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WO2025048709A1 - Composition et procédés d'identification de nouveaux variants d'aav pour l'administration de gènes différenciés à des cardiomyoctes et/ou à des myofibres squelettiques - Google Patents

Composition et procédés d'identification de nouveaux variants d'aav pour l'administration de gènes différenciés à des cardiomyoctes et/ou à des myofibres squelettiques Download PDF

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WO2025048709A1
WO2025048709A1 PCT/SG2024/050344 SG2024050344W WO2025048709A1 WO 2025048709 A1 WO2025048709 A1 WO 2025048709A1 SG 2024050344 W SG2024050344 W SG 2024050344W WO 2025048709 A1 WO2025048709 A1 WO 2025048709A1
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raav particle
peptide sequence
muscle
seq
particle according
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Choong Tat KENG
Wei Leong CHEW
Muhammad Irfan Bin HAJIS
Yanyin Kimberle SHEN
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Agency for Science Technology and Research Singapore
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • 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
    • C12N15/63—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
    • 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
    • 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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2750/14145—Special targeting system for viral vectors
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Definitions

  • the present invention relates to the provision of recombinant adeno-associated virus (rAAV) -based delivery vectors comprising short peptide inserts of at least 5 amino acid length, which show enhanced transduction efficiency in one or more muscle cells, particularly in cardiomyocytes and/or skeletal muscle cells.
  • rAAV vectors of the present disclosure are suitable for use in the treatment and/or prophylaxis of a cardiac-related and/or muscle-related disease or disorder and/or for use in gene therapy. Also provided are methods of treating cardiac-related and/or muscle-related diseases or disorders.
  • Adeno-associated viruses have attracted widespread interest and are rapidly emerging as new therapeutic delivery vectors for monogenic disorders due to their established safety profile, as exemplified by various AAV-based therapies approved by the FDA in recent years.
  • AAV1-SERCA2a As highlighted by the results from a recent clinical trial in patients with cardiac diseases, the bio-distribution analyses of AAV1-SERCA2a suggest that less than 1% of cardiomyocytes contained the vector genome, implying that the AAV1 vector applied via antegrade intracoronary infusion at a dose of 10 A13 viral genomic sequence (vgs) transduces cardiomyocytes at very low efficiency (Greenberg B et al., 2016). Furthermore, there are still concerns over the immunogenicity and toxicity of high AAV vector doses which may result in complications and failure of therapy in some patients. To improve on the safety and efficacy profile, more efficient AAV capsids which can allow lower dose usage and lower risk of complications or failure of the gene therapy in patients are needed.
  • Inherited cardiomyopathy and skeletal muscle dystrophies are some of the diseases that have yet to benefit clinically from improved performance of gene delivery vectors.
  • Monogenic cardiovascular diseases MCVDs
  • arrhythmias such as Brugada syndrome caused by mutations in the SCN5A gene
  • cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy (ARVC), caused by mutations in DSC2, DSG2, DSP, JUP, PKP2, and TMEM43.
  • ALD arrhythmogenic right ventricular cardiomyopathy
  • DMD Duchenne muscular dystrophy
  • LGMD limb girdle muscle dystrophy
  • the present invention relates to the provision of improved recombinant AAV (rAAV) based vectors suitable for use in the treatment and/or prophylaxis of cardiac-related and/or muscle- related diseases or disorders.
  • rAAV recombinant AAV
  • a recombinant adeno-associated virus (rAAV) particle comprising a viral shell comprising a modified AAV capsid protein, wherein the modified AAV capsid protein comprises an extraneous peptide sequence comprising a length of at least 5 amino acids, wherein the extraneous peptide sequence enhances/increases the transduction efficiency of the rAAV particle into one or more muscle cell(s), compared to the transduction efficiency of a wild-type AAV particle not comprising the extraneous peptide sequence.
  • rAAV adeno-associated virus
  • the rAAV particle of the present disclosure may be a recombinant adeno-associated virus serotype 1 (rAAV1) particle and the extraneous peptide sequence may substitute and replace the amino acid residue at position 590 of the AAV capsid protein.
  • rAAV1 adeno-associated virus serotype 1
  • the one or more muscle cell(s) may be one or more cardiomyocyte(s) and/or one or more skeletal myofiber cell(s).
  • the extraneous peptide sequence may preferably comprise the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 65.
  • a recombinant polynucleic acid molecule encoding a modified AAV capsid protein, wherein the modified AAV capsid protein comprises an extraneous peptide sequence having a length of at least 5 amino acids.
  • the recombinant polynucleic acid molecule may be operatively linked to a mammalian promoter sequence.
  • a pharmaceutical composition comprising the rAAV particle according to the first aspect and a pharmaceutically acceptable carrier.
  • the rAAV particle according to the first aspect for use in medicine.
  • the rAAV particle according to the first aspect for use in treating a cardiac- related and/or muscle-related disease or disorder.
  • a method of treating a disease or disorder comprises administering the rAAV particle according to the first aspect, to a subject in need thereof.
  • the disease or disorder is a cardiac-related and/or muscle-related disease or disorder.
  • the cardiac-related and/or muscle-related disease or disorder may comprise inherited cardiomyopathy, skeletal muscle dystrophy, monogenic cardiovascular diseases (MCVDs) such as arrhythmia and Brugada syndrome, cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy (ARVC), Duchenne muscular dystrophy (DMD) and limb girdle muscle dystrophy (LGMD).
  • MCVDs monogenic cardiovascular diseases
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • DMD Duchenne muscular dystrophy
  • LGMD limb girdle muscle dystrophy
  • rAAV particle according to the first aspect for the preparation of a pharmaceutical composition for gene therapy.
  • the rAAV particles of the present disclosure provide enhanced transduction efficiency into muscle cells, particularly in cardiomyocytes, skeletal myofibers or both. More advantageously, the improved rAAV particles of the present disclosure may reduce off-target transduction, and thus may allow for lower dose application and may lower the risk of adverse effects (such as immune responses against the engineered vectors) and/or failure of gene therapy.
  • FIGS. 1A-1G depict the construction of AAV1 variant libraries and diversity analysis.
  • A Workflow for the construction and production of AAV1 diverse variants library, followed by in vivo selection of variants for improved transduction performance for cardiomyocytes and skeletal myofibers.
  • B Schematic of Counting Peptide Inserts (CoPl) pipeline for short peptide insert analysis as applied for AAVs.
  • C Extracted reads from flanking anchor sequences. Highlighted sequences (grey, underlined) are the seq_start and seq_end within the reference. The sequence in-between the anchor sequence are added to the count and cached for further downstream analyses.
  • FIGS. 2A - 2B show tables comparing selected engineered AAV variants of the present disclosure against engineered AAVs of the prior art for (A) cardiomyocytes and (B) skeletal muscles.
  • the selected AAV1 variants of the present disclosure for cardiomyocytes tropism performed better, up to 102-fold over parent and with improved cardio/muscle specificity of up to 212-fold and muscle de-targeting of up to 0.17-fold.
  • the transduction performance improved by not more than 25-fold and with improved cardio/muscle specificity of not more than 6.25-fold, and without muscle de-targeting being investigated.
  • the selected AAV1 variants of the present disclosure for skeletal muscles tropism also performed better, up to 108-fold over parent and with improved muscle/cardio specificity of up to 43.21 -fold and cardiomyocytes de-targeting of up to 0.57- fold.
  • the transduction performance improved by not more than 23-fold and with improved muscle/cardio specificity of not more than 0.16-fold, and without cardiomyocytes de-targeting being investigated.
  • the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of’.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
  • extraneous peptide sequence refers to any peptide sequence which is foreign from and not found in the known wild-type AAV capsid protein, which is then introduced into said AAV capsid protein.
  • the extraneous peptide sequence may be of any length, for example, at least 5 amino acids in length or more.
  • the extraneous peptide sequence may also comprise amino acid sequences which are randomly generated.
  • the term "subject" is herein defined as vertebrate, particularly mammal, more particularly human.
  • the subject may particularly be at least one animal model, e.g., a mouse, rat and the like.
  • the subject may be a human.
  • the present invention is based, in part, on the discovery that insertion of a short peptide sequence into a viral capsid protein may mediate viral re- targeting to specific cell types and/or improve the efficiency of transduction.
  • the inventors have successfully generated a panel of recombinant AAV vectors comprising short extraneous peptide inserts with enhanced specific-tissue transduction efficacy into muscle cells, particularly cardiomyocytes and/or skeletal myofibers, suitable for use in the treatment and/or prophylaxis of cardiac-related and/or muscle-related diseases or disorders.
  • a recombinant adeno-associated virus (rAAV) particle comprising a viral shell comprising a modified AAV capsid protein, wherein the modified AAV capsid protein comprises an extraneous peptide sequence comprising a length of at least 5 amino acids, wherein the extraneous peptide sequence enhances/increases the transduction efficiency of the rAAV particle into one or more muscle cell(s), compared to the transduction efficiency of a wild-type AAV particle not comprising the extraneous peptide sequence.
  • rAAV adeno-associated virus
  • the rAAV particle described herein may be derived from an AAV particle of different serotypes, for example, AAV1 , AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype.
  • the rAAV particle may be an rAAV1 , rAAV6, rAAV7, rAAV8, or a rAAV9 particle.
  • the rAAV particle is a rAAV1 particle.
  • an AAV particle may comprise a capsid (which is the protein shell of the virus) enclosing a nucleic acid molecule(s) within.
  • the capsid protein of a rAAV particle disclosed herein may include an extraneous peptide sequence not found in a known wild-type AAV capsid sequence.
  • a rAAV1 particle of the present invention may comprise an extraneous peptide sequence not found in a wild-type AAV1 particle. Accordingly in some embodiments, the extraneous peptide sequence may substitute and replace the amino acid residue at position 590 of the wild-type AAV capsid protein.
  • the extraneous peptide sequence may substitute and replace the amino acid residue D590 of the wild-type AAV1 capsid protein (SEQ ID NO: 66).
  • the extraneous peptide sequence may vary in length.
  • the extraneous peptide sequence may comprise a length of at least 5 amino acids.
  • the extraneous peptide sequence may comprise a length of at least 7 amino acids.
  • the extraneous peptide sequence may comprise a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids.
  • the extraneous peptide sequence may comprise a length of 7 amino acids or a length of 10 amino acids.
  • the rAAV particles disclosed herein provide enhanced transduction efficiency in muscle cells, and particularly in cardiomyocytes, skeletal myofibers, or both.
  • the one or more muscle cell(s) may comprise one or more cardiomyocyte(s) and/or one or more skeletal myofiber cell(s).
  • the one or more muscle cell(s) may comprise one or more mammalian muscle cell(s).
  • the extraneous peptide sequence may comprise the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 65.
  • the rAAV particles may provide enhanced transduction efficiency in cardiomyocytes.
  • the one or more muscle cell(s) may comprise one or more cardiomyocyte(s).
  • the extraneous peptide sequence in these embodiments may comprise the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 32, or SEQ ID NO: 58 to SEQ ID NO: 62.
  • the rAAV particles may provide enhanced transduction efficiency in skeletal myofibers.
  • the one or more muscle cell(s) may comprise one or more skeletal myofiber cell(s).
  • the extraneous peptide sequence in these particular embodiments may comprise the amino acid sequence set forth in any one of SEQ ID NO: 33 to SEQ ID NO: 54, or SEQ ID NO: 63 to SEQ ID NO: 65.
  • the rAAV particles may provide enhanced transduction efficiency in both cardiomyocytes and skeletal myofibers.
  • the one or more muscle cell(s) may comprise one or more cardiomyocyte(s) and skeletal myofiber cell(s).
  • the extraneous peptide sequence in these further embodiments may preferably comprise the amino acid sequence set forth in any one of SEQ ID NO: 55 to SEQ ID NO: 57.
  • a recombinant polynucleic acid molecule encoding a modified AAV capsid protein, wherein the modified AAV capsid protein comprises an extraneous peptide sequence having a length of at least 5 amino acids.
  • the extraneous peptide sequence in the modified AAV capsid protein encoded by the recombinant polynucleic acid molecule comprises a peptide sequence having a length of at least 7 amino acids. More preferably, the extraneous peptide sequence comprises a peptide sequence having a length of 7 amino acids or a length of 10 amino acids. More preferably, the extraneous peptide sequence comprises the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 65.
  • the recombinant polynucleic acid molecule may be operatively linked to a mammalian promoter sequence.
  • nucleic acid molecule within the rAAV particle may comprise a recombinant nucleic acid sequence comprising an extraneous nucleic acid sequence encoding the capsid protein with the extraneous peptide sequence. It will also further be appreciated that the nucleic acid molecule of the rAAV particle may further comprise further extraneous nucleic acid sequence(s); for example for gene therapy.
  • a pharmaceutical composition comprising the rAAV particle disclosed herein and a pharmaceutically acceptable carrier.
  • Suitable pharmaceutical carriers typically will contain inert ingredients that do not interact with the agent or active ingredient.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% 10 mg/ml benzyl alcohol), phosphate-buffered saline, Hank’s solution, Ringer’s lactate and the like.
  • Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying agents, solubilizing agents, pH buffering agents, wetting agents). Methods of encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art.
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., anatomizer or nebulizer or pressurized aerosol dispenser).
  • the rAAV particle disclosed herein for use in medicine.
  • the rAAV particle disclosed herein for use in treating a cardiac-related and/or muscle-related disease or disorder.
  • a method of treating a disease or disorder the method comprises administering the rAAV particle disclosed herein, to a subject in need thereof.
  • the disease or disorder is a cardiac-related and/or muscle- related disease or disorder.
  • rAAV particle disclosed herein in the manufacture of a medicament for the treatment of a cardiac-related and/or muscle-related disease or disorder in a subject.
  • the cardiac-related and/or muscle-related disease or disorder may comprise inherited cardiomyopathy, skeletal muscle dystrophy, monogenic cardiovascular diseases (MCVDs) such as arrhythmia and Brugada syndrome, cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy (ARVC), Duchenne muscular dystrophy (DMD) and limb girdle muscle dystrophy (LGMD).
  • MCVDs monogenic cardiovascular diseases
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • DMD Duchenne muscular dystrophy
  • LGMD limb girdle muscle dystrophy
  • rAAV particle of the present disclosure for the preparation of a pharmaceutical composition for gene therapy.
  • the rAAV particle disclosed herein may be suitable for nucleic acid (prophylactics or therapeutics) delivery to treat human cardiac-related or muscle-related genetic disorders.
  • the rAAV particle disclosed herein may also be suitable for nucleic acid (prophylactics or therapeutics) delivery into other cell populations that are susceptible to AAV1 and its variants.
  • the rAAV of the present disclosure can be delivered to a subject in need thereof by a variety of routes of administration including, for example, oral, nasal, dietary, topical, transdermal, or parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection) routes of administration. Administration can be local or systemic. Preferably, the medicament is formulated for subcutaneous or intravenous administration.
  • the actual dose of a therapeutic amount of the pharmaceutical composition disclosed herein and treatment regimen can be determined by a skilled physician, taking into account the nature of the condition being treated, and patient characteristics.
  • Capsid libraries are generated by substitution of D590 with random 21- mer or 30-mer oligonucleotides which codes for random 7-mer or 10- mer peptides.
  • the engineered capsid sequences are packaged in AAV vectors and used for transduction of mammalian cells or tissue at low dose of 1x10 A9 total viral genomes (vgs) per mice.
  • AAV variants in the library each bearing its own capsid sequence were produced as per standard protocol. Briefly, AAV were packaged via a triple transfection of 293AAV cell line (Cell Biolabs AAV-100) that were plated in a HYPERFIask ‘M’ (Corning) in growth media consisting of DMEM + glutaMax + pyruvate + 10%FBS (Thermo Fisher), supplemented with 1X MEM non-essential amino acids (Gibco). Confluency at transfection was between 70-90%. Media was replaced with fresh pre-warmed growth media before transfection.
  • pHelper Cell Biolabs
  • 100 pg of pRep(stop) and 100 pg of pZac- CMV-P40-Cap1-PolyA self-encoding diverse capsids in the libraries] were mixed in 5 ml of DMEM, and 2 mg of PEI “MAX” (Polysciences) (40 kDa, 1 mg/ml in H2O, pH 7.1) added for PEI: DNA mass ratio of 5:1.
  • PEI “MAX” Polysciences
  • the mixture was incubated for 15 min, and transferred drop-wise to the cell media.
  • Cells were harvested 48-72 hrs after transfection by scrapping or dissociation with 1 *PBS (pH7.2) + 5 mM EDTA, and pelleted at 1500 g for 12 min. Cell pellets were resuspended in 1-5 ml of lysis buffer (Tris HCI pH 7.5 + 2 mM MgCI + 150 mM NaCI), and freeze-thawed 3x between dry-ice-ethanol bath and 37 °C water bath. Cell debris was clarified via 4000 g for 5 min, and the supernatant collected.
  • lysis buffer Tris HCI pH 7.5 + 2 mM MgCI + 150 mM NaCI
  • the collected supernatant was treated with 50 U/ml of Benzonase (Sigma-Aldrich) and 1 U/ml of RNase cocktail (Invitrogen) for 30 min at 37 °C to remove unpackaged nucleic acids. After incubation, the lysate was loaded on top of a discontinuous density gradient consisting of 6 ml each of 15%, 25%, 40%, 60% Optiprep (Sigma-Aldrich) in an 29.9 ml Optiseal polypropylene tube (Beckman-Coulter). The tubes were ultra-centrifuged at 54000 rpm, at 18 °C, for 1.5 hr, on a Type 70 Ti rotor.
  • the 40% fraction was extracted, and dialyzed with 1 xPBS (pH 7.2) supplemented with 35 mM NaCI, using Amicon Ultra-15 (100 kDa MWCO) (Millipore).
  • Amicon Ultra-15 100 kDa MWCO
  • the titer of the purified AAV vector stocks were determined using real-time qPCR with ITR sequence- specific primers and probe (Aurnhammer C. et al., 2012), referenced against the ATCC reference standard material 8 (ATCC).
  • mice Male C57BL/6J mice 10-16 weeks old from InVivos were used. Mice were injected intra-peritoneally (IP) with 10 A9 vgs of the AAV1 -variants library, sacrificed 3-4 weeks after injection, and the heart and quadriceps muscle were dissected and placed on ice. Dissociation of muscle tissue into a single-cell suspension was done with the Skeletal Muscle Dissociation Kit (Miltenyi Biotec) following manufacturer’s instructions. Briefly, muscle tissue were cut into 2mm pieces with a razor blade and enzymatically digested at 37°C for 1 h.
  • the cell suspensions were then strained through a 70pm cell strainer, and centrifuged at 300g for 20 min.
  • the cell pellets were then re-suspended in Hibernate A with 10pM Calcien-AM (Invitrogen) and 1 g/ml Propidium Iodide (PI) (Stemcell), for FACS sorting.
  • Live, single cells were selected by positive selection of Calcien-AM and negative selection of PI.
  • PI Propidium Iodide
  • cells were counted and 20,000 cells were used for generation of 10X 3’ chromium libraries.
  • Reverse transcription of the RNA transcripts followed by targeted amplification of the random peptides insert region on the capsid sequence allowed us to construct an amplicon library.
  • the amplicon libraries constructed from the cDNA were then sequenced on an iSeq (Illumina) to determine the diversity of the libraries and to assess the comparative transduction efficiency of each variant to the mammalian cells or tissue.
  • Counting Peptide Inserts is a simple text-based search and analysis program which does quality filtering and counts the occurrences of peptide inserts in a NGS dataset.
  • CoPl is written in Python 3.0 and executable on Windows or Linux platforms (FIG. 1). Users are able to easily modify variables specific to their experiment in a TOML formatted file. These variables include a reference sequence, two invariable anchor sequence flanking the intended engineered site (‘seq_start’ and ‘seq_end’), and a list of other optional options (FIGS. 1B and 1C).
  • Fastp was employed, which merges paired end reads to ease downstream analyses. Fastp also removes reads with poor Qscore.
  • CoPl is able to analyze paired ends reads without the need to merge. Analysis is strand specific. If the user is unsure of the strand direction, CoPl will identify the direction of the merged and paired fastq files and subsequently analyze accordingly. All reads with the anchor sequences are included for further analysis. If one of the two anchor sequences are not found, CoPl will search for the anchor sequence tolerating 1 mismatch or indel via a shift- or algorithm (Baeza-Yates, R. & Gonnet, G., 1992). For each read with both anchor present, CoPl will determine the length of the insertion. An amplicon containing no insertion is considered as 0 indel.
  • the insert is then translated into amino acid to count for the peptide insert occurrences.
  • the nucleotide sequence and quality for each read is cached into memory for downstream collapse of low occurrence inserts.
  • low count inserts with nucleotide sequences within 2 Levenshtein distance are collapsed to the relatively abundant inserts.
  • Peptide insert sequences are concatenated for indexing. A simple burrow-wheeler aligner was employed for quick query search with 1 mismatches tolerated (Li, H. & Durbin, R., 2009).
  • CoPl ensures that the relatively abundant insert count is at least 5 times of the low insert count as current sequencing technology has an error rate of less than 20% (1-2% for Illumina platforms) (Zorita, E. et al., 2015).
  • CoPl chooses the peptide sequence with a lower hamming distance and/or relatively lower Qscore difference to collapse on.
  • CoPl subsequently plots the distribution of the peptide lengths and the in-frame amino acid distribution of the inserts.
  • a 100mb gzipped paired ends fastq file takes approximately 10 minutes to analyze using CoPl.
  • a split Rep/Cap system was used with the AAV cargo sequences bearing self-encoding capsid sequences driven under the control of a mammalian CMV and included a polyA tail for transcript capture (FIG. 1A).
  • amino acid D590 was replaced with random 7-mer and 10- mer peptides and the final libraries consist of capsid sequences for AAV1-7mer, AAV1-10mer and AAV1-WT (FIG. 1A).
  • E.coli plasmids library was produced followed by AAV1 variants production.
  • the cargo plasmid from the AAV1 variants viruses were then extracted and used for construction of NGS library to study the diversity of the AAV1 library.
  • NGS sequencing and analysis 11453 7-mers variants and 2735 10- mer variants were identified in the AAV1 virus library after filtering out variants with only 1 read depth.
  • the inventors were able to confirm that the library consist of AAV1-WT (as internal control for normalization), AAV1-7mer and AAV1-10mer in the percentage of 26.67%, 64.33% and 8.92% respectively (FIG. 1D).
  • Further analysis to look into the amino acid diversity of the 7-mer and 10-mer peptide insertions confirmed the highly diverse nature of the constructed AAV1 variants library present in the library (FIGS. 1 E and 1 F respectively).
  • Example 3 In vivo selection of AAV1 variants library for mouse cardiomyocytes and skeletal myofibers
  • RNA transcript of the AAV1 variants in target tissue (FIG. 1A).
  • Amplicon libraries constructed from the cDNA of the RNA transcripts are then sequenced on an iSeq (Illumina) and the data analyzed to identify fold change in transduction by relative abundance of script and normalization by following a series of formulaes (FIG. 1A).
  • the present disclosure describes novel compositions of rAAV1 capsids for delivery of genes into specific muscle cell types.
  • the inventors demonstrated the empirical methodology and bioinformatics pipeline for the selection of novel rAAV capsids, which significantly improves the gene transduction efficacy into cardiomyocytes, skeletal myofibers, or both muscle tissue types.
  • Capsid libraries are generated by substitution of D590 with random 21-mer or 30-mer oligonucleotides which codes for random 7-mer or 10- mer peptides.
  • the engineered capsid sequence is then packaged in AAV1 vectors and used for transduction of mammalian cells or tissue.
  • NGS is employed to look at the diversity of the libraries and to assess the comparative transduction efficiency of each variant to the mammalian cells or tissue.
  • CoPl was developed, a bioinformatics pipeline that enables comprehensive analysis of short peptide inserts in any primary sequence of proteins.
  • the present invention provides novel rAAV particles with preferential tissue tropism towards cardiomyocytes and/or skeletal muscles.
  • the rAAV particles of the present disclosure provide at least 10- to 100-fold improvement in transduction efficiency.
  • the rAAV particles disclosed herein have at least 25-fold skewed tropism towards cardiomyocytes compared to skeletal muscle.
  • the rAAV particles disclosed herein are also muscle-detargeted to various extent. As muscles made up approximately 40% of the body mass, de-targeting from the muscle can significantly reduce unnecessary AAV off- target transduction which may allow for lower dose application as well as potentially lower the immune responses against the engineered vectors.
  • the differential improvement in delivery to the different muscle cell types will be important for a more precise and targeted gene therapy treatment, depending if the genetic disorder affect a single or both cell types.
  • Rode, L. et al. AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes. Mol. Ther. 30, 3601-3618 (2022).
  • Tabebordbar M. et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919-4938.e22 (2021).

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Abstract

La présente invention concerne la création de vecteurs d'administration à base de virus adéno-associé recombiné (rAAV) comprenant de courts inserts peptidiques d'une longueur d'au moins 5 acides aminés, qui présentent une efficacité de transduction accrue dans une ou plusieurs cellules musculaires, en particulier dans les cardiomyocytes et/ou les cellules musculaires squelettiques. Les vecteurs rAAV de la présente invention peuvent être utilisés pour le traitement et/ou la prophylaxie d'une maladie ou d'un trouble cardiaque et/ou musculaire et/ou pour la thérapie génique. La présente invention porte également sur des méthodes de traitement de maladies ou de troubles liés au cœur et/ou aux muscles.
PCT/SG2024/050344 2023-08-25 2024-05-23 Composition et procédés d'identification de nouveaux variants d'aav pour l'administration de gènes différenciés à des cardiomyoctes et/ou à des myofibres squelettiques Pending WO2025048709A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021077000A1 (fr) * 2019-10-16 2021-04-22 The Broad Institute, Inc. Compositions de ciblage musculaire modifiées
WO2022053630A1 (fr) * 2020-09-10 2022-03-17 Genethon Capside de aav modifiée par peptide
WO2022173847A2 (fr) * 2021-02-09 2022-08-18 Affinia Therapeutics Inc. Vaa recombinants ayant un tropisme et une spécificité améliorés
WO2023039476A1 (fr) * 2021-09-08 2023-03-16 The Broad Institute, Inc. Compositions modifiées pour le ciblage du système nerveux central et des muscles
WO2023060142A2 (fr) * 2021-10-05 2023-04-13 The Broad Institute, Inc. Compositions de muscle cardiaque modifiées

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021077000A1 (fr) * 2019-10-16 2021-04-22 The Broad Institute, Inc. Compositions de ciblage musculaire modifiées
WO2022053630A1 (fr) * 2020-09-10 2022-03-17 Genethon Capside de aav modifiée par peptide
WO2022173847A2 (fr) * 2021-02-09 2022-08-18 Affinia Therapeutics Inc. Vaa recombinants ayant un tropisme et une spécificité améliorés
WO2023039476A1 (fr) * 2021-09-08 2023-03-16 The Broad Institute, Inc. Compositions modifiées pour le ciblage du système nerveux central et des muscles
WO2023060142A2 (fr) * 2021-10-05 2023-04-13 The Broad Institute, Inc. Compositions de muscle cardiaque modifiées

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YU, C.Y. ET AL.: "A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery", GENE THERAPY, vol. 16, no. 8, 28 May 2009 (2009-05-28), pages 953 - 962, XP037771311, [retrieved on 20240614], DOI: 10.1038/GT.2009.59 *

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