100% complementary to the target portion within the allele comprising the mutation but lower levels of complementarity (e.g.
  • MicroRNA are from 16 to 27 nucleotides, more in particular from 20 to 24 nucleotides, even more in particular from 21 to 23 nucleotides.
  • the oligonucleotide as defined in the first aspect is an antisense oligonucleotide (AON or ASO).
  • the AONs act by binding to target pre-mRNA or mRNA via Watson-Crick base pairing and inducing downregulation of gene expression by different mechanisms such as through mRNA cleavage, RNase H- mediated mRNA degradation or steric hindrance.
  • the oligonucleotide of the present invention may contain deoxyribonucleotides or ribonucleotides.
  • the oligonucleotide comprises DNA or RNA.
  • the oligonucleotide consists of DNA or RNA.
  • the oligonucleotide of the present invention can also contain both deoxyribonucleotides and ribonucleotides.
  • the oligonucleotide is a DNA-RNA chimera.
  • the oligonucleotide comprises deoxyribonucleotides and ribonucleotides. More particularly, the oligonucleotide consists of deoxyribonucleotides and ribonucleotides.
  • the oligonucleotide of the first aspect comprises a sequence GTCTGAG of deoxyribonucleotides.
  • the oligonucleotide consists of a sequence GTCTGAG of deoxyribonucleotides.
  • the oligonucleotide consists of a sequence GTCTGAG of deoxyribonucleotides and one or more ribonucleotides.
  • Gapmers are chimerical single-stranded AONs that are compounded by a central block of DNA nucleotides (DNA gap), flanked by strands of RNA.
  • the oligonucleotide is a gapmer.
  • the oligonucleotide of the first aspect consists of nucleotides arranged in the structure 5’-A-B-C- 3', wherein “B” is a DNA gap of deoxyribonucleotides, and "A” and “C” are flanking RNA blocks of ribonucleotides.
  • the RNase H is an enzyme family which in almost all organisms degrades DNA-RNA hybrids as a defense against viral infection. Gapmers mechanism of gene-silencing action relies on the degradation through the action of Rnase-H in the protein synthesis process: the DNA sequence of a gene is first transcribed into mRNA, the gapmer binds to the mRNA target, and the "gapmer DNA”-“mRNA” duplex is degraded by the Rnase H1, which avoids the translation to protein (i.e. silencing the expression of the correspondent gene).
  • the oligonucleotide consists of a structure 5'-A-B-C-3', wherein “B” is 7, 8, 9, 10, 11, 12, or 13 deoxyribonucleotides long, and "A” and “C” are each at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 ribonucleotides long.
  • “B” is 7, 8, 9, 10, 11, 12, or 13 deoxyribonucleotides long, and "A” and “C” are each at least 3, 4, 5, or 6 ribonucleotides long.
  • “B” is 7 deoxyribonucleotides, and "A” and “C” are each at least 3 ribonucleotides long.
  • “B” is 7 deoxyribonucleotides long, and “A” and “C” are each at least 4 ribonucleotides long. More in particular, “B” is 7 deoxyribonucleotides long, and “A” and “C” are each at least 5 ribonucleotides long.
  • the oligonucleotide consists of a structure 5'-A-B-C-3', wherein “B” is from 7 to 13, or 6 to 12 deoxyribonucleotides long, and “A” and “C” are each from 1 to 9, 2 to 8, 3 to 7, or 4 to 6 ribonucleotides long.
  • “B” is from 7 to 13 deoxyribonucleotides long, and "A” and “C” are each from 3 to 6 ribonucleotides long.
  • “B” is from 7 to 8 deoxyribonucleotides long, and "A” and “C” are each from 4 to 5 ribonucleotides long.
  • the oligonucleotide consists of a structure 5'-A-B-C-3', wherein “B” is 7, 8, 9, 10, 11, 12, or 13 deoxyribonucleotides long, and "A” and “C” are each 1, 2, 3, 4, 5, 6, 7, 8 or 9 ribonucleotides long.
  • “B” is 7, 8, 9, 10, 11, 12, or 13 deoxyribonucleotides long, and "A” and “C” are each 3, 4, 5, or 6 ribonucleotides long.
  • “B” is 7 deoxyribonucleotides, and "A” and “C” are each 3 ribonucleotides long.
  • “B” is 7 deoxyribonucleotides long, and “A” and “C” are each 4 ribonucleotides long. More in particular, “B” is 7 deoxyribonucleotides long, and “A” and “C” are each 5 ribonucleotides long.
  • the oligonucleotide is arranged in a structure 5'-A-B-C-3', wherein
  • oligonucleotide consists of:
  • brackets ( ) are deoxyribonucleotides
  • nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides, wherein T* is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and
  • nucleotides in curly brackets ⁇ ⁇ are ribonucleotides
  • the oligonucleotide consists of a sequence (a), or of a fragment of this sequence (a) that are lacking certain nucleotides from the ends.
  • the oligonucleotide can consist of fragments of sequence (a) which lack consecutive nucleotides starting to delete them from the ends (5' and/or 3') of the sequence (a).
  • an oligonucleotide can consist of a sequence (a) "123456”, or of fragments of (a) such as “23456”, “12345”, “2345”, “345”, among other; but the oligonucleotide does not consist of fragments of (a) such as "13456”, “12346”, “246”, “135”, “36”, “16”, “14”, among other. All of this, provided that the oligonucleotide is arranged in the structure A-B-C described in the embodiment.
  • the oligonucleotide consists of:
  • nucleotides in the positions 13 to 20 are deoxyribonucleotides
  • nucleotides in the positions 4 to 12 and 20 to 28 are deoxyribonucleotides or ribonucleotides, wherein the nucleotide in the position 21 is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and
  • nucleotides in the positions 1 to 3 and 29 to 31 are ribonucleotides
  • the oligonucleotide is arranged in a structure 5'-A-B-C-3', wherein
  • oligonucleotide consists of
  • brackets ( ) are deoxyribonucleotides
  • nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides, wherein T* is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and
  • nucleotides in curly brackets ⁇ ⁇ are ribonucleotides
  • the oligonucleotide consists of:
  • nucleotides in the positions 10 to 16 are deoxyribonucleotides
  • nucleotides in the positions 4 to 9 and 17 to 22 are deoxyribonucleotides or ribonucleotides, wherein the nucleotide in the position 18 is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and
  • nucleotides in the positions 1 to 3 and 23 to 25 are ribonucleotides
  • the oligonucleotide is of sequence [ACCCAAC](AGGTCTGAGGT)[CCCCGGG] (SEQ ID NO: 3), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence ACCCAACAGGTCTGAGGTCCCCGGG (SEQ ID NO: 3), wherein the nucleotides in positions 8 to 18 are deoxyribonucleotides and nucleotides in positions 1 to 7 and 19 to 25 are ribonucleotides.
  • the oligonucleotide is of sequence [ACCCAACA](GGTCTGAGG)[UCCCCGGG] (SEQ ID NO: 4), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence ACCCAACAGGTCTGAGGUCCCCGGG (SEQ ID NO: 4), wherein the nucleotides in positions 9 to 17 are deoxyribonucleotides and nucleotides in positions 1 to 8 and 18 to 25 are ribonucleotides.
  • the oligonucleotide is of sequence [CCAACA](GGTCTGAGG)[UCCCCG] (SEQ ID NO: 5), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CCAACAGGTCTGAGGUCCCCG (SEQ ID NO: 5), wherein the nucleotides in positions 7 to 15 are deoxyribonucleotides and nucleotides in positions 1 to 6 and 16 to 21 are ribonucleotides.
  • the oligonucleotide is arranged in a structure 5'-A-B-C-3', wherein
  • - “B” is from 7 to 10 deoxyribonucleotides long
  • a and C are each 5 ribonucleotides long; and wherein the oligonucleotide consists of
  • brackets ( ) are deoxyribonucleotides
  • nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides
  • nucleotides in curly brackets ⁇ ⁇ are ribonucleotides
  • the oligonucleotide consists of:
  • nucleotides in the positions 8 to 14 are deoxyribonucleotides
  • nucleotides in the positions 6 to 7 and 15 are deoxyribonucleotides or ribonucleotides
  • nucleotides in the positions 1 to 5 and 16 to 20 are ribonucleotides
  • the oligonucleotide is of sequence [CCAAC](AGGTCTGAGG)[UCCCC] (SEQ ID NO: 37), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CCAACAGGTCTGAGGUCCCC (SEQ ID NO: 37), wherein the nucleotides in positions 6 to 15 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 16 to 20 are ribonucleotides.
  • the oligonucleotide is of sequence [CAACA](GGTCTGAGG)[UCCCC] (SEQ ID NO: 6), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CAACAGGTCTGAGGUCCCC (SEQ ID NO: 6), wherein the nucleotides in positions 6 to 14 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 15 to 19 are ribonucleotides.
  • the oligonucleotide is of sequence [CAACA](GGTCTGAG)[GUCCC] (SEQ ID NO: 30), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CAACAGGTCTGAGGUCCC (SEQ ID NO: 30), wherein the nucleotides in positions 6 to 13 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 14 to 18 are ribonucleotides.
  • the oligonucleotide is arranged in a structure 5'-A-B-C-3', wherein
  • oligonucleotide consists of
  • brackets ( ) are deoxyribonucleotides
  • nucleotides in square brackets [ ] are ribonucleotides
  • the oligonucleotide consists of:
  • nucleotides in the positions 10 to 16 are deoxyribonucleotides
  • nucleotides in the positions 1 to 9 and 17 to 25 are ribonucleotides
  • the oligonucleotide is of sequence SEQ ID NO: 7.
  • the oligonucleotide is of sequence [CAACAG](GTCTGAG)[GUCCCC] (SEQ ID NO: 8), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CAACAGGTCTGAGGUCCCC (SEQ ID NO: 8), wherein the nucleotides in positions 7 to 13 are deoxyribonucleotides and nucleotides in positions 1 to 6 and 14 to 19 are ribonucleotides.
  • the oligonucleotide is of sequence [AACAG](GTCTGAG)[GUCCCC] (SEQ ID NO: 9), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence AACAGGTCTGAGGUCCCC (SEQ ID NO: 9), wherein the nucleotides in positions 6 to 12 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 13 to 18 are ribonucleotides.
  • the oligonucleotide is of sequence [CAACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 10), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence CAACAGGTCTGAGGUCCC (SEQ ID NO: 10), wherein the nucleotides in positions 7 to 13 are deoxyribonucleotides and nucleotides in positions 1 to 6 and 14 to 18 are ribonucleotides.
  • the oligonucleotide is of sequence [ACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 11), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence ACAGGTCTGAGGUCCC (SEQ ID NO: 11), wherein the nucleotides in positions 5 to 11 are deoxyribonucleotides and nucleotides in positions 1 to 4 and 12 to 16 are ribonucleotides.
  • the oligonucleotide is of sequence [AACAG](GTCTGAG)[GUCC] (SEQ ID NO: 12), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence AACAGGTCTGAGGUCC (SEQ ID NO: 12), wherein the nucleotides in positions 6 to 12 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 13 to 16 are ribonucleotides.
  • the oligonucleotide is of sequence [ACAG](GTCTGAG)[GUCC] (SEQ ID NO: 13), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence ACAGGTCTGAGGUCC (SEQ ID NO: 13), wherein the nucleotides in positions 5 to 11 are deoxyribonucleotides and nucleotides in positions 1 to 4 and 12 to 15 are ribonucleotides.
  • the oligonucleotide is of sequence [AACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 14), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • the oligonucleotide is of sequence AACAGGTCTGAGGUCCC (SEQ ID NO: 14), wherein the nucleotides in positions 6 to 12 are deoxyribonucleotides and nucleotides in positions 1 to 5 and 13 to 17 are ribonucleotides.
  • the oligonucleotide of the invention is of sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 6 and SEQ ID NO: 37. More in particular, it is of SEQ ID NO: 14.
  • the gapmer disclosed herein preferably contain modified nucleotides (nucleotide analogues).
  • the DNA gap of the gapmer is usually modified with deoxyribonucleotides with phosphorothioated backbones which provide an increased resistance to nucleases.
  • some or all of the deoxyribonucleotides of the oligonucleotide as defined in the first aspect have a phosphorothioated backbone.
  • all of the deoxyribonucleotides of the oligonucleotide as defined in the first aspect have a phosphorothioated backbone.
  • the oligonucleotide comprises modifications to help enhance the properties of the oligonucleotide.
  • the most typical modified nucleotides used are 2'-0-Methyl (2’0-Me), locked nucleic acid (LNA) bases or 2'deoxy-2'-fluorobeta-D-arabinonucleic acid (2’FANA).
  • 2'-0- Methyl, 2’0-Me, or 2'-O-methylation is a nucleoside modification of RNA, where a methyl group is added to the 2' hydroxyl of the ribose moiety of a nucleoside, producing a methoxy group.
  • Other modified ribonucleotides used are 2'-MOE (2'-Methoxyethy I).
  • some or all of the ribonucleotides of the oligonucleotide as defined in the first aspect are 2'-0-Methyl (2’0-Me), locked nucleic acid (LNA) bases, 2'deoxy-2'-fluorobeta-D-arabinonucleic acid (2'FANA) or 2'-Methoxyethyl (2’-MOE) RNA nucleotides.
  • some or all of the ribonucleotides are selected from the group consisting of 2'-0-Methyl (2’0-Me) RNA nucleotides, 2’-Methoxyethy I (2'-MOE) RNA nucleotides and combinations thereof.
  • all of the ribonucleotides are 2'-0-Methyl (2’0- Me) RNA nucleotides.
  • adenine ribonucleotides contain a 2'0-Methyladenosine nucleoside
  • guanine ribonucleotides contain a 2'0-Methylguanosine nucleoside
  • cytosine ribonucleotides contain a 2'0- Methylcytidine nucleoside
  • uracil ribonucleotides contain a 2'0-Methyluridine nucleoside.
  • all of the ribonucleotides are 2'-Methoxyethyl (2'-MOE) RNA nucleotides.
  • adenine ribonucleotides contain a 2'-MOE-Adenosine nucleoside
  • guanine ribonucleotides contain a 2'-MOE- guanosine nucleoside
  • cytosine ribonucleotides contain a 2'-M0E-cytidine nucleoside
  • uracil ribonucleotides contain a 2'-M0E-uridine nucleoside
  • the deoxyribonucleotides of the oligonucleotide as defined in the first aspect are deoxyribonucleotides linked by phosphorothioate linkages.
  • the ribonucleotides of the oligonucleotide as defined in the first aspect are modified at the 2'-0 position of the ribose moiety.
  • the ribonucleotides are selected from the group consisting of 2'-0-Methyl RNA nucleotides, 2’-Methoxyethy I (2'-MOE) RNA nucleotides, and combinations thereof.
  • the oligonucleotide as definde in the first aspect comprises ribonucleotides of modified at the 2'-0 position of the ribose moiety and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide comprises ribonucleotides selected from the group consisting of 2'-0-Methyl RNA nucleotides, 2'-Methoxyethyl (2'-MOE) RNA nucleotides, and combinations thereof; and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide comprises 2'-0-Methyl RNA nucleotides and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide comprises 2'-Methoxyethyl (2'-MOE) RNA nucleotides and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as definde in the first aspect consists of ribonucleotides of modified at the 2'-0 position of the ribose moiety and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide consists of ribonucleotides selected from the group consisting of 2'-0-Methyl RNA nucleotides, 2'-Methoxyethyl (2’-M0E) RNA nucleotides, and combinations thereof; and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide consists of 2'-0-Methyl RNA nucleotides and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide consists of 2'-Methoxyethy I (2’-MOE) RNA nucleotides and deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence AACAG(GTCTGAG)GUCCC (SEQ ID NO: 15), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [AACAG]GTCTGAG[GUCCC] (SEQ ID NO: 16), wherein the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [AACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 17), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages and the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence AACAGGTCTGAGGUCCC SEQ ID NO: 15, wherein the nucleotides in positions 6 to 12 are deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence AACAGGTCTGAGGUCCC SEQ ID NO: 16, wherein the nucleotides in positions 1 to 5 and 13 to 17 are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence AACAGGTCTGAGGUCCC SEQ ID NO: 17, wherein the nucleotides in positions 6 to 12 are deoxyribonucleotides with phosphorothioate linkages and the nucleotides in positions 1 to 5 and 13 to 17 are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence CAACA(GGTCTGAG)GUCCC (SEQ ID NO: 31), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CAACA]GGTCTGAG[GUCCC] (SEQ ID NO: 32), wherein the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CAACA](GGTCTGAG)[GUCCC] (SEQ ID NO: 33), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages and the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence CAACA(GGTCTGAGG)UCCCC (SEQ ID NO: 34), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CAACA]GGTCTGAGG[UCCCC] (SEQ ID NO: 35), wherein the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CAACA](GGTCTGAGG)[UCCCC] (SEQ ID NO: 36), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages and the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence CCAAC(AGGTCTGAGG)UCCCC (SEQ ID NO: 38), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CCAAC]AGGTCTGAGG[UCCCC] (SEQ ID NO: 39), wherein the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide as defined in the first aspect comprises or consists of a sequence [CCAAC](AGGTCTGAGG)[UCCCC] (SEQ ID NO: 40), wherein the nucleotides in brackets ( ) are deoxyribonucleotides with phosphorothioate linkages and the nucleotides in square brackets [ ] are ribonucleotides with 2'-0-Methyl modifications.
  • the oligonucleotide of the invention is of sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 34 and SEQ ID NO: 38. More in particular, it is of SEQ ID NO: 17.
  • the oligonucleotide of the invention is of sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 35 and SEQ ID NO: 39. More in particular, it is of SEQ ID NO: 16.
  • the oligonucleotide of the invention is of sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 33, SEQ ID NO: 36 and SEQ ID NO: 40. More in particular, it is of SEQ ID NO: 17.
  • the oligonucleotides of the disclosure can be produced by any technique known to one of ordinary skill in the art, such as chemical synthesis, enzymatic production or biological production.
  • the oligonucleotide of the invetion is made by chemical synthesis.
  • Non- limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques or via deoxynucleoside H- phosphonate intermediate.
  • Double stranded oligonucleotides such as siRNA molecules, can be assembled from two distinct single stranded oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA.
  • each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
  • siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an siRNA duplex having hairpin secondary structure.
  • compositions comprising a delivery agent and an oligonucleotide
  • the oligonucleotide described herein may be provided on its own or together with a delivery agent, or together with other molecules which contribute to the desired therapeutic effect.
  • the inclusion of the oligonucleotide disclosed herein within a delivery agent may be convenient, for example, in order to target the desired cells or tissues, and to enter into them an interact with its target, to increase the transfection efficiency and/or to improve degradation resistance.
  • the invention also provides as a second aspect, a composition comprising a delivery agent and an oligonucleotide as defined in the first aspect, wherein the oligonucleotide is associated to the delivery agent. All embodiments described above for the oligonucleotide also apply to the composition of the second aspect.
  • delivery agent is to be understood as a pharmaceutically acceptable vehicle.
  • the delivery agent can be organic, inorganic, or both. Suitable delivery agents are well known to those of skill in the art and include, without limitation, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles. Delivery agents may also include, saline, buffer, dextrose, water, glycerol, ethanol, and the combinations thereof. In particular embodiments, the delivery agent may be a polycationic polymer, a vesicle, a liposome, or a nanoparticle.
  • the expression construct In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell.
  • oligonucleotides include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, cell sonication, gene bombardment using high velocity microprojectiles, receptor-mediated transfection, as well as the use of delivery agents such as nanoparticles, vesicles or nanovesicles, polymers, viral particles.
  • the delivery agent of the second aspect is a vesicle.
  • the vesicle is a nanovesicle.
  • the nanovesicle is an extracellular vesicle.
  • Extracellular vesicles and “EVs” are cell- derived and cell-secreted vesicles which, as a class, include exosomes, exosome-like vesicles, ectosomes (which result from budding of vesicles directly from the plasma membrane).
  • important components are "exosomes”.
  • the nanovesicle is an exosome.
  • the exosome size ranges from 30 to 500 nm, in particular from 40 to 120 nm, more in particular from 50 to 100 nm in diameter.
  • Exosomes are secreted by all types of cells and are also found abundantly in the body fluids such as: saliva, blood, urine and milk.
  • the major role of exosomes is in cell-cell communication via the functional ly-active cargo (such as miRNA, mRNA, DNA and proteins).
  • Exosomes may be isolated from biological sources, such as milk (milk exosomes), in particular bovine milk is an abundant source for isolating bovine milk exosomes.
  • the nanovesicle is a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • the delivery agent is a nanovesicle comprising a sterol and a non-lipid cationic surfactant such as those described in WO2020229469A1 (Nanovesicles and its use for nucleic acid delivery), which is herein incorporated by reference. More in particular, the delivery agent is a quatsome, as described in the same WO2020229469A1 .
  • the sterol comprises DC-cholesterol (DC-Chol) and the non-lipid cationic surfactant comprises Benzyldimethyltetradecylammonium chloride (MKC). More in particular, the sterol consists of DC- cholesterol (DC-Chol) and the non-lipid cationic surfactant consists of Benzyldimethyltetradecylammonium chloride (MKC).
  • DC-Chol DC-cholesterol
  • MKC Benzyldimethyltetradecylammonium chloride
  • An additional aspect of the invention refers to the oligonucleotide of the first aspect associated to a delivery agent. In other words, it refers to a delivery agent containing the oligonucleotide of the first aspect. All the embodiments described for the second aspect, accordingly apply to this additional aspect.
  • a third aspect of this disclosure refers to a composition comprising the oligonucleotide as defined above. All embodiments described above for the oligonucleotide and the composition comprising the oligonucleotide and a delivery agent also apply to the composition of the third aspect.
  • the composition is a pharmaceutical composition comprising a therapeutically effective amount of oligonucleotides of the first aspect and/or the composition of the second aspect together with pharmaceutically acceptable excipients and/or carriers.
  • pharmaceutically acceptable excipients or carriers refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • the election of the pharmaceutical formulation will depend upon the nature of the active compound and its route of administration. Any route of administration may be used. In some embodiments, the route of administration is parenteral, and the composition is then appropriate for parenteral administration. In a particular embodiment, the route of administration is by injection. In a more particular embodiment, the route of administration is systemic, for example, by intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, or transdermal injection. In a particular embodiment, the route of administration is local, for example, intratumoral injection. Topical administration is also contemplated, such that the pharmaceutical composition may be a topical composition.
  • compositions may be in any form, including, among others, tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, aerosols, or dry powdered forms suitable for reconstitution with water or other suitable liquid medium before use, for immediate or retarded release.
  • excipients and/or carriers can readily be determined by those skilled in the art according to the type of formulation being prepared.
  • suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like.
  • surface active agents are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like.
  • any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
  • the pharmaceutical composition is a sterile injectable solution.
  • a sterile injectable solution This may be prepared, for instance, by incorporating the oligonucleotides in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • preferred methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the pharmaceutical composition comprises a therapeutically effective amount of oligonucleotides.
  • therapeutically effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the particular dose of oligonucleotide or compositions comprising a delivery agent and an oligonucleotide administered according to this disclosure will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
  • the oligonucleotides or compositions comprising a delivery agent and an oligonucleotide of the present disclosure are used with other therapeutic agents.
  • the oligonucleotides or compositions comprising a delivery agent and an oligonucleotide are used in a combined treatment.
  • the oligonucleotides or compositions comprising a delivery agent and an oligonucleotide are used in combined treatment with palliative treatments. More in particular, the palliative treatments are physiological and/or pneumological treatments.
  • kits of parts that comprises at least one oligonucleotide as defined above.
  • the kit comprises more than one oligonucleotide as defined above.
  • the kit comprises at least two oligonucleotides, one as defined above (which reduces the expression of a pathogenic allele of COL6A1) and the other one reducing the expression of another congenital muscular dystrophy related genes.
  • the kit comprises at least two oligonucleotides, one as defined above (which reduces the expression of a pathogenic allele of COL6A1) and the other one reducing the expression of another COL-RDs related genes.
  • the kit comprises at least two oligonucleotides, one as defined above reducing the expression of COL6A1 and the other one reducing the expression of COL6A1, COL6A2 and/or COL6A3. All embodiments defined above for the oligonucleotide also apply to the kits.
  • the kits may further comprise delivery agents, excipients, carriers, means of administration, and/or instruction for use.
  • a third aspect of the present disclosure is directed to an oligonucleotides, a compositions comprising a delivery agent and an oligonucleotide and a kit, as defined above, for use as a medicament, in particular, for treating a condition associated, or caused by, the (CO/_6A/)c.877G>A mutation. All embodiments defined above for the oligonucleotide, the composition comprising a delivery agent and an oligonucleotide, and the kit also apply to their uses.
  • Treatment” or “treating” conditions associated or caused by the (CO/_6A/)c.877G>A mutation and related diseases or disorders includes a prophylactic treatment before the clinical onset of the disease or a therapeutic treatment after the clinical onset of the disease and may be achieved by arresting the development or reversing the symptom of any of these conditions, disorders or diseases.
  • the terms “treatment” and “treating” include any of following: the prevention of the disease or disorder or of one or more symptoms associated with the disease or disorder; a reduction or prevention of the development or progression of the disease or disorder or symptoms; and the reduction or elimination of an existing disease or disorder or symptoms.
  • the treatment of conditions associated to the (CO/_6A1)c.877G>A mutation may be achieved by downregulating the expression of an allele carrying a (CO/_6A1)c.877G>A mutation.
  • the oligonucleotide or compositions as defined above are for use in downregulating the expression of an allele carrying a (CO/_6A1)c.877G>A mutation.
  • the oligonucleotide or compositions as defined above are for use in patients presenting the mutation (COL6A1)c.877G>A. In a more particular embodiment, they are for use in patients presenting the mutation (COL6A1)c.877G>A in heterozygosis ((CO/_6A1)het.c.877G>A).
  • condition associated to the (CO/_6A1)c.877G>A mutation is a neuromuscular disease. More in particular it is a muscular dystrophy. In a more particular embodiment, the disease is a congenital muscular dystrophy. In a more particular embodiment, the disease is a collagen-VI related dystrophy (COL6-RD). In a more particular embodiment, the disease is selected from Ullrich Congenital Muscular Dystrophy, Bethlem myopathy and/or a COL6-RD with intermediate phenotype. In a more particular embodiment, the disease is a COL6-RD with intermediate phenotype.
  • oligonucleotide that downregulates the expression by at least 40% of a COL6A1 allele carrying a (CO/_6A1)c.877G>A mutation, wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele at the site of the mutation, and which either does not downregulate the expression of a COL6A1 wild-type allele or does downregulate the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  • B is from 7 to 13 deoxyribonucleotides long
  • a and C are each from 3 to 9 ribonucleotides long; and wherein the oligonucleotide consists of:
  • brackets ( ) are deoxyribonucleotides
  • nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides, wherein T* is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and
  • nucleotides in curly brackets ⁇ ⁇ are ribonucleotides, or
  • oligonucleotide according to any one of clauses 1-7, which comprises a sequence [AACAG] (GTCTGAG) [GUCCC] (SEQ ID NO: 14), wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  • composition comprising a delivery agent and an oligonucleotide as defined in any one of clauses 1-10, wherein the oligonucleotide is associated to the delivery agent, in particular wherein the delivery agent is a nanovesicle that comprises a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC- cholesterol.
  • a pharmaceutical composition comprising a therapeutically effective amount of one or more oligonucleotides as defined in any one of clauses 1-10, or the composition as defined in clause 11, together with a pharmaceutically acceptable excipient or carrier.
  • Dermal fibroblasts were obtained from patients and a healthy aged matched control. Cells were cultured in Dulbecco's modified Eagle medium (DMEM) with high glucose, L-Glutamine 1 :100, Fetal Bovine Serum (FBS) 10% and Penicillin-streptomycin and fungizone (PSF) 1 :100.
  • DMEM Dulbecco's modified Eagle medium
  • FBS Fetal Bovine Serum
  • PSF Penicillin-streptomycin and fungizone
  • the COL6A1 mutation of interest is found in heterozygosis in the exon 10 ((CO _6A1)c.877 G>A) in our cohort of patients, causing the missense mutation p.Gly293Arg.
  • the gapmer tested was designed to silence the mutant allele at the mRNA and to have resistance to nucleases.
  • the gapmer design presents a central gap of 7 deoxynucleotides with sequence GTCTGAG linked by phosphorotioated linkages, and two flanking regions of 5 ribonucleotides with 2'OMe modifications, with sequences AACAG and GUCCC, at the 5'end and the 3'end of the DNA gap respectively.
  • fibroblasts were seeded in a six-well plate at a concentration of 2x10 5 cells/well in growth medium for 24 hours. After this time, the medium was changed for Opti-MEM (1x) Gibco (Thermofisher®). Cells were treated with the oligonucleotide at concentrations of 25 nM, 50 nM, 100 nM, 150 nM and 200 nM, and the negative control scramble oligonucleotide at 100 nM. The dilutions were prepared at a final volume of 100 pL, using Opti-MEM as the diluent.
  • Invitrogen Lipofectamine 2000 was the transfection reagent and 5pL of it and also 100 pL of Opti-MEM were added to the oligonucleotide dilutions. After 20 minutes of incubation, the medium of the wells was removed, 1 mL of every mix was added to the corresponding well, and the samples were incubated for 24 hours at 37°C and 5% CO2.
  • Genomic DNA was extracted, using the Kit DNeasy® Blood and Tissue Kit (QIAGEN) following the manufacturer's instructions, and amplified by a polymerase chain reaction (PCR).
  • the components of the PCR were the buffer 1 X, MgSO42 mM, dNTPs 0.2 mM, the primers forward and reverse 0.2 pM, Taq, 200 ng of DNA and water up to 25 pl.
  • the primers used were of sequence CTCCTTGGCCCAAATCCTATC (SEQ ID NO: 18) (primer forward) and of sequence GGACAACATCACTGCTGAGAA (SEQ ID NO: 19) (primer reverse).
  • the PCR was run in the following conditions: 35 cycles at 95°C for 5 minutes (initial denaturation), 98°C for 20 seconds (denaturation), 55°C for 30 seconds (annealing) and 72°C at 55 seconds (elongation) and finally, a post amplification at 72°C for 5 minutes.
  • the agarose gel electrophoresis composed of 2% of agarose in TBE buffer 1 X, was used to confirm the presence of amplified DNA and it was revealed using the IBright®CL1000 (Thermofisher®). Once proved, it was purified using the QIAquick® PCR purification kit (QIAGEN) following the manufacturer's instructions and sent for Sanger sequencing to Macrogen.
  • QIAquick® PCR purification kit QIAGEN
  • the second reaction carried out was composed by water, Buffer Go Script 5x, MgCI2 2 mM, PCR nucleotide mix 0.5 mM, RNasin Ribonuclease Inhibitors and Go Script Reverse Transcriptase.
  • the conditions of the termoblock for this reaction were 5 minutes at 25°C, 45 minutes at 42°C and 15 minutes at 70°C.
  • Droplet DigitalTM PCR was used to quantify the wild-type and mutant allele expression of the exon 10 of COL6A1 and also the whole COL6A1 expression.
  • the allele specific ddPCR was composed of 11 pL ddPCR Supermix for Probes (no dUTP) (Bio-Rad, United States), 450 nM of the forward primer of sequence CCGGAGATCCTGGAAGA (SEQ ID NO: 20), 450 nM of the reverse primer of sequence TTTTTCTCCCTTCATTCCCT (SEQ ID NO: 21), 250 nM of the wild-type allele probe of sequence CGGGGACCTCGGACC, 5’ HEX-labeled (SEQ ID NO: 22) 250 nM of the mutated allele probe of sequence CGGGGACCTCAGACC, 5’ FAM-labeled (SEQ ID NO: 23) and water up to a final volume of 18 pL.
  • the ddPCR was carried out with 11 pL ddPCR Supermix for Probes (no dUTP), 450 nM of the forward primer, 450 nM of the reverse primer, the probe (reference: Hs01095585_m1 of Thermo Fished Scientific) and water up to a final volume of 17 pL. 2 ng of the cDNA was added. A volume of 20 pL of every mix was placed to the wells of a BioRad cartridge. Also, 70 pL of the droplet generator oil were added.
  • the droplets were generated using the QX200 droplet generator (Bio-Rad, United States) and were transferred to a 96-well PCR plate.
  • the PCR plate was placed in the thermocycle.
  • the conditions of the allele specific PCR were 95°C for 10 minutes, followed by 39 cycles at 95°C for 30 seconds and 50°C for 1 minute and finally 10 minutes at 98°C.
  • the conditions were 95°C for 10 minutes, followed by 39 cycles at 94°C for 30 seconds and at 60°C for 1 minute, and finally 10 minutes at 98°C.
  • the plate was placed into the WX200 droplet reader (Bio-Rad, United States) and the concentrations of the mutant and wild-type allele or the total COL6A1 were analysed using Bio-Rad QuantaSoftTM software (v1 .7.4). Results
  • a dose-response analysis of the oligonucleotide was carried out, in five different concentrations between 25 nM and 200 nM, and the relative expression in a transcriptional level of the alleles was studied in order to determine how the treatments affect them.
  • the treatment with the oligonucleotide presented surprising efficacy at 50 nM, 150 nM and 200 nM (p ⁇ 0.0001), without silencing the wild-type allele (Figure 3).
  • Extracellular collagen VI in primary patient's fibroblasts is recovered significantly after the treatment with the oligonucleotide at 150nM
  • AONs 1, 3, 4, 5, 6 and 7 are gapmers designed with a central core of deoxynucleotides linked by phosphorotioated linkages, and two flanking regions of 5 ribonucleotides with 2'OMe modifications.
  • Example 2 Lipofectamine transfections of the different AONs were carried out in fibroblasts obtained from dermal biopsies of patients carrying the pathogenic variant COL6A1 c.877G>A. Total RNA was then extracted from these cells, and retrotranscription and subsequent digital droplet PCR were performed to detect the specific expression of the mutant and wild type transcript. The methodologies followed to carry out Example 2 are described in Example 1, except that the oligonucleotides tested are different.
  • AON 1 SEQ ID NO: 27
  • AON 1 differs from the gapmer tested in Example 1 in a two deoxyribonucleotides shorter DNA core.
  • AON 3 SEQ ID NO: 28
  • AON 4 SEQ ID NO: 29
  • Fibroblasts derived from patients were also transfected with alternative AONs that, both in their total length and the length of the DNA core, were longer than AON 2.
  • AON 5 SEQ ID NO: 33
  • AON 6 SEQ ID NO: 36
  • AON 7 SEQ ID NO: 40

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Abstract

La présente invention concerne des oligonucléotides qui permettent de diminuer l'expression d'un allèle porteur d'une mutation dominante dans COL6A1, la diminution s'effectuant par hybridation dudit oligonucléotide à un transcrit d'ARN dudit allèle au site de la mutation dominante, et qui ne supprime pas l'expression de l'allèle de type sauvage ou qui diminue l'expression de l'allèle de type sauvage dans une moindre mesure qu'il ne diminue l'expression de l'allèle porteur de la mutation dominante. L'invention concerne également l'utilisation de compositions pour traiter des patients atteints de dystrophies musculaires, en particulier des dystrophies associées au collagène VI.
PCT/EP2024/067949 2023-06-27 2024-06-26 Oligonucléotides pour le traitement de maladies neuromusculaires Pending WO2025003222A1 (fr)

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