WO2025024227A2 - Variants d'aav pour une administration cardiaque - Google Patents

Variants d'aav pour une administration cardiaque Download PDF

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WO2025024227A2
WO2025024227A2 PCT/US2024/038526 US2024038526W WO2025024227A2 WO 2025024227 A2 WO2025024227 A2 WO 2025024227A2 US 2024038526 W US2024038526 W US 2024038526W WO 2025024227 A2 WO2025024227 A2 WO 2025024227A2
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amino acids
seq
capsid protein
insertion site
aav9
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WO2025024227A3 (fr
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Jonathan MELNICK
Roxanne CROZE
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4D Molecular Therapeutics Inc
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4D Molecular Therapeutics 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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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
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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
    • C12N2750/14145—Special targeting system for viral vectors
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    • 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

  • a computer readable XML file entitled “090400-5025 WO Sequence Listing” created on July 17, 2024, with a file size of about 25,300 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
  • Adeno-associated virus (AAV) vectors have been used in clinical trials for cardiac disorders.
  • AAV capsids such as AAV9 exhibit significant transduction of off-target tissues, particularly the liver.
  • variant AAV capsid sequences that specifically target cardiac tissue, with reduced transduction of liver tissue, with a favorable cardiac to liver distribution of vector genomes to non-human primates relative to parental capsid AAV9.
  • the variant AAV capsids described herein comprise a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) in the GH loop of the capsid.
  • recombinant AAV (rAAV) virions are provided, these rAAV virions comprising a variant capsid protein as described herein, wherein the rAAV virions exhibit decreased transduction of liver cells relative to the transduction of liver cells by an AAV virion comprising a native AAV capsid protein of serotype 9.
  • infectivity of a cardiac cell by an rAAV virion comprising a variant capsid protein as herein described is not significantly reduced relative to infectivity of the cardiac cell by an AAV virion comprising a native AAV capsid protein of serotype 9.
  • the heart-to-liver ratio is higher compared to that of a subject that received an AAV9 vector (native AAV9 vectors exhibit a roughly 1 : 1 heartdiver ratio).
  • the heart-to-liver ratio of an rAAV virion comprising a variant capsid protein as described herein is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, or at least 10-fold higher than the heart to liver ratio of AAV9.
  • the rAAV virion comprises a heterologous nucleic acid.
  • the heterologous nucleic acid encodes an RNA that encodes a polypeptide.
  • the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, e g. the heterologous nucleic acid sequence is an RNA interference agent, a guide RNA for a nuclease, etc.
  • compositions comprising the subject infectious rAAV virions and a pharmaceutically acceptable carrier.
  • an isolated nucleic acid comprising a sequence encoding a variant AAV capsid protein as described herein and a host cell comprising the isolated nucleic acid.
  • the isolated nucleic acid and/or isolated host cell comprises the rAAV.
  • a method for delivering a heterologous nucleic acid to a cardiac cell comprising contacting the cardiac cell with a recombinant adeno- associated virus (AAV) comprising a variant AAV capsid protein as described herein.
  • AAV adeno- associated virus
  • the method is performed in vitro.
  • the method is performed in vivo.
  • the method is performed ex vivo.
  • a method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to a cardiac cell in a mammalian subject comprising administering to the mammal an effective amount of a recombinant AAV (rAAV) comprising a variant AAV capsid protein as herein described or administering a pharmaceutical composition comprising the recombinant AAV and a pharmaceutically acceptable excipient.
  • rAAV recombinant AAV
  • a method for treating and/or preventing a cardiac disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a recombinant AAV comprising a variant AAV capsid protein as herein described or administering a pharmaceutical composition comprising the recombinant AAV and a pharmaceutically acceptable excipient.
  • a recombinant AAV comprising a variant AAV capsid protein as herein described for use in treating a cardiac disorder is provided.
  • the use of a recombinant AAV comprising a variant AAV capsid protein as herein described in the manufacture of a medicament for the treatment of cardiac disorder is provided.
  • the recombinant AAV comprises (i) a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
  • the heterologous nucleic acid comprises a nucleotide sequence that encodes a gene product, in which case contacting a cardiac cell with a variant AAV vector as described herein provides a method for delivering a gene product to a cardiac cell.
  • the gene product is an antisense RNA, a microRNA (miRNA), a short hairpin RNA (shRNA) or a small interfering RNA (siRNA) or a precursor or mimic thereof.
  • the gene product is a long non-coding RNA.
  • the gene product is a short non-coding RNA.
  • the gene product is an antisense RNA.
  • the gene product is an aptamer.
  • the gene product is a polypeptide.
  • the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function.
  • the heterologous nucleic acid comprises a nucleotide sequence that encodes a therapeutic gene product capable of ameliorating a cardiac disorder.
  • the nucleotide sequence encoding the gene is operably linked to at least one transcription control sequence, preferably a transcription control sequence that is heterologous to the nucleic acid.
  • the transcription control sequence is a cell- or tissue-specific promoter that results in cell-specific expression of the nucleic acid e g. in cardiac cells.
  • the transcription control sequence is a constitutive promoter that results in similar expression level of the nucleic acid in many cell types (e.g. a CAG, CBA (chicken beta actin) or CMV promoter).
  • the transcription control sequence comprises a CAG promoter comprising (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of chicken beta-actin gene and (iii) the splice acceptor of the rabbit beta-globin gene as described in Miyazaki et al., Gene 79(2):269-77 (1989).
  • CMV cytomegalovirus
  • the recombinant AAV (or pharmaceutical composition comprising same) is administered to the mammal by systemic (e.g., intravascular) administration.
  • Figs. 1A-B illustrates transduction of human cardiomyocytes at the specified multiplicities of infection (MOIs) by native AAV9 carrying an EGFP reporter gene operably linked to a CAG promoter compared to an rAAV comprising a capsid protein of SEQ ID NO: 11 carrying an EGFP reporter gene operably linked to a CAG promoter.
  • Fig. IB is a graph quantifying the level of transduction by detection of EGFP and cardiac troponin T (cTnT).
  • FIG. 2 illustrates transduction of primary hepatocytes at the specified multiplicities of infection (MOIs) by native AAV9 carrying an EGFP reporter gene operably linked to a CAG promoter compared to an rAAV comprising a capsid protein of SEQ ID NO: 11 carrying an EGFP reporter gene operably linked to a CAG promoter.
  • MOIs multiplicities of infection
  • isolated designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated.”
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids.
  • a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
  • a coding region typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then that a single vector can contain just a single coding region, or comprise two or more coding regions.
  • regulatory region refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • nucleic acid is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.
  • a polynucleotide which encodes a gene product can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • a coding region for a gene product e.g., a polypeptide
  • a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.
  • Transcriptional control sequences refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • transcription control regions include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissuespecific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • RNA messenger RNA
  • tRNA transfer RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • post transcriptional modifications e.g., polyadenylation or splicing
  • polypeptides with post translational modifications e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • Promoter and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.
  • Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissuespecific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
  • RNAi refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., duplex RNA such as siRNA or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.
  • RNAi single-stranded RNA
  • double-stranded RNA i.e., duplex RNA such as siRNA or pre-miRNA
  • Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand.
  • Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif).
  • the sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.
  • Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15- 25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21- 22, or 21-23 nucleotides in length, and the double- stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length).
  • siRNA small-interfering RNA” or “siRNA,” e.g., interfering RNA of about
  • siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini.
  • siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or
  • siRNA are chemically synthesized.
  • siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coll RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Nall. Acad. Sci.
  • dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length.
  • a dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer.
  • the dsRNA can encode for an entire gene transcript or a partial gene transcript.
  • siRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).
  • CRISPR encompasses Clustered regularly interspaced short palindromic repeats/CRISPR-associated (Cas) systems that evolved to provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.
  • the Cas protein naturally contains DNA endonuclease or RNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs).
  • the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)).
  • a single molecule also called a single guide RNA (“sgRNA”).
  • the Cas protein associates with a DNA-targeting or RNA-targeting RNA (which term encompasses both the two- molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas or Cas-like protein and guides the protein to a target nucleic acid sequence.
  • CRISPR agents can be found, for example in (a) Jinek et. al., Science. 2012 Aug 17;337(6096):816-21 : "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. 2013 Feb 28; 152(5): 1173-83: "Repurposing CRISPR as an RNA- guided platform for sequence- specific control of gene expression", and (c) US patent application number 13/842,859 and PCT application number PCT/US13/32589; all of which are hereby incorporated by reference in their entirety.
  • CRISPR agent encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and/or synthetic sequences, that can be used in the Cas- based system (e.g., a Cas9; any component of a DNA-targeting RNA or RNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).
  • a Cas9 any component of a DNA-targeting RNA or RNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.
  • a donor polynucleotide e.g., a donor polynucleotide; and the like.
  • ZFNs Zinc-finger nucleases
  • ZFNs artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain.
  • ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences.
  • ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • ZFN agent encompasses a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.
  • TALEN Transcription activator-like effector nucleases
  • TALENs are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain.
  • TALENs can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • target DNA in the cell e.g., the cell's genome
  • TALEN agent encompasses a TALEN and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.
  • Plasmid refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular doublestranded DNA molecules.
  • Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. Sequence similarity or sequence homology can be determined in a number of different manners. Commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • Sequence homologies may be generated by any of a number of computer programs known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/.
  • Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc.
  • Of particular interest are alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997)
  • % homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • gaps penalties assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible-reflecting higher relatedness between the two compared sequences — may achieve a higher score than one with many gaps.
  • “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).
  • the final % homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix — the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244).
  • DNASISTM Hagachi Software
  • CLUSTAL Higgins D G & Sharp P M (1988), Gene 73(1), 237-244
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups.
  • Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well.
  • the sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C. D. and Barton G.
  • Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc.
  • Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • thienylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or P-alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or P-alanine residues.
  • a further form of variation which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the a-carbon.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease (and/or symptoms caused by the disease) from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and/or symptoms caused by the disease), i.e., arresting its development; and (c) relieving the disease (and/or symptoms caused by the disease), i.e., causing regression of the disease (and/or symptoms caused by the disease), i.e., ameliorating the disease and/or one or more symptoms of the disease.
  • the subject compositions and methods may be directed towards the treatment of muscle disease.
  • Nonlimiting methods for assessing muscle diseases and the treatment thereof include measuring therapeutic protein production (e.g. muscle biopsy followed by immunohistochemistry or serum sampling followed by ELISA or enzyme activity assays), measuring symptoms of heart failure (e.g. the New York Heart Association Functional Classification or the Minnesota Living With Heart Failure Questionnaire), functional cardiac status (e.g. the 6-minute walk test or peak maximum oxygen consumption), bio marker analysis (e.g. N-terminal prohormone brain natriuretic peptide), left ventricular function/remodeling (e.g. left ventricular ejection fraction or left ventricular end-systolic volume), muscle strength (e.g.
  • therapeutic protein production e.g. muscle biopsy followed by immunohistochemistry or serum sampling followed by ELISA or enzyme activity assays
  • symptoms of heart failure e.g. the New York Heart Association Functional Classification or the Minnesota Living With Heart Failure Questionnaire
  • functional cardiac status e.g. the 6-minute walk test or peak maximum oxygen consumption
  • bio marker analysis e.g. N-terminal prohormon
  • muscle function e.g. the Vignos Scale, Timed Function Tests, the Hammersmith Motor Ability Score, timed rise from floor, walk tests, Motor Function Measure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test, or Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders
  • muscle disease symptoms e.g. the Neuromuscular Symptoms Score or Clinical Global Impressions
  • mitochondrial function e g. 31 P magnetic resonance spectroscopy
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans; non- human primates, including simians; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • an effective amount is an amount sufficient to effect beneficial or desired clinical results.
  • An effective amount can be administered in one or more administrations.
  • an effective amount of a compound e.g., an infectious rAAV virion
  • an effective amount of an infectious rAAV virion is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of (and/or symptoms associated with) a particular disease state (e.g., a disorder associated with cardiac dysfunction).
  • an effective amount of an infectious rAAV virion is an amount of the infectious rAAV virion that is able to effectively deliver a heterologous nucleic acid to a target cell (or target cells) of the individual.
  • Effective amounts may be determined preclinically by, e.g., detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques that are well understood in the art, e.g. RT-PCR, western blotting, ELISA, fluorescence or other reporter readouts, and the like. Effective amounts may be determined clinically by, e.g. detecting a change in the onset or progression of disease using methods known in the art, e.g. 6-minute walk test, left ventricular ejection fraction, hand-held dynamometry, Vignos Scale and the like as described herein and as known in the art.
  • systemic administration is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation).
  • intravascular administration refers to the administration of an agent, e.g., a composition comprising a rAAV, into the vasculature of a subject, including the venous and arterial circulatory systems of the subject.
  • agents e.g., a composition comprising a rAAV
  • Methods for intravascular administration are well known in the art and include for example, use of a hypodermic needle, peripheral cannula, central venous line, etc.
  • tropism refers to the preferential targeting by a virus (e.g., an AAV) of cells of a particular host species or of particular cell types within a host species.
  • a virus e.g., an AAV
  • a virus that can infect cells of the heart, lung, liver, and muscle has a broader (i.e., increased) tropism relative to a virus that can infect only lung and muscle cells.
  • Tropism can also include the dependence of a virus on particular types of cell surface molecules of the host. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependencies can be tested using various cells lines deficient in particular classes of molecules as potential host cells for viral infection).
  • the tropism of a virus describes the virus's relative preferences. For example, a first virus may be able to infect all cell types but is much more successful in infecting those cells with surface glycosaminoglycans.
  • a second virus can be considered to have a similar (or identical) tropism as the first virus if the second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), even if the absolute transduction efficiencies are not similar.
  • the second virus might be more efficient than the first virus at infecting every given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism as the first virus.
  • the tropism of a virion comprising a subject variant AAV capsid protein is not altered relative to a naturally occurring virion (e.g., AAV9).
  • the tropism of a virion comprising a subject variant AAV capsid protein is reduced relative to a naturally occurring virion (e.g., AAV9).
  • directed evolution refers to a capsid engineering methodology, in vitro and/or in vivo, which emulates natural evolution through iterative rounds of genetic diversification and selection processes, thereby accumulating beneficial mutations that progressively improve the function of a biomolecule.
  • Directed evolution often involves an in vivo method referred to as “biopanning” for selection of AAV variants from a library which variants possess a more efficient level of infectivity of a cell or tissue type of interest
  • AAV vectors comprising a variant capsid protein as herein described provide for a minimum transduction of off-target tissue, in particular lung.
  • the observed liver de-targeting of AAV vectors herein described resulted in a ⁇ 44-fold lower liver expression, representing a substantial improvement of the off-target profile.
  • This substantial improvement in the off-targeting profile may allow for higher dosing, increased transgene expression and therapeutic efficacy while minimizing adverse off-targeting effects in patients suffering from cardiac disorders such as heart disease.
  • helper viruses include, typically, adenovirus or herpes simplex virus.
  • the helper functions can be provided to a packaging cell including by transfecting the cell with one or more nucleic acids encoding the various helper elements and/or the cell can comprise the nucleic acid encoding the helper protein.
  • HEK 293 were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to El and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72).
  • those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them.
  • the variant AAV capsid protein comprises an insertion of from about 7 to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH-loop of a parental AAV capsid protein, wherein the peptide comprises the sequence NLTRVSG (SEQ ID NO:1).
  • the variant capsid protein when present in an AAV virion, exhibits reduced liver tropism relative to that of the corresponding parental capsid protein and/or that of a native AAV virion such as AAV9.
  • the GH loop or loop IV, of the AAV capsid protein it is meant the solvent- accessible portion referred to in the art as the GH loop, or loop IV, of AAV capsid protein.
  • the insertion site can be within about amino acids 570-611 of AAV2 VP1.
  • the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any wild-type AAV serotype or AAV variant.
  • the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590.
  • the insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580.
  • the insertion site can be between amino acids 590 and 591 , amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.
  • the insertion site is located between amino acids 587 and 591 of VP1 of AAV9.
  • the insertion site can be between amino acids
  • the insertion site is between amino acids
  • 588 and 589 or is between amino acids 589 and 590.
  • a peptide insertion disclosed herein has a length of 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.
  • the peptide insertion has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and/or carboxyl terminus of the amino acid sequence NLTRVSG (SEQ ID NO: 1).
  • Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P).
  • a peptide insertion comprises 1 to 3 spacer amino acids at the N-terminus and 1 to 3 spacer amino acids at the C-terminus. In other embodiments, a peptide insertion comprises 1 spacer amino acid at the N-terminus and 1 spacer amino acid at the C-terminus. In some embodiments, a peptide insertion comprises a serine and/or glycine at the N-terminus and an alanine at the C- terminus. In other embodiments, a peptide insertion comprises an alanine and serine at the N- terminus and an alanine, leucine and serine at the C-terminus. In some preferred embodiments, the peptide insertion comprises or consists of an amino acid sequence selected from NLTRVSG (SEQ ID NO: 1), ASNLTRVSGALS (SEQ ID NO:2) and GNLTRVSGA (SEQ ID NO:3).
  • the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and further comprises one or more amino acid substitutions relative to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in an AAV virion, exhibits reduced liver tropism relative to that of the corresponding parental capsid protein and/or that of a native AAV virion such as AAV9.
  • the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1).
  • the variant capsid protein comprises the following amino acid sequence or comprises an amino acid sequence at least 85%, at least 90%, at least
  • amino acid sequence in each case comprises an insertion peptide comprising SEQ ID NO: 1 (in each case, the core insertion is in bold underline and the spacer amino acids are in italics):
  • LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:11; Spacer AS - ALS; insertion between amino acids 589 and 590 relative to AAV9)
  • amino acid sequence of native AAV9 VP1 capsid is provided below:
  • a variant capsid as herein described comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-13 and comprises a NLTRVSG (SEQ ID NO: 1), ASNLTRVSGALS (SEQ ID N0:2) or GNLTRVSGA (SEQ ID NO:3) peptide insertion between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, between amino acids 589 and 590 or between amino acids 590 and 591 relative to AAV9.
  • a variant capsid as herein described comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:8-l 1 and 13 and comprises a NLTRVSG (SEQ ID NO:1), ASNLTRVSGALS (SEQ ID NO:2) or GNLTRVSGA (SEQ ID NO:3) peptide insertion between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, between amino acids 589 and 590 or between amino acids 590 and 591 relative to AAV9
  • the variant capsid comprises a peptide insertion of SEQ ID NO:2 and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 11.
  • a full complement of AAV Cap proteins includes VP1, VP2, and VP3.
  • the ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins may be provided.
  • the invention includes packaging cells, which are encompassed by "host cells,” which may be cultured to produce packaged viral vectors of the invention.
  • the packaging cells of the invention generally include cells with heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). Each of these component functions is discussed in the ensuing sections.
  • the vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013/063379). A preferred method is described in Grieger, etal. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production.
  • the suspension HEK293 cell line generates greater than 10 5 vector genome containing particles (vg)/cell or greater than 10 14 vg/L of cell culture when harvested 48 hours post-transfection.
  • triple transfection refers to the fact that the packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA, and another plasmid encodes the transgene and its various control elements (e.g., modified GLA gene and CAG promoter).
  • helper functions e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA
  • transgene and its various control elements e.g., modified GLA gene and CAG promoter.
  • This scalable manufacturing technology has been utilized to manufacture GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), Giant Axonal Neuropathy (scAAV9) and Retinitis Pigmentosa (AAV2), which have been administered into patients.
  • AAV2 retinal neovascularization
  • scAAV8 Hemophilia B
  • scAAV9 Giant Axonal Neuropathy
  • AAV2 Retinitis Pigmentosa
  • a minimum of a 5-fold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.
  • the packaging cells include viral vector functions, along with packaging and vector functions.
  • the viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced by the modified GLA sequence and its associated expression control sequences.
  • the viral vector functions include sufficient expression control sequences to result in replication of the viral vector for packaging.
  • the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the transgene and its associated expression control sequences.
  • the transgene is typically flanked by two AAV TRs, in place of the deleted viral rep and cap ORFs.
  • transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or a marker polypeptide.
  • the terminal repeats (TR(s)) (resolvable and non-resolvable) selected for use in the viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred.
  • Resolvable AAV TRs need not have a wild-type TR sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the TR mediates the desired functions, e.g., virus packaging, integration, and/or provirus rescue, and the like.
  • the TRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double-D sequence" as described in U.S. Pat. No.
  • the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.
  • the packaging functions include capsid components.
  • the capsid components are preferably from a parvoviral capsid, such as an AAV capsid or a chimeric AAV capsid function.
  • suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependovirus.
  • the capsid components may be selected from AAV capsids, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAVrhlO, AAVrh74, RHM4-1, RHM1 5-1 , RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV Hu 26, AAV1 .1 , AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2- TT-S312N, AAV3B-S312N, and AAV-LK03, and other novel capsids as yet unidentified or from non-human primate sources.
  • Capsid components may include components from two or more AAV capsids.
  • the packaged viral vector generally includes a gene sequence (e.g., a therapeutic gene to treat a cardiac disorder) and expression control sequences flanked by TR elements, referred to herein as the "transgene” or “transgene expression cassette,” sufficient to result in packaging of the vector DNA and subsequent expression of the gene sequence in the transduced cell.
  • the viral vector functions may, for example, be supplied to the cell as a component of a plasmid or an amplicon.
  • the viral vector functions may exist extrachromosomally within the cell line and/or may be integrated into the cell's chromosomal DNA.
  • any method of introducing the nucleotide sequence carrying the viral vector functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.
  • the viral vector functions are provided by transfection using a virus vector; standard methods for producing viral infection may be used.
  • the packaging functions include genes for viral vector replication and packaging.
  • the packaging functions may include, as needed, functions necessary for viral gene expression, viral vector replication, rescue of the viral vector from the integrated state, viral gene expression, and packaging of the viral vector into a viral particle.
  • the packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, a Baculovirus, or HSV helper construct.
  • the packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.
  • the helper functions include helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector. Examples include functions derived from adenovirus, baculovirus and/or herpes virus sufficient to result in packaging of the viral vector.
  • adenovirus helper functions will typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA.
  • the packaging functions may be supplied by infection of the packaging cell with the required virus.
  • the packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. See, e.g., pXR helper plasmids as described in Rabinowitz et al., 2002, J.
  • the packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA (e.g., El or E3 in HEK 293 cells).
  • helper virus functions may be employed.
  • the packaging cells are insect cells
  • Herpes virus may also be used as a helper virus in AAV packaging methods.
  • Hybrid herpes viruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes.
  • Any method of introducing the nucleotide sequence carrying the helper functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.
  • the helper functions are provided by transfection using a virus vector or infection using a helper virus; standard methods for producing viral infection may be used.
  • any suitable permissive or packaging cell known in the art may be employed in the production of the packaged viral vector.
  • Mammalian cells or insect cells are preferred.
  • Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenoviral El under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines.
  • the packaging cell is capable of growing in suspension culture, more preferably, the cell is capable of growing in serum-free culture.
  • the packaging cell is a HEK293 that grows in suspension in serum free medium.
  • the packaging cell is the HEK293 cell described in U.S. Pat. No. 9,441,206 and deposited as ATCC No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002/46359.
  • the packaging cell is cultured in the form of a cell stack (e.g. 10-layer cell stack seeded with HEK293 cells).
  • Cell lines for use as packaging cells include insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present invention. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line.
  • the following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed.
  • Virus capsids according to the invention can be produced using any method known in the art, e.g., by expression from a baculovirus (Brown et al., (1994) Virology 198:477-488).
  • the virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and rAAV template as described, for example, by Urabe et al., 2002, Human Gene Therapy 13: 1935-1943.
  • the present invention provides for a method of rAAV production in insect cells wherein a baculovirus packaging system or vectors may be constructed to carry the AAV Rep and Cap coding region by engineering these genes into the polyhedrin coding region of a baculovirus vector and producing viral recombinants by transfection into a host cell.
  • a baculovirus packaging system or vectors may be constructed to carry the AAV Rep and Cap coding region by engineering these genes into the polyhedrin coding region of a baculovirus vector and producing viral recombinants by transfection into a host cell.
  • the AAV DNA vector product is a self- complementary AAV like molecule without using mutation to the AAV ITR. This appears to be a by-product of inefficient AAV rep nicking in insect cells which results in a self-complementary DNA molecule by virtue of lack of functional Rep enzyme activity.
  • the host cell is a baculovirus-infected cell or has introduced therein additional nucleic acid encoding baculovirus helper functions or includes these baculovirus helper functions therein.
  • These baculovirus viruses can express the AAV components and subsequently facilitate the production of the capsids.
  • the packaging cells generally include one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line or integrated into the cell's chromosomes.
  • the cells may be supplied with any one or more of the stated functions already incorporated, e.g., a cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, or a cell line with helper functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA.
  • the rAAV vector may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients.
  • the AAV variants disclosed herein were generated through the use of in vivo directed evolution involving the use of primate cardiac screens following intravenous administration.
  • the variant capsid proteins disclosed herein when present in an AAV virion, confer decreased transduction of a liver cell compared to the transduction of the liver cell by a native AAV9 virion, when administered systemically.
  • the AAV variant virion or variant rAAV exhibits at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 500-fold or more than 500-fold, decreased transduction of a liver cell, compared to the transduction of the liver cell by a wild-type AAV virion of serotype 9.
  • the AAV variant virion preferentially transduces a cardiac cell, e.g., a subject rAAV virion infects a cardiac cell with 2-fold, 5- fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-muscle cell (e.g., a liver cell).
  • the transduced cardiac cell is cardiomyocyte, cardiac fibroblast, smooth muscle cell or a cardiac progenitor cell. Transduction of a cardiac cell, e.g. increased efficiency of transduction, broader transduction, more preferential transduction, etc. may be readily assessed in vitro or in vivo by any number of methods in the art for measuring gene expression.
  • the AAV may be packaged with a genome comprising an expression cassette comprising a reporter gene, e.g. a fluorescent protein, under the control of a ubiquitous or tissue specific promoter, and the extent of transduction assessed by detecting the fluorescent protein by, e.g., fluorescence microscopy.
  • the AAV may be packaged with a genome comprising a barcoded nucleic acid sequence, and the extent of transduction assessed by detecting the nucleic acid sequence by, e.g., PCR.
  • the AAV may be packaged with a genome comprising an expression cassette comprising a therapeutic gene for the treatment of a cardiac disease, and the extent of transduction assessed by detecting the treatment of the cardiac disease in an afflicted patient that was administered the AAV.
  • an AAV comprising a variant capsid protein comprising a peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) in the GH-loop of the capsid further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
  • the gene product is a reporter gene, non-limiting examples of which include fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme that produces a detectable product, such as luciferase (e.g., from Gaussia, Renilla, or Photinus), P-galactosidase, P-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or proteins that can be directly detected.
  • fluorescent protein e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2
  • an enzyme that produces a detectable product such as luciferase (e.g., from Gaussia, Renilla, or Photinus), P-galactosidase, P-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransfera
  • the gene product is a therapeutic protein suitable for the treatment of one or more cardiac disorders.
  • the gene product is a protein selected from the group of cardiac repair factors, calcium regulators, and pro-angiogenic factors.
  • cardiac repair factor such as the human myeloid derived growth factor (huMydgf)
  • a calcium regulator selected from the group consisting of the calcium regulator proteins sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), small ubiquitin-related modifier 1 (SUMO1), S100 calcium-binding protein Al (S100A1), and the pro-angiogenic vascular endothelial growth factor (VEGF).
  • a cardiac repair factor such as the human myeloid derived growth factor (huMydgf)
  • SERCA2a calcium regulator selected from the group consisting of the calcium regulator proteins sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), small ubiquitin-related modifier
  • the protein supplements a corresponding defective gene in the subject to be treated, including, without limitation, a protein selected from beta-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3), troponin I type 3 (TNNI3), cardiac troponin T (TNNT2), tropomyosin alpha-1 chain (TPM1), myosin light chain (MYL3), plakophilin-2 (PKP2), 5 ’-AMP-activated protein kinase subunit gamma-2 (PRKAG2), titin (TTN), myosin, light chain 2 (MYL2), actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT- like subfamily, member 1 (KCNQ1), myocyte enhancer factor 2c (MEF2C), cardiac LIM protein (CSRP3), a cardiac sarcomeric protein, beta-myosin heavy chain, myosin ventricular essential light chain 1, cardiac troponin
  • the heterologous nucleic acid may encode a functional RNA, e.g., an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. Nos.
  • a functional RNA e.g., an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. Nos.
  • RNAi interfering RNAs
  • siRNA small interfering RNAs
  • microRNA or other non-translated “functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like.
  • RNAi or antisense RNA against the multiple drug resistance (MDR) gene product e.g., to treat tumors and/or for administration to the heart to prevent damage by chemotherapy
  • MDR multiple drug resistance
  • RNAi or antisense RNA against myostatin e.g., to treat tumors and/or for administration to the heart to prevent damage by chemotherapy
  • RNAi or antisense RNA against myostatin e.g., to treat tumors and/or for administration to the heart to prevent damage by chemotherapy
  • RNAi or antisense RNA against myostatin Duchenne or Becker muscular dystrophy
  • RNAi or antisense RNA against VEGF or a tumor immunogen including but not limited to those tumor immunogens specifically described herein (to treat tumors), RNAi or antisense oligonucleotides targeting mutated dystrophins (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against the hepatitis B surface antigen gene (to prevent
  • RNAi or antisense RNA against the targets described above or any other target can also be employed as a research reagent.
  • the interfering RNA knocks-down expression of an mRNA encoding a protein associated with a cardiac disorder.
  • the gene product is an interfering RNA, more preferably an miRNA.
  • the gene product encodes a microRNA (miRNA).
  • the microRNA preferably is one that is involved in the regulation of the Mitogen-activated protein kinase (MAPK) pathway, the MYOD pathway, the FOXO3 pathway, or the ERK-MAPK pathway.
  • the microRNA encoded by the transgene of the viral vector for use in the method of the invention is selected from the group consisting of miR- 378, miR669a, miR-21 miR212, and miR132.
  • a gene product is a site-specific endonuclease that provides for sitespecific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a muscle disease.
  • a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.
  • a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele.
  • a subject rAAV virion can be used to deliver both a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional muscle protein (e.g., functional lamin A/C, functional fibrillin, functional collagen type VI, etc.).
  • a functional muscle protein e.g., functional lamin A/C, functional fibrillin, functional collagen type VI, etc.
  • a rAAV virion disclosed herein comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional muscle protein.
  • Functional muscle proteins include, e.g., lamin A/C, fibrillin 1, COL6A1, COL6A2, COL6A3, and the like.
  • Site-specific endonucleases that are suitable for use include, e.g., meganucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and Clustered regularly interspaced short palindromic repeats/CRISPR-associated (Cas), where such sitespecific endonucleases are non-naturally occurring and are modified to target a specific gene.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • Cas Clustered regularly interspaced short palindromic repeats/CRISPR-associated
  • site-specific endonucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides.
  • Such site-specific endonucleases also referred to as “INDELs” then throw the protein out of frame and effectively knock out the gene. See, e.g., U.S. Patent Publication No. 2011/0301073, the contents of which are incorporated herein by reference.
  • the nucleotide sequence encoding the gene product is operably linked to a promoter.
  • Suitable promoters may be selectively or constitutively active in heart tissue, and in particular in cardiomyocytes.
  • the promoter is a ubiquitous promoter such as a CMV promoter, a CAG promoter, a PGK-1 promoter, or an EF-la promoter.
  • the ubiquitous promoter is a CAG promoter comprising (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of chicken beta-actin gene and (iii) the splice acceptor of the rabbit beta-globin gene as described in Miyazaki et al., Gene 79(2):269-77 (1989).
  • the promoter functionally linked to the transgene is the CAG promoter.
  • the promoter functionally linked to the transgene is the CMV promoter.
  • the promoter operably linked to the transgene is a tissue- or cell-specific promoter.
  • the promoter is a cardiomyocyte-specific promoter.
  • cardiac-specific or preferred promoters include, without limitation, cardiac muscle a-actin promoter (Minty and Kedes (1986) Mol. Cell. Biol. 6:2125— 2136); cardiac troponin T (TNNT2) promoter (Farza et al. (1998) J. Mol. Cell Cardiol. 30(6): 1247-53); cardiac troponin C (TNNC1) promoter (Scheier et al.
  • cardiac myosin-binding protein C promoter (Lin et al. (2013) PLoS ONE 8(7): e69671); cardiac troponin I (TNNI3) promoter (Bhavsar et al.
  • myosin light chain 2A promoter myosin light chain 2v (MLC-2V) promoter
  • myosin light chain 2v (MLC-2V) promoter muscle creatine kinase (MCK) promoter
  • MCK muscle creatine kinase
  • CMV-MLC2 or CMV-MLC1.5, CMV-MLC260 phosphoglycerate kinase (PGK) promoter
  • the gene product is also under the control of one or more enhancers, preferably one or more cardiac-specific enhancers such as human calsequestrin 1 (CASQ1) enhancer.
  • CASQ1 human calsequestrin 1
  • methods for delivering heterologous nucleotide sequences to cardiac tissue or the heart while minimizing delivery to peripheral organs such as the liver are provided utilizing an rAAV comprising a variant capsid protein as herein described.
  • the rAAV may be employed to deliver a nucleotide sequence of interest to a cardiac cell in vitro, e.g., to produce a polypeptide or nucleic acid in vitro for ex vivo gene therapy.
  • the rAAV are additionally useful in a method of delivering a nucleotide sequence to a subject in need thereof, e g., to express a therapeutic or immunogenic polypeptide or nucleic acid.
  • the polypeptide or nucleic acid may thus be produced in vivo in the subject.
  • the subject may be in need of the polypeptide or nucleic acid because the subject has a deficiency of the polypeptide, or because the production of the polypeptide or nucleic acid in the subject may impart some therapeutic effect, as a method of treatment or otherwise.
  • the rAAV are useful to express a polypeptide or nucleic acid that provides a beneficial effect to the heart.
  • the ability to target rAAV to cardiac cell while minimizing delivery to the liver may be particularly useful to treat diseases or disorders involving cardiac dysfunction.
  • a method of delivering a nucleic acid of interest to a cardiac cell comprising contacting the cardiac cell with an AAV particle as herein described.
  • a method of delivering a nucleic acid of interest to a cardiac cell in a mammalian subject comprising administering an effective amount of the AAV particle as herein described or a pharmaceutical formulation comprising same to a mammalian subject.
  • delivery of the nucleic acid to the liver is minimized.
  • Representative cardiac cells to which the nucleic acid can be delivered include, cardiomyocytes, cardiomyoblasts, cardiac fibroblasts, smooth muscle cells and cardiac progenitor cells.
  • the nucleic acid is delivered to cardiomyocytes.
  • the rAAV virion can be delivered to cardiac tissue or the heart by intravascular (intravenous or intra-arterial) administration, direct cardiac injection (into the left atrium, right atrium, right ventricle and/or septum), antegrade or retrograde infusion into the coronary artery (via the left anterior descending or left circumflex coronary arteries), recirculation, intraperi cardial injection, trans endocardial injection, or by any other convenient mode or route of administration that will result in the delivery of the rAAV virion to cardiac tissue or the heart.
  • the rAAV virion is administered by intramyocardial injection.
  • a subject rAAV virion is delivered to cardiac tissue or the heart by systemic intravenous administration.
  • a method for the treatment of a cardiac disorder in a subject in need of such treatment by administering to the subject comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO:1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter, or administering to the mammal a pharmaceutical composition comprising said rAAV and a pharmaceutically acceptable carrier.
  • rAAV recombinant adeno-associated virus
  • the variant AAV capsid protein is at least 90%, at least 95%, at least 98%, at least 99% or is 100% identical to the amino acid sequence set forth as any one of SEQ ID NOs:4-13. In preferred embodiments, the variant AAV capsid protein is at least 90%, at least 95%, at least 98%, at least 99% or is 100% identical to the amino acid sequence set forth as any one of SEQ ID NOs:8-l 1 and 13.
  • the cardiac disorder is heart disease.
  • the method of treating or preventing a heart disease comprises transduction of primate (e.g., human) cardiomyocytes.
  • the heart disease is a cardiomyopathy, preferably selected from hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), restrictive cardiomyopathy (RCM) and left ventricular non-compaction cardiomyopathy (LVNC).
  • the cardiomyopathy may be primary cardiomyopathy, preferably inherited cardiomyopathy, cardiomyopathy caused by spontaneous mutations, and acquired cardiomyopathy, preferably ischemic cardiomyopathy caused by atherosclerotic or other coronary artery diseases, cardiomyopathy caused by infection or intoxication of the myocardium.
  • the heart disease is heart failure or chronic heart failure, optionally selected from heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mid-range ejection fraction (HFmrEF).
  • HFpEF preserved ejection fraction
  • HFrEF heart failure with reduced ejection fraction
  • HFmrEF heart failure with mid-range ejection fraction
  • the heart disease is congestive heart failure, coronary heart disease.
  • the heart disease is selected from angina pectoris, cardiac fibrosis, myocarditis, endocarditis, congenital cardiovascular defects, a valvular heart disease, myocardial infarction, atrial arrhythmia, ventricular arrhythmia, diastolic heart failure, systolic heart failure, left ventricular non-compaction, ventricular septal defect, ischemia and cardiac hypertrophy.
  • the cardiac disorder is selected from Barth syndrome, Frei dri ch’s Ataxia, Catecholaminergic polymorphic ventricular tachycardia (CPVT), Marfan syndrome, Noonan syndrome, Brugada syndrome, Long QT Syndrome, Myotonic Dystrophy 1 , Limb Girdle Dystrophy Type IB, Naxos disease, Loeys-Dietz syndrome, X-Linked Emery-Dreifuss muscular dystrophy (EDMD) and SV Aortic Stenosis.
  • CPVT Catecholaminergic polymorphic ventricular tachycardia
  • Marfan syndrome Noonan syndrome
  • Brugada syndrome Brugada syndrome
  • Long QT Syndrome Long QT Syndrome
  • Myotonic Dystrophy 1 Limb Girdle Dystrophy Type IB
  • Naxos disease Loeys-Dietz syndrome
  • EDMD X-Linked Emery-Dreifuss muscular dystrophy
  • the use of an rAAV in the treatment of a cardiac disorder or for the manufacture of a medicament for the treatment of cardiac disorder comprises (i) a capsid comprising a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO:1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter.
  • the rAAV comprises a capsid comprising a capsid protein having the amino acid sequence of SEQ ID NO: 1 1 .
  • the rAAV is administered by intravascular (e g. intravenous) injection.
  • a pharmaceutical composition for use in treating a cardiac disorder comprising a pharmaceutically acceptable carrier and an infectious rAAV comprising (i) a capsid comprising a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter.
  • the infectious rAAV comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs:4-13 or an amino acid sequence at least 85% identical thereto and (ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat (b) a CAG or CMV promoter (c) a nucleic acid encoding a gene product (d) a polyadenylation sequence and (e) an AAV2 terminal repeat.
  • the infectious rAAV comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs:8-l 1 and 13 or an amino acid sequence at least 85% identical thereto and (ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat (b) a CAG or CMV promoter (c) a nucleic acid encoding a gene product (d) a polyadenylation sequence and (e) an AAV2 terminal repeat
  • the subject to be treated is a human or non-human primate.
  • Non-human primates include, but not limited to, monkeys, squirrel monkeys, owl monkeys, baboons, chimpanzees, marmosets, gorillas, apes, lemurs, macaques and gibbons.
  • a human primate comprises a human.
  • the pharmaceutical composition comprises 1 x 10 12 to 1 x 10 15 vector particles/kg or vector genomes/kg, 1 x 10 12 to 1 x 10 15 vector particles or vector genomes, or about 1 x 10 12 , about 2 x 10 12 , 3x 10 12 , about 4 x 10 12 , about 5 x 10 12 , about 6 x 10 12 , about 7 x 10 12 , about 8 x 10 12 , about 9 x 10 12 , about 1 x 10 13 , about 2 x 10 13 , about 3 x 10 13 , about 4 x 10 13 , about 5 x 10 13 , about 6 x 10 13 , about 7 x 10 13 , about 8 x 10 13 , about 9 x 10 13 , about 1 x 10 14 , about 2 x T O 14 , about 3 x IO 14 , about 4 x 10 14 , about 5 x TO 14 , about 6 x 10 14 , about 7 x TO 14 , about
  • the pharmaceutical composition comprises about 3 x 10 12 , about 1 x 10 13 or about 5 x 10 13 vector particles/kg or vector genomes/kg. In some preferred embodiments, the pharmaceutical composition is administered to a human with a disorder associated with cardiac dysfunction via intravenous injection.
  • the composition balance and identity of the resulting plasmid library was confirmed by full-length sequencing on a PacBio Sequel lie NGS instrument.
  • the plasmid library was transfected into a 293T-derived cell line using FuGene. Following transfection, cells and supernatant were collected and lysed, endonuclease treated, clarified via sterile filtration, and frozen. Thawed harvest material was clarified and loaded onto relevant affinity resins, eluted at low pH and immediately neutralized.
  • the manufactured AAV library was sequenced on a PacBio Sequel lie NGS and tittered using digital droplet PCR. In addition, aggregation by dynamic light scattering and endotoxin composition by the limbus amebocyte lysate (LAL) method was determined.
  • LAL limbus amebocyte lysate
  • Frozen tissue was thawed, homogenized and subject to genomic DNA isolation followed by full length AAV variant Cap gene PCR amplification using serotype agnostic primers.
  • the resulting capsid PCR product was sequenced on a PacBio Sequel lie NGS instrument and output sequences were aligned to WT AAV reference sequences to identify the specific unnatural variations in each sequence. Data was normalized to total read count per sample.
  • iPSC-CMs were obtained from FujiFilm and cultured according to manufacturer’s instructions. Briefly, cells were seeded into 24-well plate at a density of 156,000 cells/cm 2 in plating medium and incubated for 48 hours. Subsequently, the cells were fed with maintenance medium, with medium changes performed every 48 hours.
  • Human iPSC-cardiomyocytes were transduced 6 days after seeding with a multiplicity of infection (MOI, vg/cell) of 5,000, 10,000 or 20,000. Samples were collected post infection for analysis.
  • MOI multiplicity of infection
  • Human primary hepatocytes were transduced one day after seeding with a multiplicity of infection (MOI, vg/cell) of 10,000, 20,000 or 50,000. Samples were collected post infection for analysis.
  • MOI multiplicity of infection
  • iPSC-CMs were dissociated into single-cell suspensions using TrypLE solution. Subsequently, cells were fixed and permeabilized using Cytofix/Cytoperm solution obtained from BD Bioscience. Cells were then stained using a cardiac Troponin T (cTnT) conjugated antibody at 4 °C for 30 min in dark following with multiple wash steps. Finally, the samples were loaded onto a BD Celesta flow cytometer for analyzing the cTnT and GFP positive populations.
  • cTnT cardiac Troponin T
  • Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100. Primary antibody was added for 18 hours at 4°C, followed by a secondary antibody and nuclear counterstain for 1 hour at room temperature. Cells were imaged on a Zeiss Axiovert fluorescent microscope in blue, green and red channels.
  • Results Table 1 depicts the sequence frequencies of top capsid variants as determined by PacBio full- length capsid sequencing compared between in vitro iPSC-cardiomyocytes and NHP selections.
  • NHP non-human primate
  • AAV adeno-associated virus
  • MOI multiplicity of infection
  • iPSC induced pluripotent stem cell
  • * indicates SEQ ID NO.l
  • Linker 1 glycine (G) - alanine (A);
  • Linker 2 alanine serine (AS) - alanine leucine serine (ALS), (dash represents the peptide insertion location); Shift 0, insertion between amino acid 588 and 589 of AAV; Shift -1, insertion between 587 and 588; Shift +1, insertion between 589 and 590; Shift -2 insertion between 586 and 587; Shift +2, insertion between 590 and 591.
  • Sequencing results identified a capsid comprising capsid protein of SEQ ID NO: 11 and other variants having a core insertion of SEQ ID NO: 1 with high frequency in NHP heart and iPSC-cardiomyocytes following transduction with Round 2 library (Table 1). The same variants also showed limited transduction in NHP liver tissue.
  • Table 1 [00140] Human iPSC-cardiomyocytes were transduced with rAAV comprising a capsid of SEQ ID NO: 11 or with wildtype AAV9 virions, each carrying an EGFP transgene operably linked to a CAG promoter, to monitor transduction efficiency. Seven days post transduction, cells were analyzed by immunocytochemistry (ICC) and flow cytometry.
  • ICC immunocytochemistry
  • capsid comprising a capsid protein of SEQ ID NO: 11 or wildtype capsid AAV9 carrying CAG-EGFP transgene to monitor transduction efficiency.
  • capsid comprising a capsid protein of SEQ ID NO: 11 or wildtype capsid AAV9 carrying CAG-EGFP transgene to monitor transduction efficiency.
  • Capsid comprising a capsid protein of SEQ ID NO: 11 transduced hepatocytes better than AAV9 by ICC images (Figure 2; immunocytochemistry of transduction efficiency of SEQ ID NO: 1 compared to AAV9 and positive control at three MOI in primary hepatocytes; EGFP, enhance green fluorescent protein (green); HNF4A, hepatocyte nuclear factor 4 alpha (red); DAPI, nuclei (blue); MOI, multiplicity of infection; AAV, adeno-associated virus).

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Abstract

L'invention concerne des variants de capsides du virus adéno-associé (AAV) qui confèrent une transduction accrue dans le cœur. L'invention concerne également des méthodes d'administration d'un transgène au cœur et des méthodes de traitement de troubles cardiaques par mise en contact de cellules cardiaques avec un AAV recombinant (rAAV) contenant un variant de capside d'AAV et un acide nucléique hétérologue codant pour un transgène.
PCT/US2024/038526 2023-07-27 2024-07-18 Variants d'aav pour une administration cardiaque Pending WO2025024227A2 (fr)

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