WO2022217366A1 - Oligonucléotides modifiés pour le traitement de la dystrophie musculaire de duchenne - Google Patents

Oligonucléotides modifiés pour le traitement de la dystrophie musculaire de duchenne Download PDF

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WO2022217366A1
WO2022217366A1 PCT/CA2022/050587 CA2022050587W WO2022217366A1 WO 2022217366 A1 WO2022217366 A1 WO 2022217366A1 CA 2022050587 W CA2022050587 W CA 2022050587W WO 2022217366 A1 WO2022217366 A1 WO 2022217366A1
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nucleotides
antisense oligonucleotide
lna
frna
alkyl
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Masad J. Damha
Daniel Timothy O'reilly
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The Royal Institution For The Advancement Of Learning/Mcgill University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present disclosure generally relates to oligonucleotides and uses thereof and in particular antisense oligonucleotides for use in modulating protein expression.
  • DMD Duchenne Muscular dystrophy
  • Dystrophin plays a vital role in connecting the cytoskeleton of muscle fibers to the extracellular matrix.
  • Dystrophin only accounts for just 0.002% of cellular proteins, but its loss of function is devastating and results in DMD. Loss of function occurs when mutations happen in the Dystrophin gene, resulting in a truncated Dystrophin protein that can no longer connect the cytoskeleton to the extracellular matrix. The end results are damaged and inflamed muscle fibers.
  • the Dystrophin gene is recessive and occurs on the X-chromosome so primarily affects males with symptoms starting as early as 2 to 5 years old with most patients. The average life expectancy is 26, while with excellent care some may live into 30-40 years of age. There is therefore a need for improved treatments for DMD.
  • an antisense oligonucleotide complementary or hybridisable to an exon of a human dystrophin pre-mRNA, wherein the antisense oligonucleotide comprises at least one locked nucleic acid (LNA), 2’-fluoro RNA (FRNA), 2’-0-alkyl sugar modified nucleotide, or 2’-0-alkoxy-alkyl sugar modified nucleotide.
  • LNA locked nucleic acid
  • FRNA 2’-fluoro RNA
  • 2’-0-alkyl sugar modified nucleotide or 2’-0-alkoxy-alkyl sugar modified nucleotide.
  • DMD Duchenne Muscular dystrophy
  • DMD Duchenne Muscular dystrophy
  • DMD Duchenne Muscular dystrophy
  • FIG. 1 shows a schematic illustration of induced exon skipping an antisense oligonucleotide (AON).
  • Fig. 2 shows structures of modified nucleotides used in the present disclosure.
  • Locked nucleic acid LNA
  • 2’-fluoro-arabinonucleic acid FANA
  • 2’-fluoro-ribonucleic acid FRNA
  • 2’-0- methyl modified RNA 2’OMe
  • 2’-0-(2-methoxyethyl) modified RNA nucleotide 2’-MOE.
  • B base
  • Fig. 4 shows gel electrophoresis results of antisense oligonucleotides from Table 2 tested for their ability to induce exon 51 skipping in KM 144 immortalized muscle cells differentiated into myotubes.
  • Fig. 5 shows a CD spectra of Exon 51 skipping antisense oligonucleotides from Table 2 hybridized to a target RNA sequence corresponding to segment of exon 51 RNA.
  • Fig. 6 shows gel electrophoresis results of antisense oligonucleotides from Table 2 tested for their ability to induce exon 53 skipping in KM 155 immortalized muscle cells that have been differentiated into mytotubes.
  • Fig. 7 shows CD spectra of Exon 53 skipping AONs hybridized to their complementary RNA oligonucleotide target; sequences are listed in Table 3. Most AONs show A-form helical character with a positive band at 270 nm and negative band at 210 nm.
  • Fig. 8 shows gel electrophoresis results of intramuscular administered antisense oligonucleotides tested for their ability to induce exon 53 skipping in vivo in in hDMD.del52 Mice.
  • Fig. 9 shows gel electrophoresis results of subcutaneous administered antisense oligonucleotides tested for their ability to induce exon 53 skipping in vivo in in hDMD.del52 Mice.
  • Fig. 10 shows quantitation of exon 53 skip levels in skeletal muscle and heart in hDMD.del52 Mice receiving weekly subcutaneous injections of 50 mg/kg for six weeks starting at 4 weeks of age.
  • Fig. 11 shows toxicity assessment of in vivo treatment of exon 53 antisense oligonucleotides (see Table 3) in hDMD.del52 mice.
  • ALP alkaline phosphatase
  • GET glutamic-oxaloacetic transaminase
  • GTT glutamic-pyruvic transaminase
  • Fig. 12 shows Exon 53 skipping efficiency of antisense oligonucleotides (AONs) delivered by gymnosis (no transfection reagent) at a concentration of 200 nM in KM155 immortalized muscle cells. Percentage Exon 53 skipping values were obtained from gel electrophoresis analysis of bands visualized after RT PCR (full length wild type: 436 bp; skipped band: 236 bp).
  • Treatment of DMD patients with mutations occurring in exon 51 include inducing exon skipping by selective binding of antisense oligonucleotides to exon 51 of dystrophin pre-mRNA, thus restoring the phase of the reading frame and enabling production of internally deleted but functional dystrophin (see Fig. 1).
  • Antisense-mediated exon skipping aims to restore the reading frame of DMD dystrophin transcripts on pre-mRNA level.
  • Antisense oligonucleotides (AONs) bind to specific exonic regions and prevent their inclusion in the mature mRNA through steric hinderance. Thus, exon skipping AONs can restore the reading frame and potentially allow the production of a BMD-like dystrophin.
  • Antisense oligonucleotides are short synthetic pieces of DNA or RNA, that are complementary to target mRNA.
  • AONs are short synthetic pieces of DNA or RNA, that are complementary to target mRNA.
  • FRNA 2'FRNA
  • 2'OMe 2'-0-methyl
  • FANA phosphorothioate
  • LNA locked nucleic acid
  • PMO phosphorodiamidate morpholino oligomer
  • exon skipping oligos For exon skipping oligos, the most clinically used chemistries have been 2OMe and PMO. There are four exon skipping therapeutics approved based on the PMO chemistry: eteplirsen (exon 51 skipping), golodirsen and viltolarsen (exon 53 skipping) and casimersen (exon 45 skipping). However, their approval is controversial as they only result in a small increase in dystrophin which may not be therapeutically relevant.
  • the present disclosure provides antisense exon skipping oligonucleotides with enhanced exon skipping capabilities utilizing novel combinations and arrangements of chemical modified nucleotides, potentially providing treatment for DMD.
  • the disclosure demonstrates that combinations of chemistries can play a pivotal role in the development of more efficient therapeutics for DMD by significantly enhancing the amount of skipped transcript.
  • antisense oligonucleotides having modified nucleotides have been developed to target exon 51 and/or exon 53 of the dystrophin gene.
  • the antisense oligonucleotides target exon 51.
  • the antisense oligonucleotides target exon 53.
  • the antisense oligonucleotides target exon 51 and/or exon 53 of human dystrophin pre-mRNA.
  • the antisense oligonucleotides are complementary to exon 51 and/or exon 53 of the dystrophin pre-mRNA.
  • the antisense oligonucleotides hybridize to exon 51 and/or exon 53 of the dystrophin pre-mRNA. “Hybridization” as used herein refers to hydrogen bonding between the nucleobases of complementary nucleotides. The degree of complementarity between an antisense oligonucleotide and its target sequence of the dystrophin pre-mRNA may be variable, and some embodiments the antisense oligonucleotide is exactly complementary to its target sequence.
  • antisense oligonucleotides are provided comprising synthetic pieces of DNA that are further modified as described herein. In other embodiments, antisense oligonucleotides are provided comprising synthetic pieces of RNA that are further modified as described herein. In yet other embodiments, antisense oligonucleotides are provided comprising synthetic pieces of both DNA and RNA that are further modified as described herein.
  • antisense oligonucleotides were synthesized having one or more modified nucleotides as shown in Fig. 2 and 3 and having phosphorothioate internucleotide linkages.
  • the nucleic acid modifications include, but are not limited to: Locked nucleic acid (LNA); 2’-fluoroarabinonucleicacid (FANA); 2’-fluoro RNA (FRNA); 2’-0-methyl RNA nucleotide (2’OMe); and 2’-0-(2-methoxyethyl) RNA (2’-MOE).
  • the LNA nucleotides were replaced with a-L-LNA nucleotides b- L-LNA is the L-stereoisomer (mirror image) variant of b-D-LNA (referred to simply as LNA), whereas a-L-LNA differs from b-L-LNA in the configuration of the N-glycosidic bond, being a (“down”) instead of b (“up”) (see Fig. 3).
  • an antisense oligonucleotide complementary or hybridisable to an exon of a human dystrophin pre-mRNA comprises at least one locked nucleic acid (LNA), 2’-fluoro RNA (FRNA), 2’-0-alkyl sugar modified nucleotide, or 2’-0-alkoxy-alkyl sugar modified nucleotide.
  • LNA locked nucleic acid
  • FRNA fluoro RNA
  • 2’-0-alkyl sugar modified nucleotide 2’-0-alkoxy-alkyl sugar modified nucleotide
  • an antisense oligonucleotide complementary or hybridisable to an exon of a human dystrophin pre-mRNA comprises at least one locked nucleic acid (LNA), 2’- fluoro RNA (FRNA), 2’-0-methyl RNA (2’OMe RNA or “2’OMe”), or 2’-0-methoxyethyl RNA (2’MOE RNA or “2’MOE”).
  • LNA locked nucleic acid
  • FRNA 2’- fluoro RNA
  • 2’-0-methyl RNA 2’OMe RNA or “2’OMe”
  • 2’MOE RNA or “2’MOE” 2’-0-methoxyethyl RNA
  • an antisense oligonucleotide complementary or hybridisable to an exon of a human dystrophin pre-mRNA consist of one or more of: locked nucleic acid (LNA), 2’-fluoro RNA (FRNA), 2’-0-methyl RNA (2’OMe RNA or “2’OMe”), and 2’-0- methoxyethyl RNA (2’MOE RNA or “2’MOE”).
  • LNA locked nucleic acid
  • FRNA 2’-fluoro RNA
  • 2’-0-methyl RNA 2’OMe RNA or “2’OMe”
  • 2’MOE RNA or “2’MOE” 2’-0- methoxyethyl RNA
  • antisense oligonucleotides are 10-50 nucleotides in length, 10-30 nucleotides in length, preferably 15-25 nucleotides in length, or more preferably about 20 nucleotides in length.
  • the antisense oligonucleotide is complementary or hybridisable to exon 53 of a human dystrophin pre-mRNA.
  • the antisense oligonucleotide comprises at most all LNA nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are LNA nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are LNA nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are LNA nucleotides. In one embodiment, the antisense oligonucleotide comprises 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides. In one embodiment, the antisense oligonucleotide comprises at most 4 LNA nucleotides.
  • the antisense oligonucleotide has all FRNA nucleotides. In one embodiment, the antisense oligonucleotide comprises at most all FRNA nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are FRNA nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are FRNA nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are FRNA. In one embodiment, the antisense oligonucleotide comprises more than 4 FRNA nucleotides.
  • the antisense oligonucleotide consists of a combination of LNA and FRNA nucleotides. In one embodiment, the antisense oligonucleotide consists of a combination of LNA and FRNA nucleotides. In one embodiment, the antisense oligonucleotide comprises 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides, and the remainder comprise one or more FRNA nucleotides (in one embodiment all remaining nucleotides are FRNA nucleotides).
  • the antisense oligonucleotide has a combination of LNA and FRNA nucleotides, and each LNA nucleotide is spaced apart by one or more FRNA nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of the FRNA nucleotides.
  • the antisense oligonucleotide comprises a sugar modified nucleotide having a modification at the 2’ position of the ribose sugar.
  • “2’-0-alkyl” refers to a sugar modified nucleotide having an alkyl group substitution at the 2’ position of the ribose sugar.
  • An “alkyl group” is a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom, having general formula: -C n Fh n +l .
  • the antisense oligonucleotide comprises one or more nucleotides having a 2’-0-methyl sugar modified nucleotide (2’OMe).
  • 2’-0-alkoxyl-alkyl refers to a sugar modified nucleotide having an alkoxy group substitution at the 2’ position of the ribose sugar, which in turn is further substituted with an alkyl group.
  • An “alkoxy group” is a functional group containing an alkyl group bonded to an oxygen atom, and having the general formula: R-O.
  • the antisense oligonucleotide comprises one or more nucleotides having a 2’-0-(2-methoxyethyl) sugar modified nucleotide (2’MOE).
  • the antisense oligonucleotide comprises at most all 2’OMe nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides.
  • the antisense oligonucleotide has all 2’MOE nucleotides. In one embodiment, the antisense oligonucleotide comprises at most all 2’MOE nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are 2’MOE nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are 2’MOE nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are 2’MOE.
  • the antisense oligonucleotide has 2’OMe nucleotides and 2’MOE nucleotides. [0041] In some embodiments, the antisense oligonucleotide comprises a combination of LNA nucleotides and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide comprises a combination of LNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl and 2’-0-(2-methoxyethyl)). In one embodiment, the antisense oligonucleotide consists of a combination of LNA nucleotides and 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each LNA nucleotide is spaced apart by the 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of the 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides.
  • the antisense oligonucleotide comprises a combination of FRNA nucleotides and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl). In some embodiments, the antisense oligonucleotide comprises a combination of FRNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl).
  • the antisense oligonucleotide consists of a combination of FRNA nucleotides and 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl).
  • each FRNA nucleotide is spaced apart by 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each FRNA nucleotide is spaced apart by 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide comprises a combination of LNA nucleotides, FRNA nucleotides, and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide comprises a combination of LNA nucleotides, FRNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide consists of a combination of LNA nucleotides, FRNA nucleotides, 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each pair of LNA and FRNA nucleotides is spaced apart by 1, 2, 3, 4, or 5 of 2’-0-alkyl or 2’-0-alkoxy- alkyl sugar modified nucleotides.
  • the antisense oligonucleotide targeting exon 53 of dystrophin pre- mRNA has a nucleotide base sequence as set forth in SEQ ID NO: 10, 11, 12, or 13 wherein at least one nucleotide is a modified nucleotide as described herein, and the rest of the nucleotides are DNA and/or RNA. Alternatively, all of the nucleotides are modified nucleotides as described herein.
  • the antisense oligonucleotide is complementary or hybridisable to exon 51 of a human dystrophin pre-mRNA.
  • the antisense oligonucleotide has all FRNA nucleotides. In one embodiment, the antisense oligonucleotide comprises at most all FRNA nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are FRNA nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are FRNA nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are FRNA. In one embodiment, the antisense oligonucleotide comprises more than 4 FRNA nucleotides.
  • the antisense oligonucleotide comprises a combination of LNA and FRNA nucleotides. In one embodiment, the antisense oligonucleotide consists of a combination of LNA and FRNA nucleotides. In one embodiment, the antisense oligonucleotide comprises 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides, and the reminder comprises one or more FRNA nucleotides (in one embodiment all remaining nucleotides are FRNA nucleotides). In one embodiment, the antisense oligonucleotide comprises at most 4 LNA nucleotides, and the reminder comprises one or more FRNA nucleotides (in one embodiment all remaining nucleotides are FRNA nucleotides).
  • the antisense oligonucleotide has a combination of LNA and FRNA nucleotides, and each LNA nucleotide is spaced apart by one or more FRNA nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of the FRNA nucleotides.
  • the antisense oligonucleotide comprises one or more aLNA nucleotide. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are aLNA nucleotides. In one embodiment, the antisense oligonucleotide comprises a combination of FRNA nucleotides and aLNA nucleotides. In one embodiment, the antisense oligonucleotide consists of a combination of FRNA nucleotides and aLNA nucleotides.
  • the antisense oligonucleotide has a combination of FRNA nucleotides and aLNA nucleotides and the aLNA nucleotide is spaced apart by the FRNA nucleotides. In one embodiment, each aLNA nucleotide is spaced apart by 2, 3, 4, or 5 of the FRNA nucleotides.
  • the antisense oligonucleotide has one or more 2’-0-alkyl or 2’-0- alkoxy-alkyl sugar modified nucleotide. In one embodiment, the antisense oligonucleotide comprises one or more nucleotides having a 2’-0-methyl sugar modified nucleotide. In one embodiment, the antisense oligonucleotide comprises one or more nucleotides having a 2’-0-(2- methoxyethyl) sugar modified nucleotide.
  • the antisense oligonucleotide comprises at most all 2’OMe nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are 2’OMe nucleotides.
  • the antisense oligonucleotide has all 2’MOE nucleotides. In one embodiment, the antisense oligonucleotide comprises at most all 2’MOE nucleotides. In one embodiment, half of the nucleotides of the antisense oligonucleotide are 2’MOE nucleotides. In one embodiment, a majority of the nucleotides of the antisense oligonucleotide are 2’MOE nucleotides. In one embodiment, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the nucleotides of the antisense oligonucleotide are 2’MOE.
  • the antisense oligonucleotide has 2’OMe nucleotides and 2’MOE nucleotides.
  • the antisense oligonucleotide comprises a combination of LNA nucleotides and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)). In some embodiments, the antisense oligonucleotide comprises a combination of LNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl and 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide consists of a combination of LNA nucleotides and 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each LNA nucleotide is spaced apart by the 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides.
  • each LNA nucleotide is spaced apart by 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides.
  • each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of the 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides. In one embodiment, each LNA nucleotide is spaced apart by 2, 3, 4, or 5 of 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides.
  • the antisense oligonucleotide comprises a combination of FRNA nucleotides and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl). In some embodiments, the antisense oligonucleotide comprises a combination of FRNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl).
  • the antisense oligonucleotide consists of a combination of FRNA nucleotides and 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-methoxy-ethyl).
  • each FRNA nucleotide is spaced apart by 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each FRNA nucleotide is spaced apart by 2’-0-alkyl sugar modified nucleotides and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide comprises a combination of LNA nucleotides, FRNA nucleotides, and 2’-0-alkyl or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide comprises a combination of LNA nucleotides, FRNA nucleotides, 2’-0-alkyl sugar modified nucleotides, and 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • the antisense oligonucleotide consists of a combination of LNA nucleotides, FRNA nucleotides, 2’-0-alkyl and/or 2’-0-alkoxy-alkyl sugar modified nucleotides (such as 2’-0-methyl or 2’-0-(2-methoxyethyl)).
  • each pair of LNA and FRNA nucleotides is spaced apart by 1 , 2, 3, 4, or 5 of 2’-0-alkyl or 2’-0-alkoxy- alkyl sugar modified nucleotides.
  • the antisense oligonucleotide targeting exon 51 of dystrophin pre- mRNA has a nucleotide base sequence as set forth in SEQ ID NOs: 5, 6, 7, 8, or 9, wherein at least one nucleotide is a modified nucleotide as described herein, and the rest of the nucleotides are DNA and/or RNA. Alternatively, all of the nucleotides are modified nucleotides as described herein.
  • antisense oligonucleotides described herein are intended for modulating splicing of a dystrophin pre-mRNA.
  • modulating splicing of a dystrophin pre-mRNA in a cell comprises contacting the cell with the antisense oligonucleotide described herein.
  • antisense oligonucleotides described herein are intended for treatment of Duchenne Muscular dystrophy (DMD) caused by mutations in the dystrophin gene resulting in a truncated Dystrophin protein.
  • treating Duchenne Muscular dystrophy comprises administering an effective amount of the antisense oligonucleotide described herein to a patient.
  • the antisense oligonucleotide described herein is used in the treatment of Duchenne Muscular dystrophy.
  • Differentiation medium consists of Dulbecco’s modified Eagle’s medium (Gibco), supplemented with 5% horse serum (Gibco), 2% GlutaMax, 0.8% glucose (Sigma-Aldrich).
  • Oligonucleotide synthesis Antisense oligonucleotides were synthesized at 1 pmol scale using either a Dr Oligo 48 (Biolytic, Fremont, CA) or Applied Biosystems synthesizer. Oligonucleotides were synthesized on a Unylinker (500 A) support using 3’-phosphoramidite derivatives of 2'-fluoro-ribonucleosides, 2'-fluoro-arabinonucleosides, 2'-methoxyethyl- ribonucleosides, 2'-0-methyl-ribonucleodies, and 2'-4'- locked-ribonucleosides, with standard protecting groups (ChemGenes).
  • Crude oligonucleotides were purified by anion exchange HPLC on an Agilent 1200 Series Instrument using a Protein-Pak DEAE 5PW column (7.5 c 75 mm) at a flow rate of 1 mL/min. The gradient was 0-24% solution 1M UCIO 4 over 30 min at 60°C. Samples were desalted either on NAP-25 desalting columns (using manufacturers protocol) or on the Agilent 1200 HPLC with a Sephadex column.
  • Antisense oligonucleotide transfection and RNA isolation Antisense oligonucleotides were transfected using lipofectamine following the manufacturer’s instructions at a concentration of 200 nM. Medium was replaced after 4 hours. In some instances, antisense oligonucleotides were delivered in the absence of transfecting agents (gymnosis, or gymnotic delivery). In the latter case, antisense oligonucleotides were added to the medium in a 200-1000 nM concentration and not replaced until RNA was isolated.
  • DEPC diethyl pyrocarbonate
  • mice Mouse model fortesting in vivo efficacy of antisense oligonucleotides. These experiments were carry out on a humanised DMD mouse model with a deletion of exon 52 of the dystrophin gene (hDMD/del52 mice). AONs were first injected intramuscularly using a 20-pg dose in 40 microliters, at day 1 and day 2. The injections were performed in both the left and right triceps and gastrocnemius, on three hDMDdel52/mcfx mice. Mice were sacrified on day 12 and exon skipping levels were determined using PCR, and gel electrophoresis. AONs were also tested in the context of weekly subcutaneous injections, administering 50 mg/kg of AON once a week for 6 weeks. Mice were sacrified on week 7, and the exon skipping levels were determined using PCR, and gel electrophoresis.
  • nested PCRs were performed using primers h50F and hex55Rin the first PCR using 3 mI cDNA, 0.5 mI of 10 mM dNTPs, 1 mI of 10 pmol/mI of each primer, 2.5 mI of 10 times supertaq buffer, and 0.125 mI of 5 U/mI Taq Polymerase and DAPC treated water in a total volume of 25 mI for each sample.
  • a PCR was performed using the following program: 5 minutes at 94°C, 25 cycles of 40 seconds at 94°C, 40 seconds at 60°C and 180 seconds at 72°C, 7 minutes at 72°C.
  • the second PCR was conducted with h52f and h54r.
  • 1.5 mI PCR product was added to 1 mI of 10 mM dNTPS, 2 mI of 10 pmol/ mI of each primer, 5 mI of supertaq buffer and 0.25 mI of 5 U/ mI Taq Polymerase and DAPC treated water in a total volume of 50 mI for each sample.
  • a PCR was performed using the following program: 5 minutes at 94°C, 35 cycles of 40 seconds at 94°C, 40 seconds at 60°C and 60 seconds at 72°C, 7 minutes at 72°C.
  • PCR fragments were loaded on a 2% agarose gel in TBE buffer to assess exon skipping.
  • AONs antisense oligonucleotides
  • Phosphorothioate AONs combining FANA with either LNA or a-L-LNA in the “1-2-1” alternating motif exhibited excellent binding to RNA targets as assessed by UV melting experiments (Table 2). The same was true for AONs combining FANA with both LNA and FRNA.
  • Thermal stability measurements revealed that oligonucleotides combining a-L-LNA and FANA (both with South sugar pucker) exhibited superior binding affinity towards RNA targets relative to oligomers combining LNA (north sugar pucker) and FANA (South sugar pucker) (Table 2).
  • LNA North sugar pucker mixes well with FRNA (North sugar pucker) and less so with a-L-LNA (South sugar pucker).
  • a-L-LNA increases binding affinity to target RNA by up to +5°C per modification, similar to LNA.
  • a-L-LNA provides similar increases in thermal stability as LNA when hybridized to RNA as was the case when combining FANA and FRNA nucleotides.
  • Table 2 Exon skipping antisense oligonucleotides targeting either exon51 (Ex51) or exon53 (Ex53).
  • RNA complement for Ex51 series 5’-AGCGACGAAGAG-3’ (SEQ ID NO: 14).
  • RNA Complement for Ex53 series 5-CAGAACCGGAGGCAACAG-3' (SEQ ID NO: 15) measure Tm values measured in 5 mM sodium phosphate buffer, 140 mM KCI, 1 mM MgCl2 (pH 7.4).
  • Circular dichroism (CD) data was obtained for AONs targeting exon 51 , where duplex concentration is 25 mM in 5 mM sodium phosphate buffer, 140 mM KCI, 1 mM MgCL at pH 7.4.
  • RNA Target 5'-AUCAAGUUAUAAAAUCACAGAGG-3' (SEQ ID NO: 16).
  • CD profiles of FANA containing AONs hybridized to RNA e.g., Ex51FANA, Ex51LNAFANA, Ex51G2B and Ex51G2A were characteristic of A/B helices, all exhibiting negative bands at 210 nm and 245 nm and a broad positive band at 260-280 n (Fig. 5).
  • AONs that promoted the desired exon51 skipping also promoted the formation of other truncated products; the exception was the 2'-0-Me RNA AON. These additional products likely originate from cryptic splice sites (CSS) - that is, regions of the mRNA that can interact with spliceosomal factors but that usually are not spliced. Additional Exon skipping antisense oligonucleotides targeting exon51 are provided in Table 3.
  • LNA represented as “NL”
  • FRNA represented as “NF”
  • N2 2’-OMe
  • NM 2’-MOE
  • any AON containing FANA produced either no (Ex53 FANA, Ex53LNAFANA and Ex53 aLNAFANA) or minimal exon skipping (Ex53G2A and Ex53G2B), likely due to subtle differences in the helical structure (e.g. groove widths) that results when AON is bound to the target RNA, preventing recruitment of splicing factors (e.g., ILF2/3) and resulting in exon 53 inclusion.
  • CD experiments were performed for AON/RNA duplexes (25 mM) in 5 mM sodium phosphate buffer, 140 M KOI, 1 M MgCI 2 at pH 7.4.
  • a synthetic RNA namely 5-CAGAACCGGAGGCAACAG- 3' (SEQ ID NO: 15) was used as target to assess the global helical characters of the AON: RNA hybrids RNA (Fig. 7).
  • FANA-containing AONs show classic CD signatures of A/B duplex formation with two negative bands at 210 nm and 240 nm and positive bands at 270 nm (Fig. 7).
  • the non-FANA containing AONs all exhibit A-form helical structures with positive and negative bands at 270 n and 210 n (Fig. 7).
  • Fig. 6 the exon skipping efficiencies
  • LNA represented as “NL”
  • FRNA represented as “NF”
  • N2 2’-OMe
  • Intramuscular injections For hDMD/del52 mouse strains administered with intramuscular injections of the antisense oligonucleotides, the intramuscular injections (20 pg in 40mI) were administered on day 1 and 2 in the triceps and gastrocnemius and sacrificed on day 12. The gel electrophoresis results, and quantitation of skip levels are shown in Fig. 8. Both the LNA-2'OMe and LNA-FRNA AONs clearly show higher levels of the skipped transcript (581 bp).
  • the LNA-2’OMe and LNA-FRNA modified AONs were the most efficient (70.9 ⁇ 10.7% and 86.5 ⁇ 7.3% skip, respectively) with comparable levels in triceps, diaphragm, and heart.
  • Table 5 provides another series of oligonucleotides we have prepared to target exon 53. This time the oligonucleotides contain combinations of a) LNA, FRNA and 2’OMe modifications; b) LNA, MOE, 2’OMe modifications; c) full MOE modifications; and d) LNA, FRNA and MOE modifications.
  • Fig. 12 shows Exon 53 skipping efficiency of antisense oligonucleotides (AONs) delivered by gymnosis (no transfection reagent) at a concentration of 200 nM in KM 155 immortalized muscle cells.
  • AONs antisense oligonucleotides
  • AON 10 is a chimera containing LNA, FRNA and 2’OMe modifications, performing as well as AON2 which exhibited pronounced exon 53 skipping levels in mice (Fig. 10).
  • AONs such as AON10 and others disclosed in this invention represents exciting oligonucleotide candidates for treating DMD.
  • Table 5 Additional Exon skipping antisense oligonucleotides targeting exon 53.
  • LNA represented as “NL”
  • FRNA represented as “NF”
  • N2 2’-OMe
  • NM 2’-MOE
  • Dystrophin The protein product of the duchenne muscular dystrophy locus. Cell 1987, 51 (6), 919-928.

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Abstract

L'invention concerne des oligonucléotides antisens modifiés pour induire un saut d'exon par ciblage de pré-ARNm de la dystrophine. Les oligonucléotides antisens sont utiles dans le traitement de la dystrophie musculaire de Duchenne.
PCT/CA2022/050587 2021-04-14 2022-04-14 Oligonucléotides modifiés pour le traitement de la dystrophie musculaire de duchenne WO2022217366A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190194656A1 (en) * 2012-01-27 2019-06-27 Biomarin Technologies B.V. RNA Modulating Oligonucleotides with Improved Characteristics for the Treatment of Duchenne and Becker Muscular Dystrophy
EP3760720A1 (fr) * 2013-03-14 2021-01-06 Sarepta Therapeutics, Inc. Compositions de saut d'exons pour le traitement de la dystrophie musculaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190194656A1 (en) * 2012-01-27 2019-06-27 Biomarin Technologies B.V. RNA Modulating Oligonucleotides with Improved Characteristics for the Treatment of Duchenne and Becker Muscular Dystrophy
EP3760720A1 (fr) * 2013-03-14 2021-01-06 Sarepta Therapeutics, Inc. Compositions de saut d'exons pour le traitement de la dystrophie musculaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN SUXIANG, LE BAO T., CHAKRAVARTHY MADHURI, KOSBAR TAMER R., VEEDU RAKESH N.: "Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro", SCIENTIFIC REPORTS, vol. 9, no. 1, 15 April 2019 (2019-04-15), pages 1 - 10, XP055982613, ISSN: 2045-2322 *
D4 SYED: "Eteplirsen: first global approval", DRUGS, vol. 76, 11 February 2016 (2016-02-11), pages 1699 - 1704, XP009510421, ISSN: 1179-1950, DOI: 10.1007/s40265-016-0657-1 *
WATANABE NAOKI, NAGATA TETSUYA, SATOU YOUHEI, MASUDA SATORU, SAITO TAKASHI, KITAGAWA HIDETOSHI, KOMAKI HIROFUMI, TAKAGAKI KAZUCHIK: "NS-065/NCNP-01: An antisense oligonucleotide for potential treatment of exon 53 skipping in Duchenne muscular dystrophy", MOLECULAR THERAPY: NUCLEIC ACIDS, vol. 13, 13 December 2018 (2018-12-13), pages 442 - 449, XP055982619, ISSN: 2162-2531 *

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