Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • 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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  • 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
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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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • 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

Definitions

• the disclosure is directed to molecular biology, gene therapy, and compositions and methods for enhancing transduction of AAV capsids comprising modified AAV capsid proteins.
• modified AAV capsids comprising modified AAV capsid proteins are provided having enhanced transduction is muscle tissue and/or reduced transduction in liver tissue.
• AAV8 and AAV9 are commonly used vectors for therapies delivered for skeletal muscle transduction via systemic injection.
• these serotypes also transduce the liver and a significant percentage of the patient population has a moderate to high NAb titer against AAV8 or AAV9.
• AAV capsids having modified AAV capsid protein having enhanced tissue-specific transduction in a desired tissue and/or reduced transduction in a non-desired tissue.
• AAV capsids comprised of modified and chimeric AAV capsid protein sequences with enhanced muscular, transduction, as well as decreased transduction in liver tissue.
• AAV capsids comprised of modified and chimeric AAV capsid protein sequences have reduced sensitivity to pre-existing neutralizing antibodies.
• the disclosure provides a modified AAV capsid protein comprising a modified variable region (VR) VIII.
• the capsid protein is a VP1, VP2, or VP3 capsid protein.
• the modified VR VIII comprises a peptide insertion.
• the peptide insertion comprises an RGD-motif peptide insertion.
• the RGD-motif insertion comprises RGDLGLS (SEQ ID NO: 303), RGDLSTP (SEQ ID NO: 304), SNSRGDYNSL (SEQ ID NO: 305), ENRRGDFNNT (SEQ ID NO:
• the RGD-motif insertion comprises a variable domain of camelid heavy-chain-only antibody (VHH) RGD peptide.
• VHH RGD peptide comprises an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence
• the peptide insertion comprises an acetylcholinesterase collagenic tail (ColQ) peptide.
• ColQ peptide comprises an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence TPFYPVGYTVKQPGTCGDGVLQPGEECDDGNPDVSDGCIDCHRAYCGDGYRHQGV EDCDGSDF GYLTCET YLPGS Y GDLRCTQ Y C SID STPCRYFT (SEQ ID NO: 302).
• the peptide insertion comprises one or more linker sequences at the N-terminus or C-terminus of the inserted peptide.
• the linker sequence comprises GGGGS (SEQ ID NO: 311), GGGGSGGGGS (SEQ ID NO: 312);
• GGGGS GGGGS GGGGS SEQ ID NO: 313; GGGGS GGGGS GGGGS (SEQ ID NO: 314); or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 315).
• the AAV serotype is AAV9 or AAVRh74.
• the modified AAV capsid protein further comprises one or more amino acid mutations.
• the one or more amino acid mutations reduce liver transduction.
• the mutations comprise: i) at least one of F503I, G507I,
• the disclosure provides an AAV capsid comprising one or more AAV capsid proteins according to any embodiment of the dislcosure.
• the AAV capsid has enhanced transduction in a targeted tissue or cell type relative to other tissue or cell types.
• the targeted tissue type is muscular tissue or muscle cells.
• the AAV capsid has enhanced transduction in muscle tissue.
• the AAV capsid has reduced transduction in non-targeted tissues or cell types.
• the non-targeted tissues include liver, lung, kidney, brain, spleen, intestine, spinal cord, or reproductive organs.
• the transduction in muscle tissue is enhanced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 100%, about 200%, or about 300% compared the parental and/or unmodified AAV capsid.
• the capsid comprises: i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53; and ii) a VP2/VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 102.
• the capsid comprises: i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75; and ii) a VP2/VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 124.
• the capsid comprises: i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 245; and ii) a VP2/VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 258.
• the capsid comprises: i) a VP1 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 249; and ii) a VP2/VP3 capsid protein comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 262.
• the disclosure provides a vector comprising a nucleic acid sequence encoding a modified AAV capsid protein of the disclosure.
• the AAV viral vector comprises a recombinant AAV (rAAV) vector encoding a therapeutic transgene or nucleotide sequence of interest (NOI).
• rAAV recombinant AAV
• the disclosure provides a cell comprising a vector of the disclosure or the AAV viral vector of the disclosure.
• the disclosure provides a method of providing a therapeutic transgene or protein to a subject, comprising administering to the subject an AAV viral vector of the disclosure or a pharmaceutical composition of the disclosure.
• the disclosure provides a method of treating a subject having a disease and/or disorder, the method comprising administering to the subject at least one therapeutically effective amount of an AAV viral vector of the disclosure or a pharmaceutical composition of the disclosure.
• the disease and/or disorder is a muscular and/or neuromuscular disorder.
• the muscular and/or neuromuscular disorder is muscular dystrophy or myotonic dystrophy.
• the AAV viral vector or the pharmaceutical composition is administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intra-aurally, intra-ocularly, or peri -ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracistemally, intranervally, intrapleurally, topically, intralymphatically, intracistemally or intranerve.
• FIG. 1 is a schematic listing modified muscle-targeted AAV7 and AAVRh74 chimeric capsid proteins.
• FIG. 2 is a schematic depicting muscle-targeted AAV Rh8 variant capsids comprising liver de-targeting amino acid mutations.
• FIG. 3 is a schematic depicting modified muscle-targeted AAV capsid proteins of the disclosure.
• FIG. 4 is a schematic depicting modified muscle-targeted AAVRh74 capsid proteins of the disclosure.
• FIG. 5 shows immunofluorescence images showing transduction efficiency of HEK293 cells with an AAV vector comprising a VP 1 -specifically displayed targeting peptide.
• FIGs. 6A-6D are immunofluorescence images showing enhanced transduction efficiency of C2C12 cells with modified muscle-targeted AAV capsids.
• FIGs. 7A-C shows results of ex vivo imaging of AAV9, LBV30, and LBV31.
• Fig. 7A is an ex vivo immunofluorescence image showing in vivo targeting of AAV9, LBV30, and LBV31 capsids in mice.
• Figs. 7B and 7C are graphs quantifying the immunofluorescence in various tissues.
• FIG. 8 is a series of schematics depicting RGD peptide insertion scaffolds and approaches.
• FIG. 9 is a graph depicting transduction of AAV viral vectors of the disclosure in the indicated mouse tissue: liver, heart, lung, spleen, kidney, intestine, testis, tongue, quadricep (quad), gastrocnemius (gc), tibialis anterior (ta), diaphragm, and brain.
• the y-axis displays luminescence of an encapsi dated luciferase reporter vector.
• the disclosure provides gene therapy compositions comprising modified and chimeric AAV capsid proteins for delivery of packaged therapeutics to muscular tissue.
• Adeno-associated virus refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae.
• Adeno-associated virus is a single- stranded DNA virus that grows in cells in which certain functions are provided by a co-infecting helper virus.
• General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).
• Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAVPHP.B.
• the AAV particle comprises, consists essentially of, or consists of three major viral proteins: VP1, VP2 and VP3.
• the AAV refers to the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13, AAVPOl, AAVPHP.B, AAVrh74 or AAVrh.10.
• Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to all serotypes (e.g ., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13, AAVPOl, AAVPHP.B, AAVrh74 and AAVrh.10).
• Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g., AAV2/5, AAV-DJ and AAV-DJ8).
• Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019; 4(22):el31610) and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).
• AAV is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two 145-nucleotide inverted terminal repeat (ITRs).
• ITRs inverted terminal repeat
• the nucleotide sequences of the genomes of the AAV serotypes are known.
• the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077
• the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983)
• the complete genome of AAV-3 is provided in GenBank Accession No.
• NC_1829 the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Then, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).
• the two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
• Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
• the cap gene is expressed from the p40 promoter and encodes the three capsid proteins, VP1, VP2, and VP3.
• Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. More specifically, after the single mRNA from which each of the VP1, VP2 and VP3 proteins are translated is transcribed, it can be spliced in two different manners: either a longer or shorter intron can be excised, resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool.
• the longer intron is often preferred and thus the 2.3-kb-long mRNA can be called the major splice variant.
• This form lacks the first AUG codon, from which the synthesis of VP1 protein starts, resulting in a reduced overall level of VP1 protein synthesis.
• the first AUG codon that remains in the major splice variant is the initiation codon for the VP3 protein.
• upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak (translation initiation) context.
• AAV AAV genome encapsidation
• some or all of the internal approximately 4.3 kb of the genome encoding replication and structural capsid proteins, rep-cap
• the rep and cap proteins may be provided in trans.
• Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized.
• AAV- infected cells are not resistant to superinfection.
• An "rAAV vector” as used herein refers to a vector comprising, consisting essentially of, or consisting of one or more transgene sequences and one or more AAV inverted terminal repeat sequences (ITRs).
• AAV vectors can be replicated and packaged into infectious viral particles, comprising modified AAV capsid proteins of the disclosure, when present in a host cell that provides the functionality of rep and cap gene products; for example, by transfection of the host cell.
• AAV vectors contain a promoter, at least one nucleic acid that may encode at least one protein or RNA, and/or an enhancer and/or a terminator within the flanking ITRs that is packaged into the infectious AAV particle.
• an rAAV vector can comprise at least one transgene nucleic acid molecule. In some aspects, an rAAV vector can comprise at least one AAV inverted terminal (ITR) sequence. In some aspects, an rAAV vector can comprise at least one promoter sequence. In some aspects, an rAAV vector can comprise at least one enhancer sequence. In some aspects, an rAAV vector can comprise at least one polyA sequence. In some aspects, an rAAV vector can comprise at least one reporter protein.
• ITR AAV inverted terminal
• an rAAV vector can comprise a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence.
• an rAAV vector can comprise, in the 5’ to 3’ direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence.
• an rAAV vector can comprise more than one promoter sequence.
• an rAAV vector can comprise at least two promoter sequences, such that the rAAV vector comprises a first promoter sequence and an at least second promoter sequence.
• the first and the at least second promoter sequences can comprise the same sequence.
• the first and the at least second promoter sequences can comprise different sequences.
• the first and the at least second promoter sequences can be adjacent to each other.
• an rAAV vector can comprise, in the 5’ to 3’ direction, a first AAV ITR sequence, a promoter sequence, an enhancer, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence.
• the at least one enhancer can be located immediately downstream of a transgene nucleic acid molecule.
• an rAAV vector can comprise, in the 5’ to 3’ direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, an enhancer, a polyA sequence, and a second AAV ITR sequence.
• rAAV vectors of the disclosure can comprise any transgene nucleic acid molecule known in the art.
• the transgene nucleic acid is a therapeutic transgene.
• a transgene nucleic acid molecule is referred to interchangeable as a nucleotide sequence of interest (NOI).
• NOI includes, without limitation, any nucleotide sequence or transgene capable of being delivered by a vector.
• NOIs can be synthetic, derived from naturally occurring DNA or RNA, codon optimized, recombinant RNA/DNA, cDNA, partial genomic DNA, and/or combinations thereof.
• the NOI can be a coding region or partial coding region, but need not be a coding region.
• An NOI can be RNA/DNA in a sense or anti- sense orientation. NOIs are also referred herein, without limitation, as transgenes, heterologous sequences, genes, therapeutic genes. An NOI may also encode a POI (protein of interest), a partial POI, a mutated version or variant of a POI. A POI may be analogous to or correspond to a wild-type protein. A POI may also be a fusion protein or nucleoprotein complex such as a CRISPR/Cas nucleoprotein complex. A POI may also be a PUF or PUMB Y protein. In some aspects, POIs can be RNA targeting or RNA-binding proteins or nucleoprotein complexes.
• the NOI is a nucleic acid encoding a target RNA-binding fusion protein which is not an RNA-guided target RNA-binding fusion protein and as such comprises at least one RNA-binding polypeptide which is capable of binding a target RNA without a corresponding gRNA sequence.
• RNA-binding polypeptides include, without limitation, at least one RNA-binding protein or RNA-binding portion thereof which is a PUF (Pumilio and FBF homology family) protein.
• a PUF protein can be designed to specifically bind most 8 to 16-nt RNA. Wang et al, Nat Methods. 2009; 6(11): 825-830. See also WO2012/068627 which is incorporated by reference herein in its entirety.
• human PUMl (1186 amino acids) contains an RNA-binding domain (RBD) in the C-terminus of the protein (also known as Pumilio homology domain PUM-FFD amino acid 828-amino acid 1175) and that PUFs are based on the RBD of human PUMl.
• RBD RNA-binding domain
• amino acids 12, 13, and 16 are important for RNA binding with 12 and 16 responsible for RNA base recognition.
• Amino acid 13 stacks with RNA bases and can be modified to tune specificity and affinity.
• the PUF design may maintain amino acid 13 as human PUMl’s native residue.
• amino acid 13 for stacking
• amino acid 13 will be engineered with an H and in other embodiments, will be engineered with a Y.
• stacking residues may be modified to improve binding and specificity.
• Recognition occurs in reverse orientation as N- to C-terminal PUF recognizes 3’ to 5’ RNA.
• PUF engineering of 8 modules (8PUF) as known in the art, mimics a human protein.
• An exemplary 8-mer RNA recognition (8PUF) would be designed as follows: RU-R1-R2-R3-R4-R5-R6-R7-R8-R8’.
• the fusion protein comprises at least one RNA-binding protein or RNA-binding portion thereof which is a PUMBY (Pumilio-based assembly) protein.
• RNA-binding protein PumHD which has been widely used in native and modified form for targeting RNA, has been engineered into a protein architecture designed to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (i.e., Pumby, for Pumilio- based assembly) are concatenated in chains of varying composition and length, to bind desired target RNAs.
• PUMBY is a more simple and modular form of PumHD, in which a single protein unit of PumHD is concatenated into arrays of arbitrary size and binding sequence specificity.
• the specificity of such Pumby-RNA interactions is high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence.
• the fusion protein disclosed herein comprises a linker between the at least two RNA-binding polypeptides.
• the linker is a peptide linker.
• the peptide linker comprises one or more repeats of the tri peptide GGS. In other embodiments, the linker is a non-peptide linker.
• the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker.
• PEG polyethylene glycol
• PPG polypropylene glycol
• POE polyoxyethylene
• polyurethane polyphosphazene
• polysaccharides dextran
• polyvinyl alcohol polyvinylpyrrolidones
• polyvinyl ethyl ether polyacryl amide
• polyacrylate polycyanoacrylates
• lipid polymers chitins, hyaluronic
• the at least one RNA-binding protein of the fusion proteins disclosed herein further comprises a sequence encoding a nuclear localization signal (NLS).
• a nuclear localization signal (NLS) is positioned at the N-terminus of the RNA binding protein.
• the at least one RNA-binding protein comprises an NLS at a C-terminus of the protein.
• the at least one RNA-binding protein further comprises a first sequence encoding a first NLS and a second sequence encoding a second NLS.
• the first NLS or the second NLS is positioned at the N-terminus of the RNA-binding protein.
• a fusion protein disclosed herein comprises the at least one RNA-binding protein as a first RNA-binding protein together with a second RNA-binding protein comprising or consisting of a nuclease domain.
• the second RNA-binding polypeptide is operably configured to the first RNA-binding polypeptide at the C-terminus of the first RNA-binding polypeptide.
• the second RNA-binding polypeptide is operably configured to the first RNA-binding polypeptide at the N-terminus of the first RNA-binding polypeptide.
• an exemplary fusion protein is a PUF or PUMBY-based first RNA-binding protein fused to a second RNA-bindng protein which is an zinc-finger endonuclease known as ZC3H12A.
• an NOI or transgenes comprising Guide RNAs for RNA-Guided RNA-Binding Proteins
• an NOI or transgene comprises a guide RNA.
• gRNA guide RNA
• sgRNA single guide RNA
• Guide RNAs of the disclosure may comprise of a spacer sequence and a “direct repeat” (DR) sequence.
• a guide RNA is a single guide RNA (sgRNA) comprising a contiguous spacer sequence and DR sequence.
• the spacer sequence and the DR sequence are not contiguous.
• the gRNA comprises a DR sequence.
• DR sequences refer to the repetitive sequences in the CRISPR locus (naturally-occurring in a bacterial genome or plasmid) that are interspersed with the spacer sequences.
• a guide RNA comprises a direct repeat (DR) sequence and a spacer sequence.
• a sequence encoding a guide RNA or single guide RNA of the disclosure comprises or consists of a spacer sequence and a DR sequence, that are separated by a linker sequence.
• the linker sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of nucleotides (nt) in between.
• a guide RNA of the disclosure comprises at least one sequence encoding a non-coding C/D box small nucleolar RNA (snoRNA) sequence.
• the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the target sequence of the RNA molecule comprises at least one 2’-OMe.
• the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the at least one sequence that is complementary to the target RNA comprises a box C motif (RUGAUGA) and a box D motif (CUGA).
• spacer sequences of the disclosure bind to pathogenic target RNA.
• the sequence comprising the gRNA further comprises a spacer sequence that specifically binds to the target RNA sequence.
• the spacer sequence has at least 50%, 55%, 60%, 65%,
• DR sequences of the disclosure bind the Cas polypeptide of the disclosure.
• the Cas protein bound to the DR sequence of the gRNA is positioned at the target RNA sequence.
• a DR sequence having sufficient complementarity to its cognate Cas protein, or nucleic acid thereof binds selectively to the target nucleic acid sequence of the Cas protein and has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in between to the sequence.
• a sequence having sufficient complementarity has 100% identity.
• DR sequences of the disclosure comprise a secondary structure or a tertiary structure.
• Exemplary secondary structures include, but are not limited to, a helix, a stem loop, a bulge, a tetraloop and a pseudoknot.
• Exemplary tertiary structures include, but are not limited to, an A-form of a helix, a B-form of a helix, and a Z-form of a helix.
• Exemplary tertiary structures include, but are not limited to, a twisted or helicized stem loop.
• Exemplary tertiary structures include, but are not limited to, a twisted or helicized pseudoknot.
• DR sequences of the disclosure comprise at least one secondary structure or at least one tertiary structure.
• DR sequences of the disclosure comprise one or more secondary structure(s) or one or more tertiary structure(s).
• a guide RNA or a portion thereof selectively binds to a tetraloop motif in an RNA molecule of the disclosure.
• a target sequence of an RNA molecule comprises a tetraloop motif.
• the tetraloop motif is a “GRNA” motif comprising or consisting of one or more of the sequences of GAAA, GUGA, GCAA or GAGA.
• a DR sequence is truncated by 1-10 nucleotides (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, to 10 nucleotides at e.g., the 5’ end in order to be expressed as mature pre-processed guide RNAs.
• a guide RNA or a portion thereof does not comprise a nuclear localization sequence (NLS).
• NLS nuclear localization sequence
• a guide RNA or a portion thereof comprises a sequence complementary to a protospacer flanking sequence (PFS).
• PFS protospacer flanking sequence
• the first RNA binding protein may comprise a sequence isolated or derived from a Casl3 protein.
• the first RNA binding protein may comprise a sequence encoding a Casl3 protein or an RNA-binding portion thereof.
• the guide RNA or a portion thereof does not comprise a sequence complementary to a PFS.
• guide RNA sequence of the disclosure comprises a promoter sequence to drive expression of the guide RNA.
• a vector comprising a guide RNA sequence of the disclosure comprises a promoter sequence to drive expression of the guide RNA.
• the promoter to drive expression of the guide RNA is a constitutive promoter.
• the promoter sequence is an inducible promoter.
• the promoter is a sequence is a tissue-specific and/or cell-type specific promoter.
• the promoter is a hybrid or a recombinant promoter.
• the promoter is a promoter capable of expressing the guide RNA in a mammalian cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA in a human cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA and restricting the guide RNA to the nucleus of the cell. In some embodiments, the promoter is a human RNA polymerase promoter or a sequence isolated or derived from a sequence encoding a human RNA polymerase promoter. In some embodiments, the promoter is a U6 promoter or a sequence isolated or derived from a sequence encoding a U6 promoter.
• the U6 promoter is a human U6 promoter. In some embodiments, the promoter is a human tRNA promoter or a sequence isolated or derived from a sequence encoding a human tRNA promoter. In some embodiments, the promoter is a human valine tRNA promoter or a sequence isolated or derived from a sequence encoding a human valine tRNA promoter.
• a promoter to drive expression of the guide RNA further comprises a regulatory element.
• a vector comprising a promoter sequence to drive expression of the guide RNA further comprises a regulatory element.
• a regulatory element enhances expression of the guide RNA.
• Exemplary regulatory elements include, but are not limited to, an enhancer element, an intron, an exon, or a combination thereof.
• a vector of the disclosure comprises one or more of a sequence encoding a guide RNA, a promoter sequence to drive expression of the guide RNA and a sequence encoding a regulatory element. In some embodiments of the compositions of the disclosure, the vector further comprises a sequence encoding a fusion protein of the disclosure.
• gRNAs correspond to target RNA molecules and an RNA-guided RNA binding protein.
• the gRNAs correspond to an RNA-guided RNA binding fusion protein, wherein the fusion protein comprises first and second RNA binding proteins.
• the first RNA-binding protein in the fusion protein is a deactivated RNA-binding protein, e.g., a deactivated Cas or catalytic dead Cas protein.
• the sequence encoding the first RNA binding protein is positioned 5’ of the sequence encoding the second RNA binding protein.
• the sequence encoding the first RNA binding protein is positioned 3’ of the sequence encoding the second RNA binding protein.
• the sequence encoding the first RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule. In some embodiments, the sequence encoding the first RNA binding protein comprises a sequence isolated or derived from a protein capable of selectively binding an RNA molecule and not binding a DNA molecule, a mammalian DNA molecule or any DNA molecule. In some embodiments, the sequence encoding the first RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule and inducing a break in the RNA molecule.
• the sequence encoding the first RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule, inducing a break in the RNA molecule, and not binding a DNA molecule, a mammalian DNA molecule or any DNA molecule. In some embodiments, the sequence encoding the first RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule, inducing a break in the RNA molecule, and neither binding nor inducing a break in a DNA molecule, a mammalian DNA molecule or any DNA molecule.
• the sequence encoding the first RNA-guided RNA binding protein comprises a sequence isolated or derived from a protein with no DNA nuclease activity.
• the sequence encoding the RNA-guided RNA binding protein disclosed herein comprises a sequence isolated or derived from a CRISPR Cas protein.
• the CRISPR Cas protein is not a Type II CRISPR Cas protein.
• the CRISPR Cas protein is not a Cas9 protein.
• the Cas9 protein is engineered to target RNA (RCas9).
• the sequence encoding the RNA- guided RNA binding protein comprises a Type VI CRISPR Cas protein or portion thereof.
• the Type VI CRISPR Cas protein comprises a Casl3 protein or portion thereof.
• Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, a bacteria or an archaea.
• Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, Leptotrichia wadei, Listeria seeligeri serovar l/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954), Lachnospiraceae bacterium, Clostridium aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Paludibacter propionicigenes WB4, Listeria weihenstephanensis FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL M6-0635 (Listeria newyorkensis), Leptotrichia w adei F 0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442 and Corynebacterium ulcerans.
• Exemplary Cas 13 proteins of the disclosure may be DNA nuclease inactivated.
• Exemplary Casl3 proteins of the disclosure include, but are not limited to, Casl3a, Casl3b, Casl3c, Casl3d and orthologs thereof.
• Exemplary Casl3b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively.
• a "viral vector” is defined as a recombinantly produced virus or viral particle that contains a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
• viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors and the like.
• Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, e.g., Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.
• An "AAV virion" or "AAV viral particle” or “AAV viral vector” or “rAAV viral vector” or “AAV vector particle” or “AAV particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector.
• production of an rAAV viral vector necessarily includes production of an rAAV vector, as such a vector is contained within an rAAV vector.
• viral capsid refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and release into the host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein ("capsid proteins"). As used herein, the term “encapsidated” means enclosed within a viral capsid.
• the viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.
• a viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proceedings of the National Academy of Sciences of the United States of America. 107 (22): 10220-5, and Rabinowitz JE, Samulski RJ (December 2000).
• the present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein; and b) an AAV capsid protein.
• An AAV capsid protein can be any AAV capsid protein known in the art.
• the AAV capsid protein is a modified AAV capsid protein.
• An AAV capsid protein can be an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein or an AAVrh.10 capsid protein.
• An AAV capsid protein can be any modified AAV capsid protein of the disclosure. Modified AAV Capsid Proteins
• viral capsid refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and release into the host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein ("capsid proteins"). As used herein, the term “encap si dated” means enclosed within a viral capsid. Provided herein are modified AAV capsid proteins which may be used to construct modified and/or chimeric AAV vectors or AAV capsids.
• An AAV capsid generally consists of a total of 60 molecules of viral proteins (VPs), VP1, VP2, and VP3 at a ratio of about 1:1:10.
• VP1, VP2, and VP3 are encoded by the cap open reading frame and are generated through alternative splicing of the mRNA and use of an alternate translational start codon.
• the VP3 sequence of about 524-544 amino acids (aa) is shared among all VPs, the VP2 sequence is approximately 57aa longer than VP3 (about 580- 601aa) and the VPl sequence is approximately 137 aa longer than VP2 (about 713-738aa).
• the VP3 common region assembles the icosahedral capsid. See Worner el al. Nature Communications Vol. 12, Article number: 1642 (2021).
• modified AAV capsid proteins of the disclosure are derived from any AAV serotype known in the art.
• An AAV capsid protein can be derived from any AAV capsid protein known in the art.
• an AAV capsid protein can be derived from an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh8 capsid protein, an AAVrh74 capsid protein or, AAV-TT (AAVv66) capsid protein, an AAV POl capsid protein, an AAVDJ, or an AAVrhlO
• the AAV capsid protein is an AAV-TT capsid protein. In some aspects, the AAV capsid protein is an AAVrhlO capsid protein. In some aspects, modified AAV capsid proteins of the disclosure can be chimeric AAV capsid proteins derived from two or more AAV capsid proteins.
• modified AAV capsid protein sequences As used herein a “modified AAV capsid protein” or a “modified capsid protein” refers to AAV capsid proteins that have been modified with respect to the wild-type AAV capsid protein sequence.
• Modified AAV capsid proteins can comprise any one of capsid proteins VPl, VP2, or VP3.
• Modifications to AAV capsid protein sequences can be any protein modification known in the art including amino acid deletions, mutations, insertions, or re-arrangements.
• Modifications to AAV capsid proteins can be the formation of chimeric AAV capsid proteins wherein regions of two or more AAV capsid proteins are spliced or combined together to form a hybrid or chimeric AAV capsid protein.
• a modified AAV capsid protein comprised of a hybrid or chimeric AAV capsid protein comprises regions of two or more AAV capsid proteins each having a unique serotype.
• a hybrid capsid protein is one where variable domain loop regions of the capsid protein has been swapped.
• a hybrid AAV capsid protein comprises variable region loops from two or more capsid sequences having different serotypes.
• Modified AAV capsid proteins of the disclosure can comprise insertions of peptides from any protein or peptide known in the art.
• the inserted peptide can be derived from a non- AAV capsid protein.
• Modified AAV capsid proteins of the disclosure can comprise any combination of modifications.
• AAV capsid proteins of the disclosure can be both chimeric and contain at least one of an amino acid deletion, mutation, insertion, or re arrangement.
• Modified AAV capsid proteins of the disclosure can used to form AAV capsids with improved properties including increased transduction in a specific tissue type (i.e. “on-target transduction” and/or reduced transduction in undesired tissue types (i.e. “off-target transduction”).
• muscle tissue-specific transduction is observed.
• ocular tissue specific transduction is observed.
• neuron or neuronal tissue specific transduction is observed.
• transduction in the liver is reduced or eliminated.
• AAV capsids comprising modified AAV capsid proteins have reduced transduction in non-targeted tissues or cell types.
• non-targeted tissues include liver, lung, kidney, brain, spleen, intestine, spinal cord, or reproductive organs.
• AAV vectors comprising modified AAV capsid proteins of the disclosure can have reduced reactivity to pre-existing neutralizing antibodies in a human subject due to the modifications to the capsid producing distinct binding epitopes not observed in commonly used AAV capsid serotypes.
• Peptides or amino acids can be inserted in any region of an AAV capsid protein. Insertions can occur at the N-terminus or C-terminus of the protein. In some aspects, insertions can occur in any variable region (VR) of the capsid protein including VR1 (VRI), VR2 (VRII), VR3 (VRIII), VR4 (VRIV), VR5 (VRV), VR6 (VRVI, VR7 (VRVII), VR8 (VRVIII), or VR9 (VRX).
• modified AAV capsid proteins of the disclosure comprise modified variable regions. In some aspects, modified AAV capsid proteins of the disclosure comprise modified VR VIII regions. In some aspects, the modification to the AAV capsid protein is an insertion into VR VIII.
• Peptides sequences were chosen to enhance transduction in a tissue-specific manner. Any sequence that enhances tissue-specific transduction is contemplated. In some aspects, muscle tissue-specific transduction is observed. In some aspects, ocular tissue specific transduction is observed. In some aspects, neuron or neuronal tissue specific transduction is observed. Inserted peptides can target tissue-specific receptors leading to increased transduction in said tissues.
• modified AAV capsid proteins comprise a peptide insertion targeting insulin receptor (INSR).
• INSR insulin receptor
• AAV vectors designed to target INSR have been shown to enhance intramuscular transduction (See Jackson et al. Molecular Therapy Methods & Clinical Development, 2020, 19, 11, 496-506 which is incorporated herein by reference in its entirety).
• the inserted INSR-targeting peptide is an insulin-mimetic peptide referred to as S519.
• the S519 peptide can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
• modified AAV capsid proteins comprise a peptide insertion targeting muscle-specific kinase (MUSK).
• MUSK expression in the liver has been shown to be either extremely low or absent.
• Acetylcholinesterase collagenic tail peptide (ColQ) is known to bind and target MUSK.
• the inserted MUSK-targeting peptide is a C-terminal portion of ColQ (ColQ CTD).
• ColQ CTD peptide can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence
• modified AAV capsid proteins comprise a peptide insertion targeting integrin.
• the integrin-targeting peptide comprises an RGD-motif.
• RGD sequences are known in the art, and include, for example, the motif RGDXXXX which may be inserted in an AAV viral vector for targeting via the integrin class of receptors see, e.g., Michelfelder et al, PLoS One. 2009; 4(4): e5122 which is incorporated herein by reference in its entirety for example of RGD sequences that may be used in modified AAV capsids described herein.
• RGD-motif peptide insertions into VR8 of AAV9 has been shown to increase mouse muscle transduction (See Weinmann et al. Nature Communications, 11 :5432 which is incorporated herein by reference in its entirety).
• the RGD peptide comprises a subsequence Y or F amino acid to produce an RGDY or RGDF motif.
• RGDY or RGDF motifs have been demonstrated to produce enhanced muscle transduction in non human primates (NHP) (See Tabebordbar et al. Cell, 184, 19, 2021, 4919-4938 which is incorporated herein by reference in its entirety).
• the RGD sequence comprises RGDLGLS (SEQ ID NO: 303).
• the RGD sequence comprises RGDLSTP (SEQ ID NO: 304), SNSRGDYNSL (SEQ ID NO: 305), ENRRGDFNNT (SEQ ID NO: 306), SRGDYNSL (SEQ ID NO: 307), RGDYNSL (SEQ ID NO: 308), RGDLST (SEQ ID NO: 309), or RGDYVGL (SEQ ID NO: 310).
• RGD sequences of the disclosure can be inserted in a scaffold (FIG. 8).
• RGD sequences of the disclosure comprise a linker on one or more of the N-terminus and C-terminus forming a linker scaffold.
• the linker scaffold comprises a flexible linker such as GGGS (SEQ ID NO: 311).
• the linker scaffold comprises a rigid scaffold such as a VHH, GP2, cyclic peptide, or knottin scaffold.
• the RGD sequence comprises a variable domain of camelid heavy- chain-only antibody (VHH) RGD peptide, for example a VHH RGD peptide comprising, consisting essentially of, or consisting of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence
• VHH camelid heavy- chain-only antibody
• RGD peptide insertions comprise a glycoprotein 2 (GP2) peptide scaffolded insertion.
• GP2 scaffolded peptide insertions comprise the amino acid sequence KFWATV GRGDLSTPFEVPVY AETLDEALELAENRRGDFNNTVTRVRP (SEQ ID NO:317) or
• GGGGSGGGGSKFWATVGRGDLSTPFEVPVYAETLDEALELAENRRGDFNNTVTRVRPGGGG S (SEQ ID NO: 318).
• RGD peptide insertions can comprise a knottin scaffolded peptide insertion.
• knottin scaffolded peptide insertions comprise the amino acid sequence NSRGDYNSLSCSQDSDCLAGCVCGPNGFC (SEQ ID NO: 319) or
• RGD peptide insertions can comprise a cyclic peptide scaffolded RGD peptide insertion.
• cyclic peptide scaffolded RGD peptide insertions comprise the amino acid sequence ACRGDYNSLCRGDLSTC (SEQ ID NO: 321) or GGGGACRGDYNSLCRGDLSTCGGGGS (SEQ ID NO: 322).
• Scaffolds can be used to insert any peptide known in the art. Therefore, VHH, GP2, cyclic peptides, knottin, or flexible linkers can be used to scaffold or flank any peptide for insertion into a modified AAV capsid protein of the disclosure.
• Inserted peptides can be flanked on the N-terminus or C-terminus by flexible linker peptides of any length.
• a flexible linker such as GGGS (SEQ ID NO: 311) is used.
• the flexible GGGS linker can be repeated multiple times to form a longer linker sequence.
• the linker is repeated one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.
• the linker sequence comprises GGGGS (SEQ ID NO: 311), GGGGSGGGGS (SEQ ID NO: 312); GGGGS GGGGS GGGGS (SEQ ID NO: 313);
• GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 314); or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 315).
• modified AAV capsid proteins may comprise amino acid mutations yielding increased transduction in a desired tissue type.
• modified AAV capsid proteins may comprise amino acid mutations yielding reduced transduction in specie tissues types.
• modified AAV capsid proteins comprise amino acid mutations that yield reduced liver tissue transduction.
• a modified AAV capsid protein comprises an amino acid sequence provided herein (e.g., a sequence selected from Table 1) with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid changes.
• the amino acid change can be the substitution of one amino acid for any other amino acid, including natural amino acids and un-natural or modified amino acids.
• the mutation is a conservative amino acid mutation.
• a conservative amino acid substitution is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity or size).
• conservative amino acid substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or the substitution of one charged or polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, and the like.
• a conservative amino acid substitution is selected from alanine to serine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine, or glutamate; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine.
• mutations at position 503 of AAVrh8 yield reduced liver transduction. In some aspects, substitution of tryptophan for alanine at position 503 of AAVrh8 yields reduced liver transduction. In some aspects, mutations at position 505 of AAVrh74 yields reduced liver transduction. In some aspects, substitution of tryptophan for alanine at position 55 of AAVrh74 yields reduced liver transduction. In some aspects, mutations at position 498 of an AAV9 capsid proteins yield reduced liver transduction. In some aspects, an AAV9 point mutation comprises an N498 mutation. In some aspects, an AAV9 point mutation comprises an N498I mutation. In some aspects, mutations at position 602 of an AAV capsid proteins yield reduced liver transduction.
• liver de-targeting mutations of AAV9 include at least one of F501I, G505R, Y706C.
• Equivalent mutations in certain vectors of the disclosure include F503I, G507R, and Y708C in LBV30 to generate LBV92.
• AAV Rh74 liver de-targeting mutations comprise mutations to any combination of residues F503, G507, Y707, and/or Y708 of AAV Rh74. On some aspects, these mutations comprise F503I, G507R, Y707C, and/or Y708C of AAV Rh74.
• point mutations of the disclosure are numbered in reference to the wild-type AAV capsid protein sequence.
• Wild type AAV capsid proteins sequences are disclosed in table XXX.
• modified AAV capsid proteins of the disclosure comprise insertions and/or deletions or variable region swaps that may alter the length of capsid protein sequence. It should be understood that point mutations referenced in table XI are in reference to the wild type or unmodified AAV capsid protein sequence.
• the disclosure provides modified AAV capsid protein amino acid sequences.
• the disclosure further provides nucleic acid sequences that encode modified AAV capsid proteins of the disclosure.
• Modified AAV capsid proteins of the disclosure are set forth in Table 1.
• Table 1 lists the ID of the assembled AAV capsid comprising VP1, VP2, and VP3 capsid proteins including modified AAV capsid proteins as disclosed herein.
• Table 1 describes the serotype of the capsid protein as well as any modifications made to the capsid sequence.
• a modified AAV capsid VP1 protein provided herein comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP1 sequence set forth in Table 1.
• a modified AAV capsid VP2/3 protein provided herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (or any percentage in between) identical to a VP2/VP3 sequence set forth in Table 1.
• a modified AAV capsid VP1 protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP1 sequence set forth in Table 1.
• a modified AAV capsid VP2/3 protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2/VP3 sequence set forth in Table 1.
• a muscle-targeted modified AAV capsid VPl protein provided herein comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VPl sequence set forth in any Table 1.
• a muscle-targeted modified AAV capsid VP2/3 protein provided herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (or any percentage in between) identical to a VP2/VP3 sequence set forth in Table 1.
• a muscle-targeted modified AAV capsid VPl protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VPl sequence set forth in Table 1.
• a muscle-targeted modified AAV capsid VP2/3 protein provided herein is encoded by a polynucleotide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to a VP2/VP3 sequence set forth in Table 1.
• modified AAV capsid proteins that are useful for in vitro transduction.
• a modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 53. In some aspects,
• SEQ ID NO: 53 is an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion.
• the modified VP1 capsid protein set forth in SEQ ID NO: 53 is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 151.
• the VP2/VP3 capsid protein set forth in SEQ ID NO: 102 is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 199.
• a modified VP1 capsid protein sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 245.
• SEQ ID NO: 245 is an Rh74 VP1 capsid protein comprising VHH RGD peptide insertion and mutations F503I, G507R, and Y707C (amino acid numbering in reference to wild-type Rh74 capsid protein).
• the modified VP1 capsid protein set forth in SEQ ID NO: 245 is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 270.
• a modified AAV capsid provided herein comprises a wild-type or modified VP2/VP3 capsid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to the VP2/VP3 sequences set forth in Table 1 and the SEQ ID NOs referenced therein.
• a modified AAV capsid of the disclosure comprises a VP1 capsid protein selected from a sequence listed in Table 1 and a VP2/VP3 capsid protein selected from a sequence listed in Table 1.
• a modified AAV capsid comprises a VP1 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 53 and a VP2/VP3 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 102.
• an AAV capsid having said sequences is referred to as LBV55.
• LBV55 comprises an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion and an AAV9 VP2/VP3 capsid protein.
• LBV93 comprises an Rh74 VP1 capsid protein comprising an mColQ peptide insertion and mutations F503I, G507R, and Y707C (amino acid numbering in reference to wild-type Rh74 capsid protein) and an Rh74 VP2/VP3 capsid protein comprising an RGD peptide insertion and mutations F503I, G507R, and Y707C (amino acid numbering in reference to wild-type Rh74 capsid protein).
• a modified AAV capsid comprises a VP1 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 245 and a VP2/VP3 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 258.
• an AAV capsid having said sequences is referred to as LB VI 10.
• LB VI 10 comprises an Rh74 VP1 capsid protein comprising VHH RGD peptide insertion and mutations F503I, G507R, and Y707C (amino acid numbering in reference to wild-type Rh74 capsid protein) and an Rh74 VP2/VP3 capsid protein comprising an RGD peptide insertion and mutations F503I, G507R, and Y707C (amino acid numbering in reference to wild-type Rh74 capsid protein).
• a modified AAV capsid comprises a VP1 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 249 and a VP2/VP3 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 262.
• an AAV capsid having said sequences is referred to as LBV114.
• LBV114 comprises an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion and mutation N498I (amino acid numbering in reference to wild-type AAV9 capsid protein) and an AAV9 VP2/VP3 capsid protein comprising an RGD peptide insertion and mutation N498L (amino acid numbering in reference to wild-type AAV9 capsid protein).
• a modified AAV capsid comprises a VP1 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 255 and a VP2/VP3 capsid protein sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 100% (or any percentage in between) identical to SEQ ID NO: 267.
• an AAV capsid having said sequences is referred to as LBV121.
• LBV121 comprises an AAV9 VP1 capsid protein comprising a VHH RGD peptide insertion and an AAV9 VP2/VP3 capsid protein comprising an RGD peptide insertion.
• a modified AAV capsid of the disclosure show improved transduction efficiency compared to the parental or wild-type AAV capsid.
• the improved transduction is in muscle tissue.
• the muscle tissue is skeletal muscle, smooth muscle, or cardiac muscle.
• the transduction efficiency of a modified muscle-targeted AAV capsid provided herein in a muscle cell is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the parental AAV capsid as determined by immunofluorescence.
• the transduction efficiency of a modified muscle-targeted AAV capsid provided herein in a muscle cell is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3- fold, at least about 1.4-fold, at least aboutl.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the parental AAV capsid as determined by immunofluorescence.
• the AAV capsids of the disclosure may be used in any suitable AAV viral vector.
• the AAV capsid surrounds a small, single-stranded DNA genome of approximately 4.8 kilobases (kb).
• the genome of an AAV contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly) flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging.
• ITRs inverted terminal repeats
• an AAV viral vectors comprising a modified AAV capsid provided herein.
• the AAV viral vector comprises a transgene.
• compositions comprising an AAV viral vector comprising a modified AAV capsid provided herein and a pharmaceutically acceptable carrier.
• a pharmaceutical composition may be formulated for any suitable route of administration, including, for example, intraveneous, intrathecal, intracranial, or intraocular administration.
• Pharmaceutical compositions for use as disclosed herein may comprise a protein(s) or a polynucleotide encoding the protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
• buffers such as neutral buffered saline, phosphate buffered saline and the like
• carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
• proteins such as glucose, mannose, sucrose or dextrans, mannitol
• proteins such as glucose, mannose, sucrose or dextrans, mannitol
• proteins such as glucose, mannose, sucrose or dextrans, mannitol
• proteins such as glucose, mannose, sucrose or dextrans, mannitol
• proteins such as glucose, mannose
• compositions of the disclosure may be formulated for routes of administration, such as e.g., oral, enteral, topical, transdermal, intranasal, and/or inhalation; and for routes of administration via injection or infusion such as, e.g., intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and/or parenteral administration.
• routes of administration such as e.g., oral, enteral, topical, transdermal, intranasal, and/or inhalation
• routes of administration via injection or infusion such as, e.g., intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and/or parenteral administration.
• the disclosure provides a method of delivering a transgene or NOI to a tissue of interest in a subject comprising administering an AAV viral vector comprising a modified AAV capsid protein of the disclosure.
• the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting an AAV viral vector of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or the fusion protein (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting an AAV viral vector of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule.
• the cell is in vivo, in vitro, ex vivo or in situ.
• the AAV viral vector of the disclosure comprises a guide RNA of the disclosure and an RNA-binding protein or fusion protein of the disclosure.
• the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting an AAV viral vector of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of treating a disease in a patient in need of such treatment comprising administering to the patient a therapeutically effective amount of an AAV viral vector or a pharmaceutical composition of the disclosure, wherein an AAV viral vector or a pharmaceutical composition comprises a vector comprising a guide RNA of the disclosure and a nucleic acid sequence encoding an RNA-binding protein or an RNA-binding protein fusion protein of the disclosure, wherein an AAV viral vector or a pharmaceutical composition modifies, reduces, destroys, knocks down or ablates a level of expression of a toxic repeat RNA (compared to the level of expression of a toxic repeat RNA treated with a non-targeting (NT) control or compared to no treatment).
• NT non-targeting
• the % elimination of the toxic repeat RNA is any of 20-99%, 25%-99%, 50%-99%, 80%-99%, 90%-99%, 95%-99%. In one embodiment, the % elimination is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment, % elimination is complete elimination or 100% elimination of the toxic repeat RNA.
• a subject of the disclosure is a neonate, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5,6, 7, 8, 9,
• a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of years or partial years in between of age.
• a subject of the disclosure is a mammal. In some embodiments, a subject of the disclosure is a non-human mammal.
• a therapeutically effective amount prevents an onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount improves a prognosis for the subject.
• a composition of the disclosure is administered to the subject via intracerebral administration. In some embodiments, the composition of the disclosure is administered to the subject by an intrastriatal route. In some embodiments, the composition of the disclosure is administered to the subject by a stereotaxic injection or an infusion. In some embodiments, the composition is administered to the brain. In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject locally.
• compositions disclosed herein are formulated as pharmaceutical compositions.
• pharmaceutical compositions for use as disclosed herein may comprise a protein(s) or a polynucleotide encoding the protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• enhanced transduction in muscle tissue occurs following delivery of AAV viral vectors comprising modified AAV capsid proteins.
• enhanced transduction in ocular tissue occurs following subretinal delivery of AAV vectors comprising modified AAV capsid proteins relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• enhanced transduction in neural tissue occurs following delivery of AAV vectors comprising modified AAV capsid proteins relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• reduced or no liver or hepatocyte transduction occurs following systemic delivery of AAV vectors comprising modified AAV capsids relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• reduced or no neutralizing antibody binding occurs following systemic delivery of AAV vectors comprising modified AAV capsids relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• neutralizing antibody titer is minimal following delivery of AAV vectors comprising modified AAV capsids relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• neutralizing antibody titer is reduced compared to delivery of AAV vectors comprising nonmodified AAV capsids relative to AAV capsids comprising unmodified, wild-type, or parental AAV capsid proteins.
• a cell of the disclosure is a prokaryotic cell.
• a cell of the disclosure is a eukaryotic cell.
• the cell is a mammalian cell.
• the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell.
• the cell is a non-human mammalian cell such as a non-human primate cell.
• a cell of the disclosure is a somatic cell. In some embodiments, a cell of the disclosure is a germline cell. In some embodiments, a germline cell of the disclosure is not a human cell.
• a somatic cell of the disclosure is a fibroblast or an epithelial cell.
• an epithelial cell of the disclosure forms a squamous cell epithelium, a cuboidal cell epithelium, a columnar cell epithelium, a stratified cell epithelium, a pseudostratified columnar cell epithelium or a transitional cell epithelium.
• an epithelial cell of the disclosure forms a gland including, but not limited to, a pineal gland, a thymus gland, a pituitary gland, a thyroid gland, an adrenal gland, an apocrine gland, a holocrine gland, a merocrine gland, a serous gland, a mucous gland and a sebaceous gland.
• an epithelial cell of the disclosure contacts an outer surface of an organ including, but not limited to, a lung, a spleen, a stomach, a pancreas, a bladder, an intestine, a kidney, a gallbladder, a liver, a larynx or a pharynx.
• an epithelial cell of the disclosure contacts an outer surface of a blood vessel or a vein.
• a somatic cell of the disclosure is a primary cell.
• a somatic cell of the disclosure is a cultured cell.
• a somatic cell of the disclosure is in vivo, in vitro, ex vivo or in situ.
• a somatic cell of the disclosure is autologous or allogeneic.
• FIG. 5 show immunofluorescence images of HEK293 cells transfected with an AAV Rh74 particle comprising a peptide insertion.
• Both eAAV Rh74 and LBV28 contain the insulin receptor targeting peptide, S519.
• LBV28 has the peptide inserted specifically in VR8 VP1 and not in VP2 or VP3.

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