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WO2025155722A1 - Associations régulatrices hybrides sélectives musculaires et procédés d'utilisation associés pour le traitement de la dystrophie myotonique de type 1 - Google Patents

Associations régulatrices hybrides sélectives musculaires et procédés d'utilisation associés pour le traitement de la dystrophie myotonique de type 1

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WO2025155722A1
WO2025155722A1 PCT/US2025/011882 US2025011882W WO2025155722A1 WO 2025155722 A1 WO2025155722 A1 WO 2025155722A1 US 2025011882 W US2025011882 W US 2025011882W WO 2025155722 A1 WO2025155722 A1 WO 2025155722A1
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seq
sequence
nucleic acid
acid sequence
puf
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Gregory Thomas NACHTRAB
Ranjan BATRA
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Astellas Gene Therapies Inc
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Astellas Gene Therapies Inc
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the disclosure is directed to molecular biology, gene therapy, and compositions and methods for modifying expression and activity of RNA molecules.
  • novel promoter combinations are disclosed herein for fine-tuning muscle specific gene therapies.
  • the novel promoter combinations disclosed herein can be used in AAV-based PUF compositions comprising such promoter combinations for treating DM1. These AAV-based PUF compositions are also provided herein.
  • the present disclosure provides recombinant adeno-associate viral (rAAV) vectors comprising: a) first inverted terminal repeat (ITR) sequence comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 32; b) an MYEPF enhancer sequence comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1; c) a CK6 promoter sequence comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 8; d) an SV40 intron sequence comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 35; e) a nucleic acid sequence encoding a PUF polypeptide, wherein the PUF polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 135; f) a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence comprising a nucleic acid sequence at
  • the rAAV vectors of the present disclosure comprise: a) a first inverted terminal repeat (ITR) sequence comprising a nucleic acid sequence at least 99% identical to SEQ ID NO: 32; b) an MYLPF enhancer sequence comprising a nucleic acid sequence at least 99% identical to SEQ ID NO: 1; c) a CK6 promoter sequence comprising a nucleic acid sequence at least 99% identical to SEQ ID NO: 8; d) an SV40 intron sequence comprising a nucleic acid sequence at least 99% identical to SEQ ID NO: 35; e) a nucleic acid sequence encoding a PUF polypeptide, wherein the PUF polypeptide comprises an amino acid sequence at least 99% identical to SEQ ID NO: 135; f) a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence comprising a nucleic acid sequence at least 99% identical to SEQ ID NO:
  • ITR inverted terminal
  • the rAAV vectors comprise a nucleic acid sequence at least 95%, preferably at least 99%, identical to the nucleic acid sequence set forth in SEQ ID NO: 47, SEQ ID NO: 139, or SEQ ID NO: 150.
  • the AAV capsid protein is a myoAAV capsid protein.
  • the AAV capsid protein comprises an amino acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 54, preferably wherein the AAV capsid protein is encoded by a nucleic acid sequence that is at least 95%, preferably at least 99%, identical to SEQ ID NO: 53.
  • the present disclosure provides nucleic acid molecules comprising a skeletal muscle selective hybrid regulatory sequence, wherein the hybrid regulatory sequence comprises: a) a myosin light chain, phosphorylatable, fast skeletal muscle gene (MYLPF) enhancer sequence or a desmin enhancer sequence, wherein the MYLPF enhancer sequence comprises a nucleic acid sequence at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein the desmin enhancer sequence comprises a nucleic acid sequence at least 95%, preferably at least 99%, identical any one of SEQ ID NO: 12 and SEQ ID NO: 14; and b) a creatine kinase (CK) promoter sequence or a desmin promoter sequence, wherein the CK promoter sequence comprises a nucleic acid sequence at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ
  • the hybrid regulatory sequences of the present disclosure comprises a nucleic acid sequence at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.
  • the hybrid regulatory sequences of the present disclosure further comprise an intron sequence.
  • the intron sequence is an SV40 (simian virus 40) intron sequence or an SIE (short intronic enhancer).
  • the intron sequence comprises a nucleic acid sequence at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 35.
  • the skeletal muscle selective hybrid regulatory sequences of the present disclosure comprise: a MYLPF enhancer sequence comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 1; a CK6 promoter sequence comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 8;
  • the nucleic acid molecules of the present disclosure comprise a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 137.
  • the skeletal muscle selective hybrid regulatory sequences of the present disclosure comprise: a MYLPF enhancer sequence comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 2; a CK7 promoter sequence comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 10; an SV40 intron comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 7; and a kozak sequence comprising a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 11.
  • the nucleic acid molecules of the present disclosure comprise a nucleic acid sequence at least 95%, preferably at least 99%, identical to SEQ ID NO: 138.
  • the hybrid regulatory sequences of the present disclosure are in operable linkage with a nucleic acid sequence encoding a PUF polypeptide.
  • the PUF polypeptide binds a CUG-repeat RNA sequence.
  • the CUG-repeat RNA sequence is & DMPK CUG-repeat sequence.
  • the PUF polypeptides of the present disclosure comprise an amino acid sequence at least 95%, preferably at least 99%, identical to the amino acid sequence of any one of SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.
  • the PUF polypeptides of the present disclosure are encoded by a nucleic acid sequence that is at least 95%, preferably at least 99%, identical to any one of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • the present disclosure provides rAAV vectors comprising any of the nucleic acid molecules of the present disclosure.
  • the present disclosure provides pharmaceutical composition comprising any of the rAAV vectors of the present disclosure or any of the nucleic acid molecules of the present disclosure.
  • the present disclosure provides a method of treating myotonic dystrophy type 1 (DM1) in a subject, the method comprising administering to the subject a pharmaceutical composition of the present disclosure.
  • DM1 myotonic dystrophy type 1
  • a subject has CUG microsatellite repeat expansion (MRE) in a DMPK RNA sequence.
  • MRE microsatellite repeat expansion
  • a cell comprises the AAV vector.
  • AAV capsid comprising LBV201 of SEQ ID NO: 54.
  • RNA splicing from the human patient-derived cardiomyocytes was analyzed across 5 different transcripts.
  • the upper panels show the splicing pattern for the given transcript and the bottom is the quantification.
  • the hybrid regulatory sequences comprised of the Mod hDesmin-SIE promoters exhibit a much more potent splicing correction compared to the hDesmin FL PUF(CUG) construct A02239.
  • Hybrid regulatory sequences of the present disclosure can comprise at least one CK6 enhancer/promoter sequence.
  • a CK6 enhancer/promoter sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%), 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 8.
  • Hybrid regulatory sequences of the present disclosure can comprise at least one CK7 enhancer/promoter sequence.
  • a CK7 enhancer/promoter sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 10.
  • the hybrid regulatory sequence comprise a MYLPF enhancer, a CK enhancer/promoter sequence, and an intronic sequence.
  • the hybrid regulatory sequence comprises a MYLPF enhancer sequence, a CK6 enhancerpromoter sequence, and an SV40 intron sequence.
  • Hybrid regulatory sequences comprising a truncated desmin promoter combined with a SIE (small intronic enhancer) are disclosed herein.
  • the truncated desmin promoter is derived from the human sequence by deleting poorly conserved regions.
  • the SIE was derived from primarily the first intron of the mouse muscle creatine kinase gene Piekarowicz et al. Mol Ther Methods Clin Dev. 15 (2019).
  • the hybrid regulatory sequence comprises a MYLPF enhancer sequence, a truncated desmin promoter sequence, and a SIE sequence.
  • a hybrid regulatory sequence can comprise at least one hMYLPF enhancer sequence, at least one CK6 enhancer/promoter sequence, at least one SV40 intron sequence, and at least one Kozak sequence. In some embodiments, a hybrid regulatory sequence can comprise, from 5’ to 3’, at least one hMYLPF enhancer sequence, at least one CK6 enhancer/promoter sequence, at least one SV40 intron sequence, and at least one Kozak sequence.
  • a hybrid regulatory sequence of the disclosure comprises a MYLPF enhancer sequence.
  • a skeletal muscle selective hybrid regulatory sequence comprises a MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle gene) enhancer sequence set forth in SEQ ID NO: 2, a CK6 (muscle creatine kinase 6) enhancer-promoter sequence set forth in SEQ ID NO: 8, an SV40 intron set forth in SEQ ID NO: 7, and a kozak sequence set forth in SEQ ID NO: 11.
  • a skeletal muscle selective hybrid regulatory sequence comprises a MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle gene) enhancer sequence set forth in SEQ ID NO: 2, a CK1 (muscle creatine kinase 1) enhancerpromoter sequence set forth in SEQ ID NO: 9, an SV40 intron set forth in SEQ ID NO: 7, and a kozak sequence set forth in SEQ ID NO: 11.
  • MYLPF myosin light chain, phosphorylatable, fast skeletal muscle gene
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 137.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 138.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 19.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 20.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 21.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 23.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 24.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 25.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 26.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 27.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 28.
  • a hybrid regulatory sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 29.
  • RNA-targeting gene therapy compositions and methods for treating myotonic dystrophy type 1 are provided here.
  • Such compositions comprise hybrid regulatory promoter-enhancer-intron combinations , hybrid regulatory sequences of the disclosure, which drive selective and preferential expression of transgenes, including CUG-repeat targeting PUF polypeptides in skeletal muscle cells over cardiac muscle cells.
  • the disclosure provides a recombinant AAV (rAAV) vector comprising a hybrid regulatory sequence of the disclosure operably linked to a transgene.
  • the disclosure also provides an rAAV vector comprising a hybrid regulatory sequence of the disclosure operably linked to a CUG-repeat RNA targeting PUF polypeptide.
  • the disclosure provides an rAAV vector comprising a first inverted terminal repeat (ITR) sequence; a hybrid regulatory sequence of the disclosure; a nucleic acid sequence encoding a transgene; and a second ITR sequence.
  • ITR inverted terminal repeat
  • the disclosure provides an rAAV vector comprising a first inverted terminal repeat (ITR) sequence; a hybrid regulatory sequence of the disclosure; a nucleic acid sequence encoding a PUF polypeptide; a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence; an SV40 polyA sequence; and a second ITR sequence.
  • ITR inverted terminal repeat
  • WPRE Woodchuck Hepatitis Virus
  • the disclosure provides an rAAV vector comprising a first inverted terminal repeat (ITR) sequence; an MYLPF enhancer sequence; a CK6 promoter sequence; an SV40 intron sequence; a nucleic acid sequence encoding a PUF polypeptide; a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence; an SV40 polyA sequence; and a second ITR sequence.
  • ITR inverted terminal repeat
  • MYLPF enhancer sequence e.g., MYLPF enhancer sequence
  • CK6 promoter sequence e.g., CK6 promoter sequence
  • an SV40 intron sequence e.g., a nucleic acid sequence encoding a PUF polypeptide
  • WPRE Woodchuck Hepatitis Virus
  • the first ITR sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 32.
  • the second ITR sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 34.
  • the MYLPF enhancer sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
  • the CK6 promoter sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 8.
  • the PUF polypeptide sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 45.
  • the WPRE sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 38.
  • the SV40 polyA sequence comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 39.
  • an rAAV vector comprising a CUG repeat targeting PUF operably linked to a hybrid regulatory sequence is set forth in Table 2.
  • the vector, A05288 comprises from 5’ to 3’ the elements set forth in Table 2.
  • Table 2 ssAAV MYLPF CK6 SV40intron PUFCUG_WPRE SV40pA A05288 elements from 5’ to 3’
  • rAAV vector A05288 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical: cctgcaggcagctgcgcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggccaactccatcactaggggttcctGCGGCCGGTCGCGTCTAGTACTAGTT TCTGAGTCCTCTAAGGTCCCTCACTCCCAACTCAGCCCCATGTCCTGTCAATTCCC ACTCAGTGTCTGATCTCCTTCTCCTCACCTTTCCCATCTCCCGTTTGACCCAGCTT
  • an rAAV vector of the present disclosure can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 150.
  • an rAAV vector comprising a CUG repeat targeting PUF operably linked to a hybrid regulatory sequence is set forth in Table 3.
  • the vector, A02239 comprises from 5’ to 3’ the elements set forth in Table 3.
  • rAAV vector A02239 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to: cctgcaggcagctgcgcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggccaactccatcactaggggttcctGCGGCCGGTCGCGTCTAGTACTAGTta cccctgcccccacagctcctctctctctctct
  • Table 4 scAAV short hDesmin SIE intron PUFCUG_WPRE3 SV40pA A04949 elements from 5’ to 3’
  • rAAV vector A04949 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
  • rAAV vector A04959 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
  • AAV vectors of the disclosure can comprise a fusion protein comprising one or more elements of the disclosure, including, but not limited to, a CUG- repeat targeting protein (such as a Cas, PUF, or PUMBY) and an endonuclease.
  • fusion proteins of the AAV vector can further comprise a linker amino acid sequence between the one or more elements of the disclosure.
  • a LBV201 capsid comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to:
  • a nucleic acid sequence encoding a MyoAAV4 capsid comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to: atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttgaaacctggagcccc tcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgcttccgggttacaaataccttggacccggcaacggactc gacaagggggagccggtcaacgcagcagacgcggcggccctcgagcacgagcacagacgcgg
  • a RepCap plasmid encoding LBV201 comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO: 149.
  • a MyoAAV4 capsid comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to:
  • a nucleic acid sequence encoding an AAV9 capsid comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to: atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttgaaacctggagcccc tcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgtgcttccgggttacaaataccttggacccggcaacggactc gacaagggggagccggtcaacgcagcagacgcggcggccctcgagcaaggaggactc gacaaggggg
  • the NLS comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the NLS comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO: 60.
  • a linker sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence set forth in any one of SEQ ID NO: 70 - SEQ ID NO: 109.
  • CUG targeting compositions of the disclosure comprise a hybrid regulatory sequence or combination of sequences disclosed herein.
  • CUG targeting compositions comprise a muscle-specific hybrid regulatory combination which is more selective for skeletal muscle than cardiac muscle in a manner such that there is a higher level of expression in skeletal muscle than in cardiac muscle.
  • the CUG targeting compositions comprise a muscle-specific hybrid regulatory combination which provides equally or balanced levels of expression for skeletal muscle and cardiac muscle.
  • RNA recognition mode of PUF proteins (named for Drosophila Pumilio and C. elegans fem-3 binding factor) that are involved in mediating mRNA stability and translation are well known in the art.
  • the PUF domain of human Pumiliol also known in the art, binds tightly to cognate RNA sequences and its specificity can be modified. It contains eight PUF modules that recognize eight consecutive RNA bases with each module recognizing a single base. Since two amino acid side chains in each module recognize the Watson-Crick edge of the corresponding base and determine the specificity of that module, a PUF protein can be designed to specifically bind most 8 to 16-nt RNA. Wang et al., Nat Methods. 2009; 6(11): 825-830. See also WO2012/068627 which is incorporated by reference herein in its entirety.
  • PumHD is a modified version of the WT Pumilio protein that exhibits programmable binding to arbitrary 8-base sequences of RNA.
  • Each of the eight units of PumHD can bind to all four RNA bases, and the RNA bases flanking the target sequence do not affect binding. See also the following for art-recognized RNA-binding rules of PUF design: Filipovska A, Razif MF, Nygard KK, & Rackham O. A universal code for RNA recognition by PUF proteins.
  • the PUF design may maintain amino acid 13 as human PUMl’s native residue.
  • amino acid 13 for stacking
  • H amino acid 13
  • Y amino acid 13
  • stacking residues may be modified to improve binding and specificity. Recognition occurs in reverse orientation as N- to C-terminal PUF recognizes 3’ to 5’ RNA. Accordingly, PUF engineering of 8 modules (8PUF), as known in the art, mimics a human protein.
  • An exemplary 8-mer RNA recognition (8PUF) would be designed as follows: R1’-R1-R2-R3-R4-R5-R6-R7-R8-R8’.
  • an 8PUF is used as the RBD.
  • a variation of the 8PUF design is used to create a 14-mer RNA recognition (14PUF) RBD, 15-mer RNA recognition (15PUF) RBD, or a 16- mer RNA recognition (16PUF) RBD.
  • the PUF can be engineered to comprise a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14- mer, 15-mer, 16-mer, 24-mer, 30-mer, 36-mer, or any number of modules between. Shinoda et al., 2018; Criscuolo et al., 2020. Repeats 1-8 of wild type human PUM1 are provided herewith at SEQ ID NOS: 110-117, respectively.
  • the nucleic acid sequence encoding the PUF domain from human PUM1 is SEQ ID NO: 118 and the amino acid sequence of the PUF domain from human PUM1 amino acids 828-1176 is SEQ ID NO: 119. See also US Patent 9,580,714 which is incorporated herein in its entirety.
  • a CUG repeat targeting PUF sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to in SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135.
  • a PUF or PUMBY RNA-binding sequence is capable of binding a toxic target CUG repeat sequence comprising UGCUGCUG (SEQ ID NO: 120).
  • the target RNA sequence is selected from the group consisting of UGCUGCUGCUGCUG (SEQ ID NO: 121), UGCUGCUGCUGCUGC (SEQ ID NO: 122), and UGCUGCUGCUGCUGCU (SEQ ID NO: 123).
  • the target RNA sequence is selected from the group consisting of CUGCUGCU (SEQ ID NO: 124), CUGCUGCUGCUGCU (SEQ ID NO: 125), CUGCUGCUGCUGCUG (SEQ ID NO: 126), and CUGCUGCUGCUGCUGC (SEQ ID NO: 127).
  • the target RNA sequence is selected from the group consisting of GCUGCUGC (SEQ ID NO: 128), GCUGCUGCUGCUGC (SEQ ID NO: 129), GCUGCUGCUGCUGCU (SEQ ID NO: 130), and GCUGCUGCUGCUGCUG (SEQ ID NO: 131).
  • RNA-binding protein or RNA-binding portion thereof is a PUMBY (Pumilio-based assembly) protein.
  • RNA-binding protein PumHD which has been widely used in native and modified form for targeting RNA, has been engineered into a protein architecture designed to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (ie., Pumby, for Pumilio-based assembly) are concatenated in chains of varying composition and length, to bind desired target RNAs.
  • PUMBY is a more simple and modular form of PumHD, in which a single protein unit of PumHD is concatenated into arrays of arbitrary size and binding sequence specificity.
  • the first RNA binding protein comprises a Pumilio and FBF (PUF) protein.
  • the first RNA binding protein comprises a Pumilio-based assembly (PUMBY) protein.
  • the fusion protein disclosed herein comprises a linker between the at least two RNA-binding polypeptides.
  • the linker is a peptide linker.
  • the linker is VDTANGS (SEQ ID NO: 71).
  • the peptide linker comprises one or more repeats of the tri-peptide GGS. In other embodiments, the linker is a non-peptide linker.
  • the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co- poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • POE polyoxyethylene
  • polyurethane polyphosphazene
  • polysaccharides dextran
  • polyvinyl alcohol polyvinylpyrrolidones
  • polyvinyl ethyl ether polyacryl amide
  • polyacrylate polycyanoacrylates
  • lipid polymers chitins, hyaluronic acid
  • nucleic acid sequences encoding PUF proteins of the disclosure are codon optimized nucleic acid sequences.
  • the codon optimized sequence encoding a PUF protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased expression in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • nucleic acid sequences encoding PUF proteins of the disclosure are codon optimized nucleic acid sequences.
  • the codon optimized sequence encoding a PUF protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased translation in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • Embodiment 28 The nucleic acid molecule of embodiment 24, wherein the intron is a SV40 intron or an SIE intron set forth in any one of SEQ ID NO; 5, SEQ ID NO: 6, SEQ ID NO: /, or SEQ ID NO: 35.
  • Embodiment 29 The nucleic acid molecule of embodiment 24, wherein the muscle selective hybrid regulatory sequence is cardiac muscle selective.
  • Embodiment 31 An AAV capsid comprising I..BV201 comprising an amino acid sequence set forth in SEQ ID NO: 54.
  • Embodiment 32 A nucleic acid molecule comprising a skeletal muscle selective hybrid regulatory sequence, wherein the hybrid regulatory sequence comprises a MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle gene) enhancer sequence set forth in SEQ ID NO: 2, a CK6 (muscle creatine kinase 6) enhancer-promoter sequence set forth in SEQ ID NO: 8, an SV40 intron set forth in SEQ ID NO: 7, and a kozak sequence set forth in SEQ ID NO: 11.
  • Embodiment 33 The nucleic acid molecule of' embodiment 32, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 136.
  • Embodiment 34 A nucleic acid molecule comprising a skeletal muscle selective hybrid regulatory sequence, wherein the hybrid regulatory sequence comprises a MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle gene) enhancer sequence set forth in SEQ ID NO: 2, a CK1 (muscle creatine kinase 1 ) enhancer-promoter sequence set forth in SEQ ID NO: 9, an SV40 intron set forth in SEQ ID NO: 7 and a kozak sequence set forth in SEQ ID NO: 1 1.
  • MYLPF myosin light chain, phosphorylatable, fast skeletal muscle gene
  • CK1 muscle creatine kinase 1
  • SEQ ID NO: 9 an SV40 intron set forth in SEQ ID NO: 7
  • a kozak sequence set forth in SEQ ID NO: 1 1.
  • Embodiment 36 A nucleic acid molecule comprising a skeletal muscle selective hybrid regulatory sequence, wherein the hybrid regulatory sequence comprises a MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle gene) enhancer sequence set forth in SEQ ID NO: 2, a CK7 (muscle creatine kinase 7) enhancer-promoter sequence set forth in SEQ ID NO: 10, an SV40 intron set forth in SEQ ID NO: 7 and a kozak sequence set forth in SEQ ID NO: 11.
  • MYLPF myosin light chain, phosphorylatable, fast skeletal muscle gene
  • CK7 muscle creatine kinase 7
  • SEQ ID NO: 10 an SV40 intron set forth in SEQ ID NO: 7
  • a kozak sequence set forth in SEQ ID NO: 11.
  • Embodiment 37 The nucleic acid molecule of embodiment 32, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 138.
  • Example 1 Modified desmin-based promoters improve expression and efficacy in vitro
  • A02239 is the standard desmin promoter control and A04949, A04959, and A04546 contain modified desmin-based promoters (FIG. 1). All AAVs had a MOI of le6.
  • Protein was harvested from transduced cardiomyocytes at 7 days post transduction (FIG. 1). The protein expression of the PUF-CUG was measured by western blot. The desmin promoter in A02239 drove minimal PUF expression while the modified desmin- based promoters, A04949 and A04546, produced robust PUF protein levels (FIG. 3).
  • RNA CUG repeats sequester MBNL proteins which results in abnormal splicing.
  • the PUF protein competes and displaces MBNL from the CUG repeats, and the free MBNL is then able to properly modulate splicing.
  • the RNA splicing from the human patient-derived cardiomyocytes was analyzed across 5 different transcripts. The splicing correction was much more potent from the modified desmin-based promoters. This demonstrates the promoters drove greater protein expression which increase efficacy in the human disease context (FIG. 4).
  • Example 2 Increased PUF expression and efficacy in vivo from novel hybrid regulatory sequences
  • AAV9 A02239 and AAV9 A04959 were intravenously injected into HSALR mice and PUF RNA expression was measured at 4 weeks post injection by ddPCR.
  • the modified desmin-based promoter increased expression 3.2x in the gastrocnemius and 7.7x in the heart (FIG. 5).

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Abstract

Sont divulguées des associations de promoteurs sélectifs des muscles et des compositions de thérapie génique ciblant l'ARN, ainsi que des procédés les comprenant pour le traitement de DM1.
PCT/US2025/011882 2024-01-16 2025-01-16 Associations régulatrices hybrides sélectives musculaires et procédés d'utilisation associés pour le traitement de la dystrophie myotonique de type 1 Pending WO2025155722A1 (fr)

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