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WO2024238684A1 - Capsides aav de pénétration de barrière hémato-encéphalique modifiées - Google Patents

Capsides aav de pénétration de barrière hémato-encéphalique modifiées Download PDF

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WO2024238684A1
WO2024238684A1 PCT/US2024/029507 US2024029507W WO2024238684A1 WO 2024238684 A1 WO2024238684 A1 WO 2024238684A1 US 2024029507 W US2024029507 W US 2024029507W WO 2024238684 A1 WO2024238684 A1 WO 2024238684A1
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capsid protein
seq
aav capsid
amino acid
aav
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Matthew TIFFANY
David OJALA
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Sangamo Therapeutics Inc
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Sangamo Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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
    • 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
    • A61P25/00—Drugs for disorders of the nervous 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
    • 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

  • This application relates to engineering AAV capsids.
  • BBB blood brain barrier
  • CNS-targeting molecules i.e., targeting peptides
  • an engineered AAV capsid protein is provided wherein a peptide sequence is inserted into a parent capsid at a peptide insertion site.
  • the peptide sequence, the parent capsid, and the peptide insertion site are as indicated in a single row of Table 1.
  • an adeno-associated virus (AAV) capsid protein comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any amino acid sequence set forth in any one of SEQ ID NO: 1-1232.
  • the amino acid sequence comprises, consists of, or consists essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 71.
  • the AAV capsid protein comprises, consists of, or consists essentially of serotype AAV2.
  • the serotype AAV2 comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1233.
  • the amino acid sequence is inserted into the AAV2 capsid protein between amino acids 588 and 589.
  • the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1233.
  • the AAV capsid having SEQ ID NO 71 inserted therein protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1233.
  • the AAV capsid protein comprises, consists of, or consists essentially of serotype AAV9.
  • the serotype AAV9 comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1234.
  • the amino acid sequence is inserted into the AAV9 capsid protein between amino acids 587 and 590.
  • the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235.
  • the AAV capsid having SEQ ID NO 71 inserted therein protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1235.
  • the AAV capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in any one of SEQ ID NO: 1-1232. In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 71.
  • the AAV capsid protein comprises, consists of, or consists essentially of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74.
  • the AAV capsid protein comprises, consists of, or consists essentially of AAV2 or AAV9.
  • the amino acid sequence is inserted into a surface exposed region of the AAV capsid.
  • an AAV capsid protein comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of an amino acid sequence set forth in any of SEQ ID NO: 1-1232, wherein the amino acid sequence is inserted between amino acid positions 450 and 600 of the AAV capsid protein.
  • the amino acid sequence is inserted between amino acid positions 588 and 589 of the AAV capsid protein.
  • the AAV capsid protein comprises, consists of, or consists essentially of the sequence forth in SEQ ID NO: 1233.
  • the amino acid sequence comprises the sequence set forth in SEQ ID NO: 71.
  • the amino acid sequence is inserted between amino acid positions 587 and 590 of the AAV capsid protein.
  • the AAV capsid protein comprises consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1234.
  • the amino acid sequence comprises, consists of, or consists essentially of SEQ ID NO: 71.
  • the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235.
  • the AAV capsid protein comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of SEQ ID NO: 71.
  • the AAV capsid protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1235.
  • a CNS-targeting molecule comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of an amino acid set forth in any of SEQ ID NO: 1-1232.
  • the amino acid sequence comprises, consists of, or consists essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 71.
  • the amino acid sequence is fused or conjugated to a small molecule, antibody, scFV, ASO, siRNA, lipid, polymer, or recombinant protein.
  • FIG. 1 illustrates an example schematic of the barriers and selective pressure to identify AAV variants that exhibited enrichment in CNS tissue.
  • FIG. 2 illustrates examples of SIFTER (Selecting in vivo for transduction and expression of RNA) capsid libraries linked to barcodes.
  • SIFTER Selecting in vivo for transduction and expression of RNA
  • capsid libraries linked to barcodes.
  • Each capsid is linked to multiple barcodes defined in a look-up table and created by pooled oligo synthesis. Different oligo pool designs allow for multiplexing libraries varying parental capsid identity or mutational strategy.
  • UMIs unique molecular identifiers
  • Barcodes and UMIs are expressed under the control of either a neuron specific human synapsin I or ubiquitous CMV promoter, allowing the read out of capsid performance in multiple cell types.
  • FIG. 3 shows the diversities of the five round 2 sub-libraries (Library A-E) that were multiplexed and cloned under expression of a neuron specific human synapsin 1 (hSynl) promoter or a ubiquitous CMV promoter.
  • the resulting 10 libraries were administered via intravenous injection, and an endpoint analysis to recover library transcripts was performed.
  • FIG. 4 provides data showing that the production of libraries is relatively uniform with most variants found +/- 32-fold change from the cloned library. As expected, variants that produce well are associated with more UMIs recovered. Bubbles circled in black outlines are variants that were selected for a third-round evaluation from NHP tissue samples. The position of the AAV9 in the library is highlighted.
  • FIGs. 5 and 6 The library was administered to two cynomolgus macaques and barcodes were recovered from CNS and peripheral tissues to assess biodistribution and expression of the library transcript.
  • FIG. 5 shows data from in vitro induced pluripotent stem cell (iPS) neuron transduction. Overall, the performance of the library in vitro is not predictive of the performance in vivo as many recovered variants from the NHP samples are less enriched in cultured neurons.
  • FIG. 6 shows data of in vivo library recovery from NHP samples. After filtering out variants that were poorly recovered, a total of 6,728 variants were identified from the human synapsin I library, and 4,005 variants from the CMV library. Based on consistency in recovery across animals and fold enrichment, 810 variants were selected from the hSynl library and 426 variants from the CMV library, for a final evaluation in NHP.
  • Bubble Plot Legend for FIGs. 5 and 6.
  • the graphs show the fold-change in variant enrichment normalized to the administered viral library on the y-axis and the coefficient of variation in the detection on the x-axis.
  • the bubble size corresponds to the fraction of replicates the variant was recovered from.
  • the color scale indicates the number of unique molecular identifiers (UMI) associated with each variant.
  • the top graphs are the variants’ performance under the control of the neuron specific hSynl promoter and the bottom graphs are under the control of the ubiquitous CMV promoter. Bubbled circles in black outlines are variants that were selected for a third-round evaluation from NHP tissue samples.
  • FIG. 7 The round 3 library was administered to three cynomolgus macaques and barcodes were recovered from CNS tissues to assess on-target biodistribution and expression of the library transcript.
  • the figure shows the performance of the 1236 variants in the round 3 library evaluation.
  • the log2 fold change (log2FC) in expression of each variant from the hSynl and CMV promoters is shown on the y-axis and the coefficient of variation of the log2 fold change measurement is shown on the x- axis.
  • the data represents assessment of whole brain transduction and mRNA expression mediated by each capsid in the library.
  • FIG. 8. The figure shows the performance of the 1236 variants in the round 3 library evaluation. The vector genome biodistribution of each variant is shown. The data represents assessment of whole brain transduction mediated by each capsid in the library.
  • FIG. 9 The graph shows the log2 fold-change in neuronal (hSynl) mRNA expression of CNSRCV300 (also referred to as SEQ ID NO: 1235, STAC-BBB, STAC/BBB, or STACBBB) and AAV9 across six brain slices analyzed per animal.
  • the box and whisker plots contains the rest of the round 3 library performance.
  • FIG. 10 The graph shows the log2 fold-change in ubiquitous (CMV) mRNA expression of CNSRCV300 and AAV9 across six brain slices analyzed per animal. The box and whisker plots contains the rest of the round 3 library performance.
  • FIG. 11 The graph shows the log2 fold-change in vector genome biodistribution of CNSRCV300 and AAV9 across four brain slices analyzed per animal. The box and whisker plots contains the rest of the round 3 library performance.
  • FIG. 12 The graph shows the log2 fold-change in mRNA expression and vector genome biodistribution of CNSRCV300 and AAV9 across all animals included in the study.
  • the box and whisker plots contains the rest of the round 3 library performance.
  • FIG. 13 The graph shows the log2 fold change in neuronal (hSynl) mRNA expression of CNSRCV300 and AAV9 in all tissues punches analyzed across the cortex, hippocampal region, deep brain regions, cerebellum and brain stem.
  • the neuronal (hSynl) mRNA expression in cervical, thoracic and lumbar spinal cord and dorsal root ganglia levels is also shown for CNSRCV300 and AAV9.
  • FIG. 14 The graph shows the log2 fold change in ubiquitous (CMV) mRNA expression of CNSRCV300 and AAV9 in all tissues punches analyzed across the cortex, hippocampal region, deep brain regions, cerebellum and brain stem.
  • CMV ubiquitous
  • the ubiquitous (CMV) mRNA expression in cervical, thoracic and lumbar dorsal root ganglia levels is also shown for CNSRCV300 and AAV9.
  • peripheral tissues mRNA expression in peripheral tissues is also shown for CNSRCV300 and AAV9.
  • FIG. 15 The image shows that CNSRCV300 drives widespread and robust transgene expression throughout the brain at a dose of 2el3 vg/kg. Negative control tissue without AAV treatment shows no signal.
  • FIG. 16 The images show that CNSRCV300 exhibits widespread transduction across all cortical regions.
  • FIG. 17 The images show that CNSRCV300 mediates efficient transduction of neurons in a variety of cortical and subcortical regions.
  • FIG. 18 The images show that CNSRCV300 mediates efficient transduction of neurons in the dentate nucleus.
  • FIG. 19 The images show that CNSRCV300 mediates efficient transduction of neurons in the Thalamus.
  • FIG. 20 The images show that CNSRCV300 mediates efficient transduction of neurons in the Putamen.
  • FIG. 21 The images show that CNSRCV300 mediates efficient transduction of neurons in the substantia nigra.
  • FIG. 22 The images show that CNSRCV300 mediates efficient transduction of neurons in the lateral geniculate nucleus.
  • FIG. 23 The images show that CNSRCV300 mediates efficient transduction of neurons in the pons.
  • FIG. 24 The images show that CNSRCV300 mediates efficient transduction of neurons in the precentral gyrus.
  • CNS-targeting molecules i.e., targeting peptides
  • CNS-targeting molecules comprising a targeting peptide seq indicated in Table 1
  • engineered AAV capsid proteins are provided.
  • a targeting peptide is inserted into an AAV capsid, for example an AAV9 capsid protein or an AAV2 capsid protein.
  • the targeting peptide is any of the targeting peptide disclosed in Table 1.
  • the peptide sequence is inserted into the AAV9 or AAV2 capsid at any of the peptide insertion sites disclosed in Table 1.
  • the targeting peptide comprises a targeting peptide that functions to target the CNS-targeting molecule to a specific target tissue (e.g., CNS tissue).
  • the desired characteristic is enhanced cell or tissue tropism as compared to the natural/wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural/wild-type AAV serotype
  • the desired characteristic is increased penetrance through the blood brain barrier following administration to a subject.
  • the desired characteristic is wider distribution throughout the multiple brain regions, e g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus.
  • the desired characteristic is elevated genetic material expression in multiple brain regions.
  • the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.
  • each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising a targeting peptide inserted into a hypervariable and/or surface exposed loop of the capsid protein: b) a nucleic acid sequence encoding barcode: c) one or more nucleic acid sequences encoding one or more promoters: and d) a nucleic acid sequence encoding a unique molecular identifier (UMI).
  • each member of the library also includes genetic material to be delivered to a cell or tissue of interest.
  • each member of the library also includes a polyA sequence.
  • the genetic material encodes one or more peptides or a polypeptides. In some embodiments, the genetic material encodes one or more antibodies or antibody fragments. In some embodiments the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNA.
  • the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are noncoding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.
  • mRNA is encoded in the genetic material.
  • the mRNA is codon optimized.
  • the genetic material encodes a gene therapy product.
  • a gene therapy product can include a peptide, a polypeptide, a recombination donor template, or an RNA molecule that when expressed carries out a desired therapeutic effect.
  • the therapeutic effect is treating any one more diseases or disorders described herein.
  • each member of the library is fused or coupled to an active agent.
  • each member of the library is fused or coupled to an active agent through conjugation.
  • the active agent comprises a therapeutic agent.
  • the therapeutic agent comprises a DNA-binding and/or targeting composition, for example, a zinc finger protein (ZFP), TAL-effector domain, and/or a sgRNA of CRISPR/Cas system.
  • the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region).
  • the peptide is fused to a Fc region of an antibody.
  • the peptide is fused to the C-terminus of the Fc region. In some embodiments, the peptide is fused to the N-terminus of the Fc region.
  • the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA).
  • the peptide is fused or coupled directly to at least one strand of the RNAi. In some embodiments, the peptide the peptide is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the peptide is fused or coupled to the sense strand of RNAi.
  • the peptide is fused or coupled to the antisense strand of RNAi.
  • the active agent comprises a diagnostic agent.
  • the diagnostic agent comprises a detectable moiety such as a fluorophore.
  • the active agent is a small molecule.
  • the promoter is operably linked to the genetic material to be delivered to the cell.
  • the one or more promoters comprises a tissue and/or cell specific promoter.
  • the one more promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others.
  • the one or more promoters comprise a cell type and/or tissue specific type promoter.
  • Exemplary cell type and/or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes.
  • Other non-limiting cell type and/or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific.
  • Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), CAMK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes).
  • tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS.
  • tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and/or Schwann cells.
  • the one or more promoters are naturally occurring promoters. In some embodiments, the one or more promoters are synthetic. In some embodiments, the one or more promoters is derived from mammals, humans, viruses, or plants. In some embodiments, the one or more promoters is truncated. In some embodiments, the one or more promoters is mutated.
  • a nucleic acid comprising a barcode is added to the genome of each AAV capsid proteins in a library.
  • the barcode is bioinformatically linked to the targeting peptide introduced into the capsid protein.
  • the DNA sequences encoding the targeting peptide are synthesized to further comprise a random or specified barcode.
  • the barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides.
  • each targeting peptide is linked to at least 2 distinct barcodes.
  • each barcode is linked to one or more UMI.
  • each member of the library comprises a nucleic acid comprising more than one barcode sequences. In some embodiments, each member of the library comprises two or more nucleic acids each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter.
  • the AAV capsid proteins are derived from AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9 serotypes.
  • the AAV variant capsid proteins are derived from less well characterized AAV serotypes, including but not limited to AAV4, AAV7, AAVrhlO, AAVrh39, and AAVrh74.
  • a library of AAV variants comprises AAV variant capsid proteins derived from a single AAV serotype.
  • a library of AAV variants comprises AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes.
  • the AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined once individual serotype libraries are developed.
  • combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.
  • the AAV serotype includes the AAV2 serotype. In some embodiments, the AAV2 serotype includes a mutation of arginine to alanine at amino acid position 588. In some embodiments, the AAV2 serotype includes the sequence SEQ ID NO: 1233. In some embodiments, the AAV serotype includes the AAV9 serotype. In some embodiments, AAV9 serotype includes the sequence of SEQ ID NO: 1234.
  • targeting peptides are introduced into aDNA sequence encoding an exposed loop in the capsid protein.
  • the targeting peptide is inserted into exposed loops (e.g. hypervariable regions) in the AAV capsid.
  • the targeting peptide comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids.
  • the targeting peptide comprises between 9 and 16 amino acids.
  • the targeting peptide is 9 amino acids in length.
  • the targeting peptide is 16 amino acids in length.
  • the HI loop is targeted (mutated) while in others, the DE loop is targeted (mutated).
  • mutations are made in both loops.
  • targeting peptides are introduced into the VR region of a surface loop, including into VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and or VR-IX.
  • targeting peptides are made in VR-I, VR-IV, and or VR-VIII.
  • targeting peptides are introduced into the AAV capsid proteins VP1, VP2 or VP3, or in two of the capsid proteins in any combination, or in all three. In some embodiments, the targeting peptides are introduced into VP1.
  • the targeting peptides are introduced into VP2. In some embodiments, the targeting peptides are introduced into VP3. In some embodiments, the targeting peptides are introduced into VP1 and VP2. In some embodiments, the targeting peptides are introduced into VP1 and VP3. In some embodiments, the mutations are introduced into VP2 and VP3. In some embodiments, the targeting peptides are introduced into VP1, VP2, and VP3. In some embodiments, the targeting peptide is introduced at a single site in a gene encoding a capsid protein.
  • a targeting peptide is introduced into the variable regions VR-I, VR-IV, or VR-VIII of the capsid protein.
  • the targeting peptide is introduced at a location between positions 450 and 600 of the capsid protein.
  • the targeting peptide is inserted between positions 587 and 590 of the capsid protein.
  • the targeting peptide is inserted between positions 587 and 590 of the AAV9 capsid protein.
  • the targeting peptide is inserted between positions 588 and 589 of the capsid protein.
  • position 588 has been altered from an arginine to an alanine.
  • the targeting peptide is inserted between positions 588 and 589 of the AAV2 capsid protein.
  • the targeting peptide comprises any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises at least 5, 6, 7, 8, or 9 contiguous amino acids of any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises a sequence that is at least 80% identical to any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises a sequence that is at least 80% identical, least 85% identical, at least 90% identical, at least 95%, or at least 99% identical to or comprises any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises SEQ ID NO: 71. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.
  • compositions comprising an engineered AAV capsid proteins and methods of making and using the same.
  • capsid proteins are useful in delivering peptides (e g., targeting peptides) across the blood brain barrier following administration to a subject.
  • the engineered AAV capsid proteins are useful in achieving is wider distribution of the genetic material throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus.
  • the engineered AAV capsid proteins are useful in elevating genetic material expression in multiple brain regions.
  • the engineered AAV capsid proteins are used to deliver genetic material of interest to a desired tissue, cell, or organelle.
  • the genetic material of interest comprises a gene editing system or portions of a gene editing system.
  • the gene editing system is capable of inducing single or double-stranded breaks into nucleic acid sequences.
  • the gene editing system is capable of inserting, substituting, or deleting a base or a sequence of bases into nucleic acid sequences.
  • the gene editing system includes a CRISPR-Cas system.
  • the gene editing system includes a TALEN.
  • the gene editing system includes a zinc finger nuclease.
  • engineered AAV capsid proteins are contained within a cell.
  • the cell is derived from the CNS.
  • the cell is derived from the PNS.
  • the cell is derived from the brain.
  • the cell is derived from the spinal cord.
  • the cell is derived from any of the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the cerebral cortex, the brain stem, the hippocampus, or the thalamus, amongst others.
  • the engineered AAV capsid proteins may be delivered to one or more target cells, tissues, organs, or organisms.
  • the engineered AAV capsid proteins demonstrate enhanced tropism for a target cell type, tissue or organ.
  • the engineered AAV capsid proteins may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (PNS), or cells and tissues of a muscle.
  • PNS central or peripheral nervous systems
  • the engineered AAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.
  • the engineered AAV capsid proteins may have enhanced tropism for B cells, hematopoietic cells, leukocytes, platelets, macrophages, megakaryocytes, monocytes and/or T cells.
  • a method of identifying an engineered AAV capsid protein with a desired characteristic compared to a natural/wild-type AAV serotype comprising: (i) contacting a cell, cell line, or tissue in vitro or in vivo with any one of the libraries of engineered AAV capsid proteins, (ii) allowing the engineered AAV capsid proteins in said library to transduce the cell, cell line, or tissue; (iii) recovering from the cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.
  • the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural/wild-type AAV serotype comprise (i) insertion of targeting peptides into hypervariable and/or surface-exposed loops in capsid proteins from one or more AAV serotypes creating libraries of modified variant capsids for each AAV serotype; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation
  • the desired characteristic includes enhanced tissue tropism as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism of the central nervous system as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced ability to cross the blood brain barrier as compared to the natural/wild-type AAV serotype. In some embodiments, the targeting peptides that are inserted into the hypervariable and/or surface-exposed loops include any of the sequences set forth in SEQ ID NO: 1-1232. In some embodiments, SEQ ID NO: 71 is inserted into the hypervariable and/or surface exposed loop.
  • capsid proteins for example AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9 are chosen for starting points.
  • capsid proteins from less well characterized AAV serotypes are chosen, including but not limited to AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, and AAVrh74.
  • a library of AAV variants comprises AAV variant capsid proteins derived from a single AAV serotype.
  • a library of AAV variants comprises AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes.
  • the AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined once individual serotype libraries are developed.
  • combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.
  • the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, E1A and E1B) are supplied in trans.
  • the AA V rep function comprises rep78, rep68, rep52, and rep40 genes.
  • the rep genes are supplied in trans.
  • the start codon of the rep78 and/or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield.
  • the cap genes are supplied as genetic material to the manufactured AAVs.
  • the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.
  • CNS Central Nervous System
  • Targeting Molecules Targeting Peptides
  • CNS-targeting molecules i.e, targeting peptides.
  • the CNS-targeting molecules have enhanced tropism for a cell or tissue, such as the delivery of genetic material of interest to said cell or tissue, for example a CNS tissue or PNS tissue or a CNS cell or PNS cell.
  • the CNS-targeting molecule comprises a sequence set forth in SEQ ID NO: 1-1232.
  • the targeting peptide comprises at least 5, 6, 7, 8, or 9 contiguous amino acids of a sequence set forth in SEQ ID NO: 1-1232.
  • the targeting peptide comprises the sequence set forth in SEQ ID NO: 71.
  • the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.
  • the CNS-targeting molecule comprises variants of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant comprises 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant comprises 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant comprises 1, 2, 3, or 4 insertions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In some embodiments, the variant comprises any combination of the substitutions, deletions, or insertions described above.
  • a variant refers to a variant in the nucleotide sequence that encodes any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant in the nucleotide sequence results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant in the nucleotide sequence encodes 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232.
  • the variant in the nucleotide sequence encodes 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 insertions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes any combination of the substitutions, deletions, or insertions described above.
  • the CNS-targeting molecule is fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein.
  • any of SEQ ID NO: 1-1232 are fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein.
  • SEQ ID NO: 71 is fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein.
  • CNS-targeting molecules may be utilized to enable a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.
  • the CNS-targeting molecules are part of an engineered AAV capsid protein.
  • the engineered capsid protein comprises any of the serotypes of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9.
  • the capsid protein comprises the serotype AAV2 or AAV9.
  • the AAV2 serotype comprises SEQ ID NO: 1233.
  • the AAV2 serotype comprises a variant of SEQ ID NO: 1233.
  • the AAV9 serotype comprises SEQ ID NO: 1234.
  • the AAV9 serotype comprises a variant of SEQ ID NO: 1234.
  • a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids in either of the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions of any of the amino acids in either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 deletions of any of the amino acids in either of the amino acid sequences set forth of SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 insertions of any of the amino acids in either of the amino acid sequences set forth SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant comprises an amino acid sequence that comprises at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234 or SEQ ID NO: 1233, and at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of SEQ ID NOS: 1- 1232.
  • the AAV2 serotype comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO:
  • the AAV9 serotype comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO:
  • a variant refers to a variant in the nucleotide sequence that encodes the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant in the nucleotide sequences results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of either of the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234
  • the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions of any of the amino acids in either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234.
  • the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234 or SEQ ID NO: 1233, and at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of SEQ ID NOS: 1-1232
  • a nucleotide sequence encodes an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1233. In some embodiments, a nucleotide sequence encodes an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1234.
  • any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into an AAV.
  • any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into AAV2.
  • AAV2 comprises the sequence set forth in SEQ ID NO: 1233.
  • SEQ ID NO: 71 is inserted into SEQ ID NO: 1233.
  • SEQ ID NO: 1233 is AAV2 with an amino acid substitution.
  • any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into AAV9.
  • AAV9 comprises the sequence set forth in SEQ ID NO: 1234.
  • SEQ ID NO: 71 is inserted into SEQ ID NO: 1234.
  • SEQ ID NO: 1234 is AAV9.
  • SEQ ID NO: 71 inserted into SEQ ID NO: 1234 can result in SEQ ID NO: 1235.
  • insertion of SEQ ID NO: 71 into AAV9 results in the sequence set forth in SEQ ID NO: 1235 (CNSRCV300).
  • insertion of SEQ ID NO: 71 into AAV9 results in a variant sequence of SEQ ID NO: 1235.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions, insertions, and/or deletions of any of the amino acids in the amino acid sequence of SEQ ID NO: 1235.
  • the variant comprises at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with SEQ ID NO: 1235 or comprises SEQ ID NO: 1235. In embodiments, the variant comprises at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.
  • the variant refers to a variant in the nucleotide sequence that encodes the amino acid sequences set forth in SEQ ID NO: 1235.
  • the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions, insertions, and/or deletions of any of the amino acids in the amino acid sequence of SEQ ID NO: 1235.
  • the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with SEQ ID NO: 1235. In embodiments, the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.
  • the CNS-targeting molecules are part of an engineered AAV capsid protein, and the engineered AAV capsid proteins are packaged into AAV particles.
  • the AAV particles that have enhanced tropism for a target tissue e.g., CNS and PNS
  • CNS-targeting molecules may be inserted into an AAV capsid protein sequence to alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro.
  • the AAV particles are capable of penetrating the blood brain barrier.
  • the AAV particles may be delivered to one or more target cells, tissues, organs, or organisms.
  • the AAV particles demonstrate enhanced tropism for a target cell type, tissue or organ.
  • the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively), or cells and tissues of a muscle.
  • the AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.
  • the AAV particles are used to deliver a viral genome to a tissue or cells such as CNS or PNS cell or tissue.
  • the delivered viral genome may include genetic material of interest, such as, for example, an antibody, an enzyme, or regulatory RNA, amongst others.
  • the viral genome includes at least one ITR sequence.
  • the viral genome includes 2 ITR sequences.
  • the ITR sequences flank the genetic material of interest.
  • the ITR sequences are complementary to each other.
  • the ITR regions are derived from the same serotype as the capsid protein. ITR regions may be between 100 and 150 nucleotides in length.
  • the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating
  • the genome of the virus contains the components required for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver genetic material of interest to the particular tissue.
  • AAV capsid proteins comprising CNS-targeting molecules (targeting peptides)
  • the CNS-targeting molecules i.e., targeting peptides
  • the CNS-targeting molecules are part of a recombinant AAV capsid protein.
  • AAV capsid proteins described herein may be produced recombinantly and may be based on adeno-associated virus (AAV) wild type sequence.
  • CNS-targeting molecules may be inserted into an AAV capsid protein sequence to alter tropism relative to the natural AAV capsid protein, to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. Stated another way, CNS-targeting molecules, which refer to the targeting peptides, that are inserted into the capsid protein, allow the capsid protein to penetrate the blood brain barrier.
  • the targeting peptide is used for enhanced or improved transduction of a target cell or tissue (e.g., cells or tissues of the central nervous system (CNS) or peripheral nervous system (PNS)).
  • a target cell or tissue e.g., cells or tissues of the central nervous system (CNS) or peripheral nervous system (PNS)
  • the targeting peptide is used to facilitate the AAV capsid protein across the blood brain barrier following administration to a subject.
  • the targeting peptide is used for enhanced or improved distribution of the genetic material throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus.
  • the targeting peptide is used for enhanced or improved genetic material expression in multiple brain regions.
  • the targeting peptide is used for enhanced or improved delivery of genetic material of interest to a desired tissue, cell, or organelle.
  • the targeting peptide increases tropism of the AAV capsid to a cell, region, or tissue of the CNS.
  • CNS cells include but are not limited to neurons (e.g., excitatory neurons, inhibitory neurons, and motor neurons) and glial cells (e.g., ependymal cells, astrocytes, oligodendrocytes.
  • CNS tissue include but are not limited to the cortex (e.g., frontal cortex, parietal cortex, occipital cortex, temporal cortex), thalamus, hypothalamus, striatum, hippocampus, entorhinal cortex, and basal ganglia.
  • the AAV capsid protein comprising a targeting peptide is capable of increased tropism by at least 1.1 -, 1.2-, 1.3-, 1.4-, 1.5-fold, relative to an AAV capsid protein that lacks a targeting peptide. In some embodiments, the AAV capsid protein comprising a targeting peptide is capable of increased tropism by over 1.5-fold, relative to an AAV capsid protein that lacks a targeting peptide.
  • the AAV capsid protein comprising the targeting peptide facilitates increased expression of delivered genetic material (e.g., a therapeutic cargo) by at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-fold in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a targeting peptide.
  • the AAV capsid protein comprising the targeting peptide facilitates increased expression of delivered genetic material (e.g., a therapeutic cargo) by more than 1.5-fold in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a targeting peptide.
  • the targeting peptide is between 6 amino acids and 20 amino acids in length, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some examples, the targeting peptide is between 9 and 16 amino acids in length. In some examples, the targeting peptide is 9 amino acids in length. In some examples, the targeting peptide is 16 amino acids in length.
  • the targeting peptide comprises an amino acid sequence of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71. In some embodiments, the targeting peptide is part of an AAV vector. In some embodiments, the targeting peptide is part of a capsid protein of the AAV vector.
  • nucleic acid sequences encode targeting peptides.
  • the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises an amino acid sequence of any sequence set forth in SEQ ID NO: 1-1232.
  • the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises an amino acid sequence of SEQ ID NO: 71.
  • the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any sequence set forth in SEQ ID NO: 1-1232.
  • the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.
  • a targeting peptide is a part of a capsid protein, and the targeting peptide is inserted at a location between amino acid residues 450 and 600 of the capsid protein. In some embodiments, the amino acid sequence is inserted at a location between amino acid residues 587 and 590 of the AAV9 capsid protein. In some embodiments, the amino acid sequence is inserted at location between amino acid residues 588 and 589 of the AAV2 capsid protein.
  • a peptide sequence that comprises any of the sequences set forth in SEQ ID NO: 1-1232 is inserted into the capsid protein.
  • the peptide sequence comprises 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of the sequences set forth in SEQ ID NO: 1-1232.
  • disclosed is a peptide sequence that comprises the sequence set forth in SEQ ID NO: 71.
  • the peptide sequence comprises 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.
  • a targeting peptide is inserted into any of the AAV capsid protein comprises any of the AAV serotypes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9.
  • the AAV capsid protein comprises the AAV2 serotype.
  • the AAV2 serotype includes a mutation at position 588 from arginine to alanine.
  • the AAV2 serotype comprises the sequence of SEQ ID NO: 1233.
  • an amino acid sequence is inserted between positions 450 and 600 of SEQ ID NO: 1233.
  • an amino acid sequence is inserted between positions 588 and 589 of SEQ ID NO: 1233.
  • the AAV capsid protein comprises the AAV9 serotype.
  • the AAV9 serotype includes the sequence of SEQ ID NO: 1234.
  • an amino acid sequence is inserted between positions 450 and 600 of SEQ ID NO: 1234.
  • the amino acid sequence is inserted between positions 587 and 590 of SEQ ID NO: 1234.
  • the amino acid sequence inserted into the AAV2 or AAV9 serotypes comprises an amino acid sequence set forth in SEQ ID NO: 1-1232.
  • the amino acid sequence inserted into the AAV2 or AAV9 serotypes comprises an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, insertion of the amino acid sequence set forth in SEQ ID NO: 71 into an AAV9 serotype results in the sequence set forth in SEQ ID NO: 1235 (CNSRCV300).
  • a targeting peptide is inserted into an AAV capsid protein. Any targeting peptide described herein may be inserted into a parent AAV capsid protein in any location that results in fully functional AAV particles.
  • the targeting peptide may be inserted into capsid proteins VP1, VP2 and/or VP3.
  • a targeting peptide is inserted in a hypervariable region of the AAV capsid protein. Non-limiting examples of such hypervariable and/or surface exposed loop of the AAV capsid protein.
  • the targeting peptide is inserted into the Hl loop.
  • the targeting peptide is inserted into the DE loop.
  • the targeting sequencing is inserted into the variable region of the surface exposed loop, for example any of VR-I, VR-II, VR-III, VR- IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX.
  • the targeting peptide comprises any of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises SEQ ID NO: 71.
  • the AAV capsid proteins described herein have enhanced tropism for a specific cell or tissue, for example, a CNS or PNS cell or tissue.
  • the enhanced tropism for a specific cell or tissue is due to the insertion of any of the sequences set forth in SEQ ID NO: 1-1232 into the AAV capsid protein.
  • the enhanced tropism for a specific cell or tissue is due to the insertion of SEQ ID NO: 71 in to the AAV capsid protein.
  • the AAV capsid proteins are capable of penetrating the blood brain barrier.
  • the AAV capsid proteins described herein are capable of penetrating the blood brain barrier due to the insertion of any of the sequences set forth in SEQ ID NO: 1-1232 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of penetrating the blood brain barrier due to the insertion of SEQ ID NO: 71 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions including, but not limited to the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, and hippocampus.
  • the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions due to the insertion of any of the SEQ ID NO: 1-1232 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions due to the insertion of SEQ ID NO: 71 into the AAV capsid protein.
  • compositions described herein including CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV particles, and AAV vectors can be included in pharmaceutical compositions.
  • the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material, (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and/or (7) allow for regulatable expression of the genetic material.
  • compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period.
  • pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, every three years, every five years, every 10 years, every 20 years, or a one-time administration.
  • compositions described herein can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical.
  • Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients.
  • the compositions described herein can also be formulated for injection, insufflation, infusion, or intradermal exposure.
  • an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity.
  • the compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.
  • the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and/or alter the release profile.
  • compositions described herein CNS -targeting molecules (targeting peptides), AAV capsid proteins, AAV particles, and AAV vectors) into cells and/or tissues are provided.
  • the methods comprise introducing into cells and/or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and/or protein in the cells and/or tissues.
  • the compositions described herein are delivered via a localized delivery route.
  • the localized delivery route includes any one or more of intramuscular administration, intraparenchymal administration, and intracerebral administration, amongst others.
  • the compositions described herein are administered via a localized delivery route through a bolus infusion.
  • compositions described herein are administered through systemic administration.
  • systemic administration includes intravenous administration.
  • intravenous administration includes subcutaneous administration.
  • systemic administration includes intraventricular administration.
  • compositions described herein are administered to the central nervous system of via intraventricular administration and/or intravenous administration.
  • compositions described herein are administered to the central nervous system via systemic administration.
  • systemic administration is intravenous (IV) injection.
  • CNS-targeting molecules described herein are administered to the central nervous system via intraventricular administration.
  • compositions can be delivered to target cell or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • target cell or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • the target cell or tissue includes, but is not limited to CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • the target cell or target tissue is a CNS cell or tissue.
  • the target cell or tissue is liver cell or tissue.
  • the target cell includes, but is not limited to, neurons, glial cells, astrocytes, oligodendroglia, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat storing cells, Kupffer cells, liver endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.
  • compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via inj ection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.
  • a method of delivering a nucleic acid to a target cell or tissue of a subject comprising: administering a composition comprising an AAV vector, wherein the AAV vector further comprises a capsid protein comprising a targeting peptide comprising at least 5, 6, 7, 8, or 9 contiguous amino acids of an amino acid sequence set forth in SEQ ID NO: 1-1232.
  • the capsid protein comprises the targeting peptide set forth in any one of SEQ ID NO: 1-1232.
  • the compositions described herein are administered as part of a composition that allows for extended release.
  • the compositions comprise a formulation that includes a depot.
  • compositions described herein CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV vectors, and AAV particles).
  • the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases/disorders, and neurodegenerative disorders, amongst others.
  • the disclosed compositions can be used to treat any one or more of Alzheimer's disease, Huntington's disease; autism; Parkinson's disease; Spinal muscular atrophy, Friedreich's ataxia.
  • the disclosed compositions are used in treatments through any of the methods of delivery described herein.
  • compositions described herein comprising administering to the subject any effective amount of at least one of the compositions described herein (CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV vectors, and AAV particles), delivering the compositions described herein into targeted cells, inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject.
  • CNS-targeting molecules targeting peptides
  • AAV capsid proteins AAV capsid proteins
  • AAV vectors and AAV particles
  • Capsid variants for library screening were synthesized as an oligo pool. Each capsid peptide was synthesized with unique nucleotide sequences encoding the peptide, and each peptide was linked to at least two distinct barcodes.
  • the oligo pool was cloned into a linearized intermediate plasmid, followed by cloning of a constant donor sequence to separate the barcode and peptide region and generate the full AAV vector construct. Two separate constant donor sequences were used, generating the barcoded library transcript under the control of a neuron specific promoter or a ubiquitous promoter. This enables an assessment of which capsids drive the most functional mRNA expression in neurons as well as all cell types transduced.
  • each barcode is linked to a unique molecular identifier (UMI). Based on the overall size of the cloned library each barcode is appended to hundreds to thousands of UMIs. The utility of the UMI is to additionally assess how many distinct AAV transduction events give rise to the NGS read counts that are measured.
  • UMI unique molecular identifier
  • peptide sequences listed in Table 1 were inserted into variable region 8 of AAV serotypes 2 and 9. Where indicated in Table 1, peptides are inserted into AAV9 (SEQ ID 1234) between 587/590, the insertions are after amino acid 587 and before 590 replacing amino acids 588 and 589. Other peptides inserted into AAV9 indicated in Table 1 are inserted between amino acids 588/589. Peptides inserted into AAV2, were inserted between positions 588/589 and the wildtype arginine at position 588 is altered to alanine (R588A) (Seq ID 1233).
  • AAV plasmid libraries were manufactured in HEK293 cells. Briefly, libraries were produced by transient transfection including supplementation of Rep in trans, capsids were purified by cesium density centrifugation, and buffer exchanged into PBS by Amicon filtration. DNase-resistant viral genomic titers were measured by quantitative real time PCR.
  • the brain was removed and placed in a coronal brain matrix in an ice-cold nuclease free PBS bath for approximately 10 minutes.
  • the brain was sliced at a 4 mm coronal slice thickness. All slices were hemisected along the mid-sagittal plane.
  • the brain slices were processed and stored according to the following table.
  • Brain slices collected for mRNA and DNA molecular analysis (NGS) were placed in chilled RNA Later and refrigerated (1 to 8°C) for approximately 24 to 72 hours to preserve mRNA integrity. After storage, 2 mm punches were collected according to the Sponsor- provided brain template. The brain punch template is maintained in the raw data. Following removal from RNA Later and prior to obtaining the punches, the brain slices were placed on a flat surface and cross reference labels added to the brain slice and photographed (photographs are maintained with the raw data). All brain punch samples were placed into clean vials, frozen on dry ice and stored at -60°C or below until shipment. The residual brain slices, following sample collection, were frozen and stored at -60°C or below until shipment.
  • the spinal cord with dorsal root ganglia (DRG) attached were divided into four segments (cervical, thoracic, lumbar and sacral). From the central portion of each segment, a single 2-2.5 cm cross-sections were collected for potential evaluation. The cross-sections were placed in RNA Later and refrigerated (1 to 8°C) for approximately 24 to 72 hours after which the samples were frozen at -60°C or below until shipment. Right and left DRG pairs associated with the portion of each segment was removed and frozen (left and right saved together) on dry ice and stored at -60°C or below until shipment.
  • Biodistribution-Other Tissues An approximate 500-600 mg sample of tissue from the liver, testes, pancreas, lung, skeletal muscle (quadriceps), heart, kidney, lymph node, and spleen were collected for analysis. Samples were frozen on dry ice and stored at -60°C or below until shipment.
  • Brain punches to be processed were placed on dry ice. For spinal cord, DRG and liver, approximately 35 mg of each tissue sample was excised on dry ice. Each tissue sample was then transferred to an Eppendorf tube pre-filled with 600 pL of TRIZOL and two 3.2 mm steel beads Sample tubes were placed into a Retsch MM300 Tissue-Lyser and 8 rounds of homogenization were performed at a frequency of 25.1 Hz for 1 1/2 minutes with a 2-minute pause between rounds to prevent overheating.
  • RNA isolation and purification of total RNA from homogenized tissue was performed using the MagMAXTM-96 Total RNA Isolation Kit in conjunction with the KingFisherTM Flex Purification System according to the manufacturer’s protocol. Briefly, bromochloropropane (BCP) was added and then centrifugation was performed to separate the homogenate into aqueous and organic phases. The aqueous phase containing partially purified RNA was then transferred to a 96 well KingFisher processing plate. Isopropanol (100%) was added to each well followed by addition of magnetic RNA binding beads. Subsequent processing was performed on the KingFisherTM Flex Purification System.
  • BCP bromochloropropane
  • RNA binding beads were magnetically captured and an on-bead DNase digestion and several washes were performed. Purified RNA was eluted in 100 pL of low salt elution buffer. The KingFisher processing plate was then transferred to a magnetic stand on the benchtop. The eluants ( ⁇ 90 pL) were transferred away from any residual beads into a 96-well PCR plate for downstream processing. The yield and purity of the RNA was determined using a NanoDrop 8000 spectrophotometer.
  • Hemisected brain slices in cassettes to be processed were placed on dry ice. Each brain slice was removed from the cassette, weighted (up to 3 g), and placed into 50 mL Bigprep Lysing Matrix D tubes. The tubes were filled with TRIZOL Reagent at 10 mL per g of tissue. The samples were then placed in a CoolBigPrep adapter for 2x 50 mL tubes and into a FastPrep- 2 Classic bead beating grinder and lysis system. The tissue samples were homogenized at 4.0 meters/second for 30 seconds with 2 minutes pause. The tissue homogenization was repeated 4 times. The lysate was centrifuged at 4,300 xg for 5 minutes at 4°C.
  • the clarified lysate was removed to a new tube and a second round of centrifugation and removal of clarified lysate to a fresh tube was conducted. Two 5 mL aliquots were transferred to separate tubes. The remaining sample was frozen at -80°C if present. The 5 mL aliquots were processed by adding 0.2 mL per mL Trizol of molecular grade chloroform. The sample was mixed by inversion and vortexing for 30 seconds then centrifuged at 4,300 xg for 30 minutes at 4°C. The aqueous phase, approximately 50 % of the lysate volume or ⁇ 2.5 mL was removed equally to 2 mL microcentrifuge tubes.
  • RNA pellet was resuspended in 75% ethanol at 1 mL per mL Trizol, vortexed, and centrifuged at 12,000 xg for 5 minutes at 4°C. The RNA pellet was air dried for 5 minutes and dissolved in 0.2 mL per mL Trizol with RNAse free DEPC treated water. The yield and purity of the RNA was determined using a NanoDrop 8000 spectrophotometer. Samples were spot checked for RNA integrity using the Agilent RNA 6000 Nano kit and Bioanalyzer 2100.
  • mRNA was purified from select brain slice derived total RNA using Dynabeads mRNA Purification Kit following the manufacturer’s instructions. Samples were spot checked for RNA integrity using the Agilent RNA 6000 Nano kit and Bioanalyzer 2100.
  • RNA input per kit was used following the manufacturer’s protocol. QuantiTect Reverse Transcription Kit was used with a library transcript specific RT oligo using total RNA or purified mRNA at template. The manufacturer’s protocol was used.
  • Illumina plate level i5 and well level i7 indices were added to the amplicons with 12 cycles of amplification: 98°C for 30 sec; 12 cycles at 98°C for 15 sec; 60°C for 25 sec, 72°C for 30 sec followed by 72°C for 10 minutes. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit kit following the manufacturer’s protocol. Samples were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2.
  • RNA-round SIFTER Selecting in vivo for transduction and expression of RNA
  • the round 2 library of -65,000 variants each under the control of neuron specific human synapsin 1 (hSynl) promoter or a ubiquitous CMV promoter was administered to two NHPs.
  • Library barcodes were recovered from cDNA reverse transcribed from total RNA extracted from CNS tissue. Approximately 14,000 variants from this round 2 SIFTER library were recovered. After filtering out variants that were poorly recovered, a total of 6,728 variants were identified from the hSynl library, and 4,005 variants from the CMV library.
  • a total of 1,236 variants were selected for evaluation in a final selection to nominate lead capsids.
  • Several of the variants exhibited substantial improvement over AAV9 in CNS transduction.
  • Seq ID 1233 AAV2 R588A
  • Seq ID 1234 AAV9
  • the 1,236 capsid variants selected for further evaluation from the round 2 library were administered to three cynomolgus macaques in a final selection to nominate lead capsids.
  • the fold change enrichment in brain wide CNS transduction was determined by next-generation sequencing and normalized to the abundance in the administered test article.
  • the average log2 fold enrichment across all CNS tissue samples and all cynomolgus macaques is shown in Table 2.
  • Enrichment in neuronal mRNA expression was assessed using the neuron specific human synapsin 1 (hSynl) promoter.
  • Enrichment in mRNA expression in all CNS cells was assessed using the ubiquitous cytomegalovirus (CMV) promoter. #N/A indicates that the capsid was not detected.
  • CMV ubiquitous cytomegalovirus
  • Brain tissue was collected at necropsy on Day 19 after intravenous administration of CNSRCV300. Brain slices of 4 mm thickness were designated for immunohistochemistry evaluation and placed in labelled cassettes, immersion fixed in 10% paraformaldehyde, refrigerated at 4° C for 16-24 hrs, transferred to phosphate buffered saline (lx PBS) with 0.01% sodium azide, and refrigerated at 4°C until processing.
  • lx PBS phosphate buffered saline
  • I l l To prepare brain slices for processing, brains were treated overnight with 20% glycerol and 2% dimethyl sulfoxide to avoid freeze artifacts. Slices were embedded in a gelatin matrix. Blocks were rapidly frozen, after curing by immersion in 2-methylbuteane chilled with crushed dry ice and mounted on a freezing stage of a AO 860 sliding microtome.
  • Blocks were sectioned coronally at 40 pM thickness on the microtome. All sections were cut through the entire length of the specimen segment and collected sequentially into a series of 24 containers, pre-filled with Antigen Preserve solution (50% PBS, pH 7.0, 50% ethylene glycol, 1% polyvinyl pyrrolidone).
  • ABC solution avidin-biotin-HRP complex; VECTASTAIN® Elite ABC, Vector Labs, Burlingame, CA
  • DAB diaminobenzidine tetrahydochloride
  • Nickel sulphate Nickel sulphate
  • FIGs. 15-24 show that capsid CNSRCV300 is able to cross the blood-brain barrier and mediate transgene expression throughout the central nervous system.
  • Seq ID 1235 CNSRCV300

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Abstract

La présente demande concerne l'ingénierie de capsides d'AAV. Dans certains modes de réalisation, les capsides AAV modifiées peuvent pénétrer dans la barrière hémato-encéphalique et transduire des cellules dans le système nerveux central.
PCT/US2024/029507 2023-05-15 2024-05-15 Capsides aav de pénétration de barrière hémato-encéphalique modifiées Pending WO2024238684A1 (fr)

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Cited By (3)

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US12296025B2 (en) 2021-11-02 2025-05-13 Voyager Therapeutics, Inc. AAV capsid variants and uses thereof
WO2025231400A1 (fr) 2024-05-03 2025-11-06 Sangamo Therapeutics, Inc. Facteurs de transcription synthétiques et leurs procédés de fabrication et d'utilisation
US12467046B2 (en) 2018-10-02 2025-11-11 Voyager Therapeutics, Inc. Redirection of tropism of AAV capsids

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WO2013102674A2 (fr) * 2012-01-05 2013-07-11 Novartis International Pharmaceutical Ltd. Cellules fongiques filamenteuses déficientes en protéase et procédés d'utilisation de celles-ci
US20180066285A1 (en) * 2015-03-24 2018-03-08 The Regents Of The University Of California Adeno-associated virus variants and methods of use thereof
US20200370137A1 (en) * 2019-04-26 2020-11-26 Sangamo Therapeutics, Inc. Engineering aav

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WO2013102674A2 (fr) * 2012-01-05 2013-07-11 Novartis International Pharmaceutical Ltd. Cellules fongiques filamenteuses déficientes en protéase et procédés d'utilisation de celles-ci
US20180066285A1 (en) * 2015-03-24 2018-03-08 The Regents Of The University Of California Adeno-associated virus variants and methods of use thereof
US20200370137A1 (en) * 2019-04-26 2020-11-26 Sangamo Therapeutics, Inc. Engineering aav

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12467046B2 (en) 2018-10-02 2025-11-11 Voyager Therapeutics, Inc. Redirection of tropism of AAV capsids
US12296025B2 (en) 2021-11-02 2025-05-13 Voyager Therapeutics, Inc. AAV capsid variants and uses thereof
US12419969B2 (en) 2021-11-02 2025-09-23 Voyager Therapeutics, Inc. AAV capsid variants and uses thereof
WO2025231400A1 (fr) 2024-05-03 2025-11-06 Sangamo Therapeutics, Inc. Facteurs de transcription synthétiques et leurs procédés de fabrication et d'utilisation

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