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WO2023201277A1 - Virus adéno-associés recombinants pour administration tropique au snc - Google Patents

Virus adéno-associés recombinants pour administration tropique au snc Download PDF

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Publication number
WO2023201277A1
WO2023201277A1 PCT/US2023/065692 US2023065692W WO2023201277A1 WO 2023201277 A1 WO2023201277 A1 WO 2023201277A1 US 2023065692 W US2023065692 W US 2023065692W WO 2023201277 A1 WO2023201277 A1 WO 2023201277A1
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Prior art keywords
capsid
seq
aav
aav9
raav
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PCT/US2023/065692
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English (en)
Inventor
Olivier Danos
Samantha YOST
Andrew Mercer
Ye Liu
Joseph Bruder
Subha KARUMUTHIL-MELETHIL
Elad FIRNBERG
Randolph QIAN
April R. TEPE
Jennifer M. EGLEY
Jared B. SMITH
Bradley HOLLIDGE
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Regenxbio Inc.
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Publication of WO2023201277A1 publication Critical patent/WO2023201277A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to recombinant adeno-associated viruses (rAAVs) having a transgene operably linked to a syn promoter for expression in specific regions of the CNS and, in embodiments, capsid proteins with one or more amino acid substitutions and/or peptide insertions that confer and/or enhance desired properties, including persistent expression and tissue tropisms.
  • rAAVs adeno-associated viruses
  • the invention provides engineered capsid proteins comprising one or more amino acid substitutions or peptide insertions that enhance the tropism of an AAV serotype for one or more tissue types as well as capsids that are not engineered but are found to confer muscle or CNS tropisms on rAAVs.
  • the one or more amino acid substitutions and/or insertions in the AAV capsid improve transduction, genome integration and/or transgene expression in heart and/or muscle tissue or the central nervous system while reducing tropism for the liver and/or the dorsal root ganglion and/or peripheral nerve cells.
  • engineered capsids which when administered to the striatum, for example by intraparenchymal administration, exhibit a tropism for dopaminergic neurons.
  • rAAVs having the capsid proteins disclosed herein are useful for delivering a transgene encoding a therapeutic protein for treatment of CNS or muscle disease.
  • rAAVs having a transgene operably linked to a hSyn promoter as disclosed herein are useful for persistent expression of a transgene encoding a therapeutic protein for treatment of CNS or muscle disease.
  • AAVs belong to the parvovirus family and are single-stranded DNA viruses with relatively small genomes and simple genetic components. Without a helper virus, AAV establishes a latent infection.
  • An AAV genome generally has a Rep gene and a Cap gene, flanked by inverted terminal repeats (ITRs), which serve as replication and packaging signals for vector production.
  • ITRs inverted terminal repeats
  • the capsid proteins form capsids that carry genome DNA and can determine tissue tropism to deliver DNA into target cells.
  • rAAVs recombinant AAVs
  • AAV9 recombinant AAVs
  • Delivery to muscle and/or heart tissue is also desirable.
  • Reduction of transduction of liver and/or dorsal root ganglion cells may also be desirable to reduce toxicity.
  • attempts to enhance the neurotropic or muscle/heart tropic properties of rAAVs in human subjects have met with limited success.
  • rAAV vectors with enhanced neurotropic properties for use, e g., in treating disorders associated with the central nervous system are desirable, with minimal transduction in liver and/or dorsal root ganglion cells and/or peripheral nerve cells to minimize adverse effects.
  • rAAV vectors with enhanced tissue-specific targeting and/or enhanced tissue-specific transduction to deliver therapies, such that decreasing dose by several fold (or increasing transgene expression several fold at an equivalent dose) would be highly beneficial.
  • expression cassettes with regulatory elements that promote tissue specific and persistent expression in specific tissue types, including specific tissue types within the CNS are also be highly beneficial.
  • AAV particles (capsids packaging a transgene) administered intraparenchymally to the striatum resulting in delivery of such capsids to other areas of the brain via retrograde and/or anterograde transport. Also disclosed herein are AAV particles that persistently express a transgene under the control of the tissue-specific promoter, hSyn. 3. SUMMARY OF THE INVENTION
  • rAAV vectors comprising expression cassettes in which a transgene encoding a therapeutic protein is operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103). Also provided are methods of administering rAAV vectors comprising expression cassettes in which a transgene encoding a therapeutic protein is operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103).
  • rAAVs recombinant adeno-associated viruses
  • capsid proteins engineered to have one or more amino acid substitutions and/or peptide insertion that enhance tissue targeting, transduction and/or integration of the rAAV genome in CNS and/or muscle tissue relative to a reference capsid, for example, the parent capsid or an AAV8 or AAV9 capsid, while having reduced biodistribution in certain tissues, such as liver and dorsal root ganglion cells, relative to the distribution in CNS and/or muscle and/or relative to the parent capsid or a reference capsid, such as AAV8 or AAV9 capsid, to reduce toxicity.
  • a reference capsid for example, the parent capsid or an AAV8 or AAV9 capsid
  • rAAVs with enhanced or increased biodistribution, including transduction, genome integration, transgene transcription and expression, in CNS tissues (including frontal cortex, hippocampus, cerebellum, midbrain) relative to a reference capsid (for example the unengineered, parental capsid that has been modified or AAV8 or AAV9), with reduced distribution, including transduction, genome integration, transgene transcription and expression in the liver and/or dorsal root ganglion cells (cervical, thoracic, and/or lumbar) compared to the biodistribution in CNS tissue and/or relative to an AAV with a reference capsid, such as the parental capsid or AAV8 or AAV9.
  • rAAVs with enhanced or increased biodistribution, including transduction, genome integration, transgene transcription and expression, in specific CNS tissues, including dopaminergic neurons and tissues including cortex, globus pallidus, amygdala, intralaminar thalamus, and substantia nigra, including when administered to the striatum, relative to a reference capsid (for example the unengineered, parental capsid that has been modified or AAV8 or AAV9), in embodiments, with reduced distribution, including transduction, genome integration, transgene transcription and expression in the liver and/or dorsal root ganglion cells (cervical, thoracic, and/or lumbar) compared to the biodistribution in CNS tissue and/or relative to an AAV with a reference capsid, such as the parental capsid or AAV8 or AAV9.
  • Such rAAVs may be useful to deliver therapeutic proteins for the treatment of CNS disease.
  • rAAV vectors to the striatum (or caudate or putamen) of a subject by intraparenchymal administration of a rAAV vector having a transgene encoding a therapeutic protein.
  • the rAAV incorporates a modified capsid protein, including an AAV8 or AAV9 capsid protein, that has enhanced transduction and/or transgene transcription or expression of the putamen or striatum, hippocampus, frontal cortex and/or midbrain compared to a rAAV incorporating an unmodified wild type capsid, such as an AAV8 or AAV9 capsid, for example, as measured by relative transgene RNA abundance (or transgene expression or DNA transgene copies).
  • a modified capsid protein including an AAV8 or AAV9 capsid protein, that has enhanced transduction and/or transgene transcription or expression of the putamen or striatum, hippocampus, frontal cortex and/or midbrain compared to a rAAV incorporating an unmodified wild type capsid, such as an AAV8 or AAV9 capsid, for example, as measured by relative transgene RNA abundance (or transgene expression or DNA transgene copies).
  • transduction is enhanced in the putamen, caudate ipsi, amydgala, globus pallidus (external) (GPe), or substantia nigra tissue relative to an AAV9 or AAV8 parental capsid (see, e.g., Example 19).
  • transduction (including transgene transcription or expression) is enhanced 2 fold, 5 fold, 10 fold, 50 fold, or 100 fold relative to transduction of an AAV9 or AAV8 parental capsid.
  • the capsid is AAV9.S454.TFR3 (SEQ ID NO: 42) or other engineered capsids disclosed herein.
  • rAAV vectors and methods of administration including intraparenchymal administration, of rAAV vectors having a recombinant AAV genome in which a transgene encoding a therapeutic protein is operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103).
  • the recombinant AAV genomes may be packaged in any of the engineered capsids disclosed herein.
  • Intraparenchymal administration of rAAV9 vectors in which the transgene is operably linked to the hSyn or CAMKII promoter exhibit durable transgene expression in neuronal cells for over 6 months, including increasing transgene expression over at least the 6 month period of time (see Example 20).
  • the expression is specific for neurons and is not detectable in astrocytes or microglia. Further, expression is localized to dopaminergic and/or GAB Anergic neurons relative to cholinergic neurons.
  • identifying a dose for intraparenchymal administration of an AAV9 vector for delivery of a transgene including a transgene operably linked to an hSyn promoter, encoding a therapeutic protein
  • rAAVs with enhanced or increased biodistribution, including transduction, genome integration, transgene transcription and expression, in skeletal muscle and/or cardiac muscle tissues relative to a reference capsid (for example the unengineered, parental capsid or AAV8 or AAV9), with reduced distribution, including transduction, genome integration, transgene transcription and expression in the liver and/or dorsal root ganglion cells (cervical, thoracic, and/or lumbar) compared to the biodistribution in muscle tissue and/or relative to an AAV with a reference capsid, such as the parental capsid or AAV8 or AAV9.
  • a reference capsid such as the parental capsid or AAV8 or AAV9.
  • Such rAAVs may be useful to deliver therapeutic proteins for the treatment of muscle disease.
  • AAV9 capsid proteins or AAV8 capsid proteins (SEQ ID NO:74 or 73, respectively, and as numbered in FIG. 3) or having one or more amino acid substitutions (including, 2, 3 or 4 amino acid substitutions) that preferentially (in particular, at a greater level than rAAVs with the wild type AAV8 or AAV9 capsid) transduce (target) cells of the CNS, and, in certain embodiments, do not target or transduce, or have reduced transduction compared to rAAVs with wild type AAV8 or AAV9 capsids, the liver and/or the dorsal root ganglion and/or peripheral nerve cells.
  • amino acid substitutions including, 2, 3 or 4 amino acid substitutions
  • AAV9 capsid proteins or AAV8 capsid proteins (SEQ ID NO:74 or 73, respectively, and as numbered in FIG. 3) having one or more amino acid substitutions (including, 2, 3 or 4 amino acid substitutions) that preferentially (in particular, at a greater level than rAAVs with the wild type AAV9 capsid) transduce (target) cells of the heart and/or skeletal muscle, and, in certain embodiments, do not target or transduce, or have reduced transduction compared to rAAVs with wild type AAV8 or AAV9 capsids, the liver and/or the dorsal root ganglion and/or peripheral nerve cells.
  • amino acid substitutions including, 2, 3 or 4 amino acid substitutions
  • Such amino acid modifications include S263F/S269T/A273T of AAV9, and corresponding substitutions in other AAV type capsids (for example according to the alignment in FIG. 3), or W530R, Q474A, N272A, or G266A of AAV9, and corresponding substitutions in other AAV type capsids or A269S of AAV8 and corresponding substitutions in other AAV capsids (for example, according to the alignment in FIG. 3).
  • capsids particularly AAV9 capsids having a peptide TEAAPFK (SEQ ID NO: 1) inserted between Q588 and A589 (herein PHP.hDYN) or alternatively between S268 and S269 or between S454 and G455) or inserted in another AAV capsid at a corresponding position (see, e.g., FIG. 3).
  • the capsid is an AAV9 PHP.eB capsid (which has the modifications A587D and Q588G and insertion of the peptide TLAVPFK (SEQ ID NO:20) between G588 and A589) and the peptide TILSRSTQTG (SEQ ID NO: 15) between position 138 and 139, or the corresponding.
  • Additional capsids have a Kidney 1 peptide LPVAS (SEQ ID NO: 6) inserted into the capsid, for example between S454 and G455 of AAV9, or alternatively between S268 and S269 or between Q588 and A589, or the corresponding position of a different capsid.
  • capsids having a TFR3 peptide RTIGPSV (SEQ ID NO: 12) inserted into the capsid, for example between S454 and G455 of AAV9, or alternatively between S268 and S269 or between Q588 and A589, or the corresponding position of a different capsid.
  • the capsids can comprise R697W substitution of AAV rh64Rl.
  • the capsids having these amino acid substitutions and insertions may further have substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) of the AAV9 capsid or at positions 498 to 500 of the AAV8 capsid, or corresponding substitutions in other AAV type capsids.
  • Engineered capsids include AAV8.BB.LD (A269S,498-NNN/AAA-500 substitutions in the amino acid sequence of AAV8, SEQ ID NO: 73), AAV9.BB.LD (S263G/S269T/A273T, 496-NNN/AAA-498 substitutions in the amino acid sequence of AAV9, SEQ ID NO: 74), AAV9.496-NNN/AAA-498 (SEQ ID NO: 31), AAV9.496-NNN/AAA-498.W503R (SEQ ID NO: 32), AAV9.W503R (SEQ ID NO: 33), or AAV9.Q474A (SEQ ID NO: 34).
  • AAV8.BB.LD A269S,498-NNN/AAA-500 substitutions in the amino acid sequence of AAV8, SEQ ID NO: 73
  • AAV9.BB.LD S263G/S269T/A273T, 496-NNN/AAA-4
  • the capsid can be AAV9.N272A.496-NNN-498 (SEQ ID NO:49) or AAV9.G266A.496-NNN-498 (SEQ ID NO: 50).
  • the capsid is AAV9.S454.TFR3 (SEQ ID NO: 42).
  • the capsid is not an engineered capsid, but is an AAVrh.10 capsid (SEQ ID NO: 76), an AAVrh.46 capsid (SEQ ID NO: 97), an AAVrh.64.Rl capsid (SEQ ID NO: 48) or an AAVrh.73 capsid (SEQ ID NO: 79).
  • transduction is measured by detection of transgene, such as GFP fluorescence.
  • the capsid protein to be engineered may be an AAV9 capsid protein but may also be any AAV capsid protein, such as AAV serotype 1 (SEQ ID NO:63); AAV serotype 2 (SEQ ID NO:64); AAV serotype 3 (SEQ ID NO: 65), AAV serotype 3B (SEQ ID NO: 67), AAV serotype 4 (SEQ ID NO:68); AAV serotype 5 (SEQ ID NO:70); AAV serotype 6 (SEQ ID NO:71); 451-461 of AAV7 capsid (SEQ ID NO:72); 451-461 of AAV 8 capsid (SEQ ID NO: 73); AAV serotype 9 (SEQ ID NO: 74); AAV serotype 9e (SEQ ID NO:75); AAV serotype rhlO (SEQ ID NO:76); AAV serotype rh20 (SEQ ID NO:77); and AAV serotype hu.37 (SEQ ID NO:63);
  • the capsids of these vectors are not engineered.
  • unmodified AAV serotype rh64Rl SEQ ID NO:48
  • AAV serotype rh.10 ((SEQ ID NO: 76) AAV serotype rh46 (SEQ ID NO: 97), and AAV serotype rh73 (SEQ ID NO: 79) can be used in the disclosed methods and compositions.
  • rAAVs incorporating the engineered capsids described herein including rAAVs with genomes comprising a transgene of therapeutic interest, including a transgene encoding a therapeutic protein for treatment of a muscle, heart or CNS disease.
  • Packaging cells for producing the rAAVs described herein are provided.
  • Method of treatment by delivery of, and pharmaceutical compositions comprising, the engineered rAAVs described herein are also provided. Also provided are methods of manufacturing the rAAVs with the engineered capsids described herein.
  • the invention is illustrated by way of examples infra describing the construction of rAAV9 capsids engineered with amino acid substitutions and assaying of tissue distribution when administered to non-human primates.
  • Embodiment 1 An rAAV comprising a recombinant capsid protein and a recombinant AAV genome comprising a transgene encoding a therapeutic protein operably linked to an hSyn promoter (SEQ ID NO: 102) flanked by AAV ITRs, in which the rAAV capsid protein comprises an AAV9 capsid protein (SEQ ID NO: 74) with one or more of the following mutations i) S263G/S269R/A273T substitutions, ii) a G266A substitution, iii) an N272A substitution, iv) a W503R substitution, v) a Q474A substitution, vi) 496-NNN/AAA-498 substitutions, vii) an insertion of the peptide TLAAPFK (SEQ ID NO: 1) between a) Q588 and A589, b) S268 and S269, or c) S454
  • Embodiment 2 The rAAV of embodiment 1 which is an AAV8.BBB.LD capsid (SEQ ID NO: 27), an AAV9.BBB.LD capsid (SEQ ID NO: 29), an AAV9.496-NNN/AAA-498 capsid (SEQ ID NO: 31), an AAV9.496-NNN/AAA-498.W503R capsid (SEQ ID NO: 32), an AAV9.W503R capsid (SEQ ID NO: 33), an AAV9.Q474A capsid (SEQ ID NO: 34), an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42), an AAV9.N272A.496-NNN/AAA-498 capsid (SEQ ID NO: 91) or an AAV9.N266A.496-NNN/AAA-498 capsid (SEQ ID NO: 92).
  • Embodiment 3 The rAAV of embodiment 2, which is an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42).
  • Embodiment 4 The rAAV vector of any one of embodiments 1 to 3, wherein the rAAV vector has increased targeting, transduction or genome integration into CNS cells, relative to a reference rAAV vector incorporating the corresponding wild type capsid protein without the amino acid substitutions or peptide insertions.
  • Embodiment 5 The rAAV vector of any one of embodiments 1 to 4, wherein the rAAV vector has decreased targeting, transduction or genome integration into liver cells, relative to the reference rAAV vector incorporating the corresponding wild type capsid protein without the amino acid substitutions or peptide insertions.
  • Embodiment 6 The rAAV vector of any one of embodiments 1 to 5, wherein the recombinant AAV genome further comprises an intron.
  • Embodiment 7 The rAAV vector of embodiment 6, wherein the intron is a chicken [3- actin intron, a minute virus of mice (MVM) intron, a human factor IX intron (e.g., FIX truncated intron 1), a [3-globin splice donor/immunoglobulin heavy chain spice acceptor intron, an adenovirus splice donor /immunoglobulin splice acceptor intron, a SV40 late splice donor /splice acceptor (19S/16S) intron or a hybrid adenovirus splice donor/IgG splice acceptor intron.
  • the intron is a chicken [3- actin intron, a minute virus of mice (MVM) intron, a human factor IX intron (e.g., FIX truncated intron 1), a [3-globin splice donor/immunoglobul
  • Embodiment 8 The rAAV vector of any one of embodiments 1 to 7, wherein the recombinant AAV genome further comprises a poly A signal.
  • Embodiment 9 The rAAV vector of embodiment 8, wherein the poly A signal is a rabbit ⁇ -globin polyA signal, a human growth hormone (hGH) polyA signal, a SV40 late polyA signal, a synthetic polyA (SPA) signal, or a bovine growth hormone (bGH) polyA signal.
  • the poly A signal is a rabbit ⁇ -globin polyA signal, a human growth hormone (hGH) polyA signal, a SV40 late polyA signal, a synthetic polyA (SPA) signal, or a bovine growth hormone (bGH) polyA signal.
  • Embodiment 10 The rAAV vector of any one of embodiments 1 to 9, wherein the recombinant AAV genome is self-complementary.
  • Embodiment 11 The rAAV vector of any one of embodiments 1 to 9, wherein the recombinant AAV genome is single-stranded.
  • Embodiment 12 A nucleic acid composition comprising a nucleic acid sequence encoding the rAAV genome of any one of embodiments 1 to 11.
  • Embodiment 13 A method of administering a therapeutic product to neuronal tissue for treatment of a neurological disorder in a subject in need thereof, said method comprising administering to the subject an rAAV comprising a recombinant capsid protein and a recombinant AAV genome comprising a transgene encoding a therapeutic protein operably linked to an hSyn promoter (SEQ ID NO: 102) flanked by AAV ITRs, wherein said transgene is expressed at a therapeutically effective amount in neuronal tissue of the subject for at least 6 months.
  • SEQ ID NO: 102 hSyn promoter
  • Embodiment 14 The method of embodiment 13, wherein the capsid protein comprises an AAV8 or AAV9 capsid protein.
  • Embodiment 15 The method of embodiment 14, wherein the the rAAV capsid protein comprises an AAV9 capsid protein (SEQ ID NO: 74) with one or more of the following mutations i) S263G/S269R/A273T substitutions, ii) a G266A substitution, iii) an N272A substitution, iv) a W503R substitution, v) a Q474A substitution, vi) 496-NNN/AAA-498 substitutions, vii) an insertion of the peptide TLAAPFK (SEQ ID NO: 1) between a) Q588 and A589, b) S268 and S269, or c) S454 and G455, or viii)an insertion of the peptide RTIGPSV (SEQ ID NO: 12) between S454 and G455, or is an AAV8 capsid (SEQ ID NO: 73) with an A269S substitution or 498- N
  • the recombinant capsid protein is an AAV8.BBB.LD capsid (SEQ ID NO: 27), an AAV9.BBB.LD capsid (SEQ ID NO: 29), an AAV9.496-NNN/AAA-498 capsid (SEQ ID NO: 31), an AAV9.496- NNN/AAA-498.W503R capsid (SEQ ID NO: 32), an AAV9.W503R capsid (SEQ ID NO: 33), an AAV9.Q474A capsid (SEQ ID NO: 34), an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42), an AAV9.N272A.496-NNN/AAA-498 capsid (SEQ ID NO: 91) or an AAV9.N266A.496- NNN/AAA-498 capsid (SEQ ID NO: 92).
  • Embodiment 17 The method of embodiment 16, wherein the recombinant capsid protein is an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42).
  • Embodiment 18 The method of any one of embodiments 13 to 17 wherein said rAAV is administered intraparenchymally.
  • Embodiment 19 The method of any one of embodiments 13 to 18 wherein the transgene is transcribed or expressed in neurons but not astrocytes or microglia.
  • Embodiment 20 The method of any one of embodiments 13 to 19 wherein the transgene is expressed in dopaminergic neurons but not cholinergic neurons.
  • Embodiment 21 The method of any one of embodiments 13 to 20 wherein the neurological disorder is Parkinson’s disease, ALS, tardive dyskinesia, levodopa-induced dyskinesia (LD) or schizophrenia.
  • Parkinson’s disease ALS
  • tardive dyskinesia ALS
  • levodopa-induced dyskinesia LD
  • Embodiment 22 The method of any one of embodiments 13 to 21 wherein the subject is a human subject and the dosage of the rAAV is proportional to a therapeutically effective dosage that is durable over 6 months in a mouse, rat or NHP model.
  • Embodiment 23 A pharmaceutical composition for treatment of a neurological disorder in a subject in need thereof comprising an rAAV comprising a recombinant capsid protein and a recombinant AAV genome comprising a transgene encoding a therapeutic protein operably linked to an hSyn promoter (SEQ ID NO: 102) flanked by AAV ITRs, wherein said transgene is expressed at a therapeutically effective amount in neuronal tissue of the subject for at least 6 months.
  • SEQ ID NO: 102 hSyn promoter
  • Embodiment 24 The pharmaceutical composition of embodiment 23 wherein the AAV capsid protein comprises an AAV8 or AAV9 capsid protein.
  • Embodiment 25 The pharmaceutical composition of embodiment 23, wherein the the rAAV capsid protein comprises an AAV9 capsid protein (SEQ ID NO: 74) with one or more of the following mutations i) S263G/S269R/A273T substitutions, ii) a G266A substitution, iii) an N272A substitution, iv) a W5O3R substitution, v) a Q474A substitution, vi) 496-NNN/AAA-498 substitutions, vii) an insertion of the peptide TLAAPFK (SEQ ID NO: 1) between a) Q588 and A589, b) S268 and S269, or c) S454 and G455, or viii)an insertion of the peptide RTIGPSV (SEQ ID NO: 12) between S454 and G455, or is an AAV8 capsid (SEQ ID NO: 73) with an A269S substitution or 4
  • Embodiment 26 The pharmaceutical composition of any one of embodiments 23 to 25, wherein the recombinant capsid protein is an AAV8.BBB.LD capsid (SEQ ID NO: 27), an AAV9.BBB.LD capsid (SEQ ID NO: 29), an AAV9.496-NNN/AAA-498 capsid (SEQ ID NO: 31), an AAV9.496-NNN/AAA-498.W503R capsid (SEQ ID NO: 32), an AAV9.W503R capsid (SEQ ID NO: 33), an AAV9.Q474A capsid (SEQ ID NO: 34), an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42), an AAV9.N272A.496-NNN/AAA-498 capsid (SEQ ID NO: 91) or an AAV9.N266A.496-NNN/AAA-498 capsid (SEQ ID NO: 92).
  • Embodiment 27 The pharmaceutical composition of embodiment 26, wherein the recombinant capsid protein is an AAV9.S454.Tfr3 capsid (SEQ ID NO: 42).
  • Embodiment 28 The pharmaceutical composition of any one of embodiments 23 to 27 wherein said pharmaceutical composition is formulated for intraparenchymal administration.
  • Embodiment 29 The pharmaceutical composition of any one of embodiments 23 to 28 wherein the transgene is transcribed or expressed in neurons but not astrocytes or microglia.
  • Embodiment 30 The pharmaceutical composition of any one of embodiments 23 to 29 wherein the transgene is expressed in dopaminergic neurons but not cholinergic neurons.
  • Embodiment 31 The pharmaceutical composition of any one of embodiments 23 to 30 wherein the neurological disorder is Parkinson’s disease, ALS, tardive dyskinesia, levodopa- induced dyskinesia (LD) or schizophrenia.
  • the neurological disorder is Parkinson’s disease, ALS, tardive dyskinesia, levodopa- induced dyskinesia (LD) or schizophrenia.
  • Embodiment 32 The pharmaceutical composition of any one of embodiments 23 to 31 wherein the subject is a human subject and the dosage of the rAAV is proportional to a therapeutically effective dosage that is durable over 6 months in a mouse, rat or NHP model.
  • Embodiment 33 A plasmid comprising a cis expression cassette, wherein the cis expression cassette comprises the nucleic acid composition of embodiment 12.
  • Embodiment 34 A host cell comprising the plasmid of embodiment 33.
  • Embodiment 35 A host cell comprising: an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a therapeutic protein operably linked to an hSyn promoter (SEQ ID NO: 102); a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep protein and AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep protein and AAV capsid protein in the host cell in culture and supply the rep and cap proteins in trans, wherein the AAV capsid protein comprises an AAV9 capsid protein (SEQ ID NO: 74) with one or more of the following mutations i) S263G/S269R/A273T substitutions, ii) a G266A substitution, iii) an N272A substitution, iv) a W503R substitution, v) a Q474A substitution, vi)
  • Embodiment 36 A method of producing recombinant AAVs comprising: culturing a host cell containing: an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises the nucleic acid of embodiment 12; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep protein and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep protein and AAV capsid protein in the host cell in culture and supply the rep and cap proteins in trans, and wherein the AAV capsid protein comprises an AAV9 capsid protein (SEQ ID NO: 74) with one or more of the following mutations i) S263G/S269R/A273T substitutions, ii) a G266A substitution, iii) an N272A substitution, iv) a W503R substitution, v) a Q474A substitution, vi) 496
  • Embodiment 37 A method of delivering a transgene to a cell, said method comprising contacting said cell with the rAAV vector of any one of embodiments 1 to 1 1, wherein said transgene is delivered to said cell.
  • Embodiment 38 The method of embodiment 34, wherein said cell is a CNS cell.
  • Embodiment 39 A method of delivering a transgene to a target tissue of a subject in need thereof, said method comprising administering to said subject the rAAV vector of any one of embodiments 1 to 11, wherein the transgene is delivered to said target tissue.
  • Embodiment 40 The method of embodiment 36, wherein the tissue is CNS tissue.
  • Embodiment 41 The method of embodiment 36 or embodiment 37, wherein the rAAV vector is administered systemically, including intravenously or intramuscularly.
  • Embodiment 42 The method of embodiment 36 or embodiment 37, wherein the rAAV vector is administered intrathecally or intracerebroventricularly or intraparenchymally.
  • Embodiment 43 The method of any one of embodiments 36 to 39 wherein the transgene is a CNS disease therapeutic.
  • Embodiment 44 The method of any one of embodiment 37 to 40, wherein the number of circular DNA units containing AAV genomes per cell in the CNS tissue increases over the first six months after administration.
  • Embodiment 45 The method of any one of embodiments 37 to 41, wherein the total number of vector genomes in circular DNA per cell in the CNS tissue increases over the first six months after administration of the rAAV vector.
  • Embodiment 46 The method of any one of embodiments 37 to 42, wherein the average number of vector genomes per piece of circular DNA in the CNS tissue increases over the first six months after administration of the rAAV vector.
  • Embodiment 47 The rAAV of any one of embodiments 1 to 11, wherein the rAAV genome does not encode a tau-specific antibody.
  • Embodiment 48 The rAAV of any one of embodiments 1 to 11 or 47, wherein the transgene does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • Embodiment 49 The nucleic acid of embodiment 12, wherein the nucleic acid sequence does not encode a tau-specific antibody.
  • Embodiment 50 The nucleic acid of embodiment 12 or embodiment 49, wherein the nucleic acid sequence does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • Embodiment 51 The method of any one of embodiments 13 to 22, wherein the rAAV genome does not encode a tau-specific antibody.
  • Embodiment 52 The method of any one of embodiments 13 to 22 or 51, wherein the transgene does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • Embodiment 53 A pharmaceutical composition of any one of embodiments 24 to 32, wherein the rAAV genome does not encode a tau-specific antibody.
  • Embodiment 54 A pharmaceutical composition of any one of embodiments 24 to 32 or 53, wherein the transgene does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • Embodiment 55 The host cell of embodiment 35, wherein the artificial genome does not encode a tau-specific antibody.
  • Embodiment 56 The host cell of embodiment 35 or embodiment 55, wherein the transgene does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • Embodiment 57 The method of any one of embodiments 37 to 46, wherein the transgene does not encode a tau-specific antibody.
  • Embodiment 58 The method of any one of embodiments 37 to 46 or 57, wherein the transgene does not encode a 4P3 antibody, a 31B6 antibody, or an 8H1 antibody.
  • FIG. 1 depicts sequence comparison of the capsid amino acid sequences including the VR-IV loop of the adeno-associated virus type 9 (AAV9 VR-IV) from residues L447 to R476, (with residues 451-459 bracketed) to corresponding to regions of other AAVs.
  • Figure discloses SEQ ID NOS:53-62, respectively, in order of appearance. The top sequence is the consensus sequence, SEQ ID NO 52
  • FIG. 2 depicts a protein model of an AAV capsid structure, showing capsid variable regions VR-IV, VR-V and VR-VIII. The box highlights the loop region of VR-IV which provides surface-exposed amino acids as represented in the model.
  • FIG. 3 depicts alignment of AAVs l-9e, 3B, rhlO, rh20, rh39, rh73, rh74 version 1 and version 2, hu!2, hu21 , hu26, hu37, hu51 and hu53 capsid sequences with insertion sites for heterologous peptides after the initiation codon of VP2, and within or near variable region 1 (VR- I), variable region 4 (VR-IV), and variable region 8 (VR-VIII), all highlighted in grey; a particular insertion site within variable region eight (VR-VIII) of each capsid protein is shown by the symbol “#” (after amino acid residue 588 according to the amino acid numbering of AAV9).
  • FIG. 4 depicts in vivo kidney to liver transduction efficiency ratio of genetically engineered AAV9 vectors containing insertions of homing peptides immediately after amino acid 454. Details on peptides used in this study are outlined in Table 6.
  • FIG. 5 depicts the relative abundance (RA) of the viral genomes (normalized to input) in the frontal cortex of the cynomolgus monkey model.
  • FIGS. 6 and 7 depict the Relative Abundance of the viral genomes (normalized to input) in the hippocampus and the cerebellum of the cynomolgus monkey model, respectively.
  • the RA of AAV.rh34 is shown by the shaded column on the left side of the graphs and the RA of AAV9 reference is showed by the shaded column in the middle of the graphs.
  • FIGS. 6 and 7 show that AAV.rh34 is a top performing capsid in the intravenous administration pool.
  • FIG. 8 depicts the RA of the viral genomes (normalized to input) in the frontal cortex of the cynomolgus monkey model.
  • FIG. 9 depicts the RA of the viral genomes (normalized to input) in the hippocampus of the cynomolgus monkey model.
  • FIG. 10 depicts the RA of the viral genomes (normalized to input) in the midbrain of the cynomolgus monkey model.
  • FIG. 11 depicts the RA of the viral genomes (normalized to input) in the cerebellum of the cynomolgus monkey model.
  • FIG. 12 depicts the RA of the viral genomes (normalized to input) in the cervical DRGs of the cynomolgus monkey model.
  • FIG. 13 depicts the RA of the viral genomes (normalized to input) in the lumbar DRGs of the cynomolgus monkey model.
  • FIG. 14 depicts a Venn diagram of the top performing 15 capsids transducing the frontal cortex, hippocampus, midbrain and cerebellum following ICV administration. As indicated in the diagram, AAV6, AAV8.BBB, AAV.rh.46, and AAV1 were the only AAVs represented in each of the top performing groups.
  • FIG. 15 depicts a Venn diagram of the top performing 45 capsids transducing the hippocampus and the 45 capsids with the lowest transduction values for DRG, to identify hippocampus-targeting DRG friendly capsids.
  • AAV.hu.60, AAV.rh.21, AAV.PHP.hB, AAV.rh.15, AAV.rh.24, AAV9.W503R, hu.5, AAV9.Q474A, and AAV.hu.10 were the only AAVs represented in each of the groups.
  • FIG. 16 depicts a Venn diagram of the top performing 40 capsids transducing the heart, biceps, and gastrocnemius and the 40 capsids with the lowest transduction values for the liver, to identify muscle-targeting capsids that have reduced targeting to the liver.
  • AAV.PHPeB.VP2Herp was the only AAVs represented in each of the groups.
  • FIG. 17 depicts a Venn diagram of the top performing 15 capsids transducing the heart, biceps, and gastrocnemius muscle.
  • FIGS. 18A and 18B depict the RA of the viral genomes (normalized to input) in the gastrocnemius and the liver of the cynomolgus monkey model, respectively.
  • FIG. 19A and 19B depict the number of genome copies of DNA (A) or RNA (B) of select “liver-detargeting” (LD) vectors as detected in the liver of NHPs following IV administration of the capsid library.
  • FIG. 20 depicts the biodistribution of select “liver-detargeting” (LD) vectors compared to their parental AAV9 capsid in various tissues, in NHPs following IV administration of the capsid library.
  • LD liver-detargeting
  • FIG. 21 depicts the biodistribution of select LD vectors compared to their parental AAV8 capsid in various tissues, in NHPs following IV administration of the capsid library.
  • FIGs 22A and 22B depict the change in relative abundance for point mutations affecting AAV9 transduction (or liver transduction) as compared to AAV9.
  • the four mutants depicted in this study demonstrate retention in the blood when compared to wild-type AAV9.
  • FIG 22B shows the change in relative abundance for the AAV8 and AAV9 mutants combining the NNN/AAA mutation with the transport motif BBB (A269S, in AAV8; S263G/S269T/A273T, in AAV9), as compared to parental capsid (AAV8 or AAV9, respectively).
  • FIG. 23A and FIG. 23B FIG. 23A shows the increase in blood retention 3-24 hr relative to AAV9.
  • FIG. 23B shows the increase in blood retention 3-24 hr relative to parental AAV.
  • FIG. 24 depicts the biodistribution of select AAV vectors in muscle tissues, including cardiac muscle, as well as cerebrum, liver and pancreas in wild-type B6 mice following IV administration of the capsid library.
  • FIGs. 25A-25H depicts biodistribution of various capsids in wild-type B6 mice compared to that of mdx mouse tissue.
  • FIG. 26 depicts qPCR analysis which revealed high copy number (RNA cp/ug) of total vector transcripts localized to putamen, caudate, GPe and interestingly, SNc, as well as other tissues (RNA copy number/ug, adjusted to log scale).
  • FIG. 27 depicts on overview of the relationship between vector spread from posterior compartments to anterior compartments, as an absolute measure of RNA copy number vs. log scale. Localization was observed in the putamen, as well as the caudate regions. Vector expression was strongest in injection site (e.g. putamen anterior).
  • FIG. 28A and 28B depict absolute RNA copy number/ug distribution by qPCR (A) and results adjusted to log scale (B), including peripheral tissues. Expression in the periphery and in unrelated brain regions was negligible, which comports with known lack of anatomical connectivity from cerebellum or hippocampus to putamen.
  • FIG. 29 depicts NGS analysis to identify vector DNA from the capsid pool in putamen.
  • the BC029 barcode indicates the mutated AAV9 capsid, AAV9.S454.Tfr3 is the highest transduced capsid identified in the putamen ipsilateral (relative to the location of injection) of this particular injected subject.
  • FIG. 30 depicts RNA abundance adjusted to input (normalized to 1 per ug ).
  • qPCR was performed to identify vector transcripts relative to high expressing capsids in the selected tissues: putamen ipsilateral (sample “punch” 1 or 2), caudate ipsilateral (punch 1 or 2), extreme capsule ipsilateral, golubus pallidus (external) (GPe) ipsilateral, amygdala ipsilateral, substantia nigra ipsilateral.
  • AAV9.S454.Tfr3 demonstrates improved transduction relative to AAV9 following intraparenchymal delivery, for example a 4 to 5 fold increase RNA expression compared to AAV9 is observed in most brain regions analyzed.
  • FIGs. 31A and 31B show biodistribution of AAV9 and AAV9.454.Tfr3 vector RNA and DNA following IV administration of pooled capsids intravenously in NHPs.
  • FTGs. 32A and 32B illustrate the biodistribution of AAV9 and AAV9.454.Tfr3 vector RNA and DNA in selected tissues following IV administration of pooled capsids intravenously in mice.
  • FIGs. 33A and 33B illustrate the correlation between DNA abundances (FIG. 33A) and RNA abundances (FIG. 33B) in NHP and mouse at the injection site following intraparenchymal delivery to striatum (mouse) or putamen (NHP) of a barcoded library of rAAV having modified capsids.
  • AAV9.S454.Tfr3 (BCO29) identified as top hit in NHP and mouse.
  • FIG. 34 illustrates AAV9 and AAV9.S454.Tfr3 (BC029) RNA levels (FIG. 34A) and AAV9 and AAV9.S454.Tfr3 (BC029) DNA levels (FIG. 34B) in NHP following intraparenchymal administration.
  • FIG. 35 illustrates AAV9 and AAV9.S454 TFR3 (BC029) RNA levels (FIG. 35A) and AAV9 and AAV9.S454 TFR3 (BC029) DNA levels (FIG. 35B) following intraparenchymal administration in mouse.
  • FIGs. 36A and 36B depict genome copy per pg DNA (left y-axis) and genome copy of BC029 (AAV9.S454.Tfr3 capsid) per pg DNA relative to genome copy per pg DNA AAV9 (right y-axis) in putamen (FIG. 36A) and caudate (FIG. 36B) in NHP administered PAVE118 library delivered by MRI-guided injection to the putamen.
  • FIGs. 37A and 37B depict RNA transcripts from BC029 (AAV9.S454.Tfr3 capsid) (left y-axis) and RNA transcripts from BC029 relative to RNA transcripts from AAV9 (right y-axis) in putamen (FIG. 37A) and caudate (FIG. 37B) in NHP administered PAVE118 library delivered by MRI-guided injection to the putamen.
  • FIGs. 38A and 38B show the ratio of RNA (transcript) :DNA (genome copy) of BC029 (AAV9.S454.Tfr3 capsid) (FIG. 38A) and the ratio of RNA:DNA of BC029 relative to the RNA:DNA ratio of AAV9 (FIG. 38B) in putamen (solid bars) and caudate (hatched bars) in NHP administered PAVE118 library delivered by MRI-guided injection to the putamen.
  • FIGs 39A and 39B show genome copies (GC/ug DNA) of BC029 (AAV9.S454.Tfr3 capsid) relative to AAV9 (FIG.
  • transcript copies from BC029 relative to AAV9 in punch samples from the NHP central nervous system (substantia nigra 1, substantia nigra 2, rostral intralaminar thalamus, caudal intralaminar thalamus and frontal cortex) from NHP administered the PAVE118 library to the putamen.
  • NHP central nervous system substantially nigra 1, substantia nigra 2, rostral intralaminar thalamus, caudal intralaminar thalamus and frontal cortex
  • FIGs. 40A-40C show GC/cell of BC029 (AAV9.S454.Tfr3 capsid) (left y-axis) and GC/cell BC029 DNA relative to GC/cell AAV9 (right y-axis) in the striatum (FIG. 40A), thalamus (FIG. 40B), and frontal cortex (FIG. 40C) of mice administered PAVE118 library by injection into the striatum.
  • FIGs. 41A-41C show transcripts/pg RNA from BC029 (AAV9.S454.Tfr3 capsid) (left y-axis) and transcripts/pg RNABC029 relative to AAV9 (right y-axis) in the striatum (FIG. 41A), thalamus (FIG. 41B), and frontal cortex (FIG. 41C) of mice administered PAVE118 library by injection into the striatum.
  • FIGs. 42A and 42B show RNA:DNA ratio (transcripts/pg RNA per GC/cell DNA) for BC029 (FIG. 42A) and RNA:DNA ratio for BC029 relative to AAV9 (transcripts/pg RNA per GC/cell DNA) (FIG. 42B) in the striatum (solid bars), thalamus (hatched bars) and frontal cortex (checked bars) in mice administered PAVE118 library by injection into the striatum.
  • FIGs. 43A-43C show ddPCR analysis of GFP transgene from isolated striatum samples at 3 weeks, 3 months and 6 months after injection of AAV9 having a GFP transgene under control of the CAG promoter (circles), the hSyn promoter (square) or the CamKII promoter (triangle) in mice.
  • Bar graphs show GFP gene copy number per cell (FIG. 43A), GFP RNA expression (FIG. 43B) and GFP RNA/DNA ratio (FIG. 43C).
  • FIGs. 44A-44C shows quantitative luminance analysis of GFP expression in brain tissue from injected mice.
  • FIG. 44A is a plot of GFP luminance under the control of the CAG promoter (circles), the hSyn promoter (squares), and the CamKII promoter (triangles) at 3 weeks, 3 months and 6 months following injection.
  • FIG. 44B is a line graph showing transgene expression over time.
  • FIG. 44C is a dot plot showing the quantitative analysis of GFP luminance under control of the CAG promoter (circles), the hSyn promoter (squares), and the CamKII promoter (triangles) at 6 months.
  • FIGd. 45A-45C shows the percentage of neurons (FIG.
  • FIG. 45A the percentage of astrocytes (FIG. 45B) and the percentage of microglia (FIG. 45C) expressing GFP under the CAG promoter (circles), the hSyn promoter (squares) and CAMK11 promoter (triangles) in injected mouse brain tissue.
  • FIGs 46A-46B show relative abundance of genome copy (FIG. 46A) and transcript copy (FIG. 46B) of AAV9 (light bars) and BC029 (dark bars) in CNS (frontal cortex, striatum and hippocampus) in mice administered PAVE118 library by ICV infusion to the left ventricle.
  • FIG. 46C shows “off target” biodistribution after ICV administration (GC/pg DNA) of AAV9 (light bars) and BC029 (dark bars) in mouse liver, NHP liver, NHP heart, NHP lumbar DRG and NHP thoracic DRG.
  • FIGs. 47 A-F show long-term kinetics of rAAV9-CAG-GFP in the striatum in mice. Quantification of luminance from native GFP fluorescence (500 ms exposure) during the first 6 months after rAAV9-CAG-GFP administration (FIG. 47A). ddPCR analysis measuring the amount of GFP mRNA relative to TBP mRNA (FIG. 47B) and GFP DNA copies per cell (FIG. 47C) as determined by copy numbers of GFP to two glucagoncopies per cell during the first 6 months after administration of rAAV9-CAG-GFP. Data from 3 weeks, 3 months and 6 months was previously presented in Figure 2.
  • FIG. 47D The number of circular DNA units that contain an AAV genome (not considering concatemers) per cell (FIG. 47D), the total number of AAV genomes in circular DNA per cell (diploid genome) (FIG. 47E) and the average number of AAV genomes per circular episome (FIG. 47F) (values from FIG. 47E divided by values from FIG. 47D) in the striatum of mice during the first 6 months after administration of rAAV9-CAG-GFP. Data was analyzed using a one-way ANOVA followed by Tukey’s multiple comparisons test. * p ⁇ 0.05; ** p ⁇ 0.01, *** p ⁇ 0.001, # p ⁇ 0.0001. Significance symbols in parentheses are compare with Day 1.
  • FIGs. 48A-48C show long-term kinetics of rAAV9 concatemers in the striatum in mice.
  • the number of circular DNA units that contain an AAV genome (not considering concatemers) per cell (FIG. 48A), the total number of AAV genomes in circular DNA per cell (diploid genome) (FIG. 48B) and the average number of AAV genomes per circular episome (FIG. 48C) (values from FIG. 48B divided by values from FIG.
  • rAAV vectors and methods of administration including intraparenchymal administration, of rAAV vectors having a recombinant AAV genome in which a transgene encoding a therapeutic protein is operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103).
  • Intraparenchymal administration of rAAV9 vectors in which the transgene is operably linked to the hSyn or CAMKII promoter exhibit durable transgene expression in neuronal cells for over 6 months, including increasing transgene expression over at least the 6 month period of time (see Example 20).
  • the expression is specific for neurons and is not detectable in astrocytes or microglia. Further, expression is localized to dopaminergic and/or GAB Anergic neurons relative to cholinergic neurons.
  • the methods may be useful in the treatment of neurological disease, for example, movement disease such as Parkinson’s Disease or levodopa- induced dyskinesia (LD) or tardive dyskinesia or other neurological disorders.
  • movement disease such as Parkinson’s Disease or levodopa- induced dyskinesia (LD) or tardive dyskinesia or other neurological disorders.
  • rAAVs recombinant adeno-associated viruses
  • capsid proteins engineered relative to a reference capsid protein, such that the rAAV has enhanced desired properties, such as increased tissue targeting, including transduction, genome integration and transgene expression, particularly, preferentially, relative to the reference capsid protein (e.g., the unengineered or wild type capsid), to CNS, including dopaminergic neurons, or to heart and/or skeletal muscle tissue.
  • the engineered capsid has reduced tropism (i.e., tissue targeting, transduction and integration of the rAAV genome) relative to the reference capsid for liver, dorsal root ganglion and/or peripheral nervous tissue to reduce toxicity of the AAV gene therapy.
  • the modifications include amino acid substitutions (including 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions) and/or peptide insertions (4 to 20, or 7 contiguous amino acids, and in embodiments no more than 12 contiguous amino acids from a heterologous protein) as described herein.
  • the AAV capsid protein to be engineered is, in certain embodiments, an AAV9 capsid protein or an AAV8 capsid protein.
  • the AAV capsid to be engineered is an AAV rh.34, AAV4, AAV5, AAV hu.26, AAV rh.31, AAV hu.13, AAV hu.56, AAV hu.53, AAV7, AAV rh64Rl, AAV rh46 or AAV rh73 capsid protein. (See FIG.
  • engineered capsids having one or more amino acid substitutions that promote transduction and/or tissue tropism, particularly for enhanced, relative to an unengineered capsid, targeting for heart and/or skeletal muscle and, in embodiments, reduced, relative to an unengineered capsid, targeting for liver, dorsal root ganglion, and/or peripheral nervous tissue.
  • injection of one AAV serotype into the striatum will be preferentially transported to e.g. the cortex, and a different AAV serotype injected into the striatum will be preferentially transported to e.g. the substantia nigra.
  • the amino acid substitutions are S263F/S269T/A273T of AAV9, and corresponding substitutions in other AAV type capsids (for example according to the alignment in FIG. 3), or W53OR, Q474A, N272A, or G266A of AAV9, and corresponding substitutions in other AAV type capsids or A269S of AAV8 and corresponding substitutions in other AAV capsids (for example, according to the alignment in FIG. 3).
  • capsids particularly AAV9 capsids having a peptide TLAAPFK (SEQ ID NO:1) inserted between Q588 and A589 (herein PHP.hDYN) or alternatively between S268 and S269 or between S454 and G455) or inserted in another AAV capsid at a corresponding position (see, e.g., FIG. 3).
  • capsids particularly AAV9 capsids having a peptide RTIGPSV (SEQ ID NO: 12) inserted between S454 and G455 (herein AAV9.S454-TFR3; SEQ ID NO: 42) or alternatively between S268 and S269 or between Q588 and A589) or inserted in another AAV capsid at a corresponding position (see, e.g., FIG.
  • capsids have a tropism and exhibit increased transcriptional activity in the putamen and caudate, including for dopaminergic neurons and exhibit retrograde or anterograde transport to the substantia nigra, and to the rostral intralaminar thalamus, caudal intralaminar thalamus and frontal cortex including when administered to the striatum.
  • capsids particularly AAV9 capsids having S263G/S269T/A273T, and 496-NNN/ AAA-498 substitutions in the amino acid sequence of AAV9 (herein AAV9.BBB.LD; SEQ ID NO: 74) (see, e.g., FIG.
  • capsids have a tropism for amygdala and/or cortex and exhibit retrograde or anterograde transport to the amygdala and/or cortex, including when administered to the striatum.
  • the capsid is an AAV9 PHP.eB capsid (which has the modifications A587D and Q588G and insertion of the peptide TLAVPFK (SEQ ID NO:20) between G588 and A589) and the peptide TILSRSTQTG (SEQ ID NO: 15) between position 138 and 139, or the corresponding.
  • Additional capsids have a Kidney 1 peptide LPVAS (SEQ ID NO: 6) inserted into the capsid, for example between S454 and G455 of AAV9, or alternatively between S268 and S269 or between Q588 and A589, or the corresponding position of a different capsid.
  • the capsids can comprise R697W substitution of AAV rh64Rl .
  • the capsids having these amino acid substitutions and insertions may further have substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) of the AAV9 capsid or at positions 498 to 500 of the A AV8 capsid, or corresponding substitutions in other AAV type capsids.
  • Engineered capsids include AAV8.BB.LD (A269S,498-NNN/AAA-500 substitutions in the amino acid sequence of AAV8, SEQ ID NO: 73), AAV9.BB.LD (S263G/S269T/A273T, 496-NNN/AAA-498 substitutions in the amino acid sequence of AAV9, SEQ ID NO: 74), AAV9.BBB (SEQ ID NO: 28), AAV9.496-NNN/AAA-498 (SEQ ID NO: 31), AAV9.496-NNN/AAA-498.W503R (SEQ ID NO: 32), AAV9.W503R (SEQ ID NO: 33), AAV9.Q474A (SEQ ID NO: 34), AAV9.S454.Tfrl (SEQ ID NO: 59) or AAV9.S454-TFR3 (SEQ ID NO: 42).
  • AAV8.BB.LD A269S,498-NNN/AAA-500
  • the capsid can be AAV9.N272A.496-NNN-498 (SEQ ID NO:49) or AAV9.G266A.496-NNN-498 (SEQ ID NO: 50).
  • the capsid is not an engineered capsid, but is an AAV7 (SEQ ID NO: 72), AAVrh.10 capsid (SEQ ID NO: 76), an AAVrh.46 capsid (SEQ ID NO: 97), an AAVrh.64.Rl capsid (SEQ ID NO: 48) or an AAVrh.73 capsid (SEQ ID NO: 79).
  • transduction is measured by detection of transgene, such as GFP fluorescence.
  • the capsids having these amino acid substitutions and insertions may further have substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) of the AAV9 capsid, or corresponding substitutions in other AAV type capsids.
  • This engineered capsid may exhibit preferential targeting for heart and/or skeletal muscle, and reduced targeting (compared to an AAV bearing the unengineered capsid) for liver and/or dorsal root ganglion cells and/or peripheral nervous system tissue, and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a muscle disease.
  • a recombinant capsid protein including an engineered AAV9 capsid protein, and an rAAV comprising the capsid protein, in which the peptide TLAVPFK (SEQ ID NO:20) is inserted between G588 and A589 of AAV9, and, in particular, the capsid protein also has amino acid substitutions A587D/Q588G (PHP.eB) and further has the peptide TILSRSTQTG (SEQ ID NO: 15) inserted after position 138 of AAV9 (collectively, AAVPHPeB.VP2Herp; see Table 17), or in the corresponding positions of another AAV.
  • Kidneyl peptide LPVAS (SEQ ID NO:6) (or alternatively CLPVASC (SEQ ID NO:5)) inserted into the capsid, for example between S454 and G455 of AAV9 (see Table 17), or alternatively between S268 and S269 or between Q588 and A589, or the corresponding position of a different capsid.
  • Such an engineered capsid may exhibit preferential targeting for heart and skeletal muscle, and reduced targeting (as compared to an AAV having the unengineered capsid) for liver and/or dorsal root ganglion cells and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a muscle disease (such as, but not limited to a muscular dystrophy).
  • the engineered rAAV exhibits at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater transduction in cardiac muscle and/or skeletal muscle cells compared to a reference AAV capsid, including an AAV9 capsid or an AAV8 capsid, or the parental capsid.
  • the muscle is gastrocnemius muscle, bicep, tricep and/or heart muscle.
  • the engineered rAAV exhibits of 50%, 60%, 70%, 80%, 90%, 95% or 99% less transduction in liver compared to the reference AAV capsid compared to a reference AAV capsid, including an AAV9 capsid or an AAV8 capsid, or the parental capsid.
  • the rAAV exhibits of 50%, 60%, 70%, 80%, 90%, 95% or 99% less transduction in dorsal root ganglion cells (including in cervical, thoracic or lumbar DRG cells) compared to the reference AAV capsid.
  • the enhanced and/or reduce transduction may be with any mode of administration, by intravenous administration, intramuscular administration, or any type of systemic administration, intrathecal administration or ICV administration.
  • engineered capsids having one or more amino acid substitutions that promote transduction and/or tissue tropism, particularly for enhanced, relative to an unengineered capsid, targeting for CNS and, in embodiments, reduced, relative to an unengineered capsid, targeting for liver, dorsal root ganglion, and/or peripheral nervous tissue.
  • the amino acid substitutions areA269S of AAV8 (or at a corresponding position in a different AAV serotype capsid), S263G/S269T/A273T of AAV9 (or at a corresponding position in a different AAV serotype capsid), N272A or N266A of AAV9 (or at a corresponding position in a different AAV serotype capsid), Q474A of AAV9 (or at a corresponding position in a different AAV serotype capsid), or W503R of AAV9 (or at a corresponding position in a different AAV serotype capsid), or R697W of rh64Rl (or at a corresponding position in a different AAV serotype capsid) or an insertion of the peptide RTIGPSV (SEQ ID NO: 12) after S454 of AAV9 (or at a corresponding position in a different AAV serotype capsid).
  • the capsids having these amino acid substitutions and insertions may further have or alternatively have substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) of the AAV9 capsid (SEQ ID NO: 74) or have substitutions of the NNN (asparagines) at 498 to 500 with AAA (alanines) of the AAV8 capsid (SEQ ID NO: 73), or corresponding substitutions in other AAV type capsids.
  • This engineered capsid may exhibit preferential targeting for CNS, and reduced targeting (compared to an AAV bearing the unengineered capsid) for liver and/or dorsal root ganglion cells and/or peripheral nervous system tissue, and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a CNS disease.
  • capsid proteins and rAAVs comprising them, that have inserted peptides that target and/or promote rAAV cellular uptake, transduction and/or genome integration in CNS tissue and, in embodiments, reduced, relative to an unengineered capsid, targeting for liver, dorsal root ganglion, and/or peripheral nervous tissue, for example, the peptide RTIGPSV (SEQ ID NO: 12), TILSRSTQTG (SEQ ID NO: 15); TLAVPFK (SEQ ID NO:20); or TLAAPFK (SEQ ID NO: 1).
  • the peptide RTIGPSV is inserted between S454 and G455 of AAV9 (see Fig.
  • the peptide TLAAPFK (SEQ ID NO: 1) is inserted between Q588 and A589 of AAV9 (AAV9.hDyn; see Table 17), or the corresponding position of another AAV (see FIG. 3).
  • the capsid is rh.34, rh.10, rh.46, rh.73, or rh64.Rl (Fig. 3 or Table 17 for sequence), or an engineered form of rh.34, rh.10, rh.46, rh.73, or rh64.Rl.
  • engineered capsids may exhibit preferential targeting for CNS, and reduced targeting (compared to an AAV bearing the unengineered capsid) for liver and/or dorsal root ganglion cells and/or peripheral nervous system tissue, and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a CNS disease.
  • the engineered capsids particularly capsids with the TFR3 peptide (RTIGPSV (SEQ ID NO: 12) inserted, such as AAV9.S454-TFR3 capsid) may exhibit preferential targeting for areas of the CNS, including when locally administered to the striatum (or intracerebroventricularly), such as dopaminergic neurons.
  • capsids may exhibit retrograde or anterograde transport to the substantia nigra and may target the caudate and external globus pallidus.
  • AAV9.S454-Tfr3 capsids exhibit, upon intraparenchymal or intracerebroventricular administration, enhanced transduction efficiency and transcriptional activity compared to, for example, AAV9 (may be 2 fold, 4 fold, 5 fold, 8 fold or 10 fold better) in the putamen and/or caudate, and may include regions of interest, including the substantia nigra, intralaminar thalamus, and frontal cortex.
  • the capsids further exhibit reduced targeting (compared to an AAV bearing the unengineered capsid) for liver and/or dorsal root ganglion cells and/or peripheral nervous system tissue, and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a CNS disease, particularly a disease involving dopaminergic neurons, including movement disorders, such as Parkinson’s Disease, levodopa-induced dyskinesia (LD) and tardive dyskinesia, or alternatively, schizophrenia.
  • Parkinson’s Disease levodopa-induced dyskinesia (LD) and tardive dyskinesia
  • LD levodopa-induced dyskinesia
  • tardive dyskinesia or alternatively, schizophrenia.
  • the engineered rAAV exhibits at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater transduction or transgene transcription (mRNA abundance) or transgene expression in CNS tissue compared to a reference AAV capsid, such as the parental capsid or AAV8 or AAV9.
  • the CNS tissue may be one or more of the frontal cortex, hippocampus, cerebellum, midbrain and/or hindbrain or, in alternative embodiments, may be dopaminergic neurons, putamen, caudate, substantia nigra, and/or globus pallidus (external).
  • the engineered rAAV exhibits of 50%, 60%, 70%, 80%, 90%, 95% or 99% less transduction in liver compared to the reference AAV capsid such as the parental capsid or AAV8 or AAV9.
  • the rAAV exhibits of 50%, 60%, 70%, 80%, 90%, 95% or 99% less transduction in dorsal root ganglion cells (including in cervical, thoracic or lumbar DRG cells) compared to the reference AAV capsid such as the parental capsid or AAV8 or AAV9.
  • the enhanced and/or reduce transduction may be with any mode of administration, by intravenous administration, intramuscular administration, or any type of systemic administration, intrathecal administration or ICV administration.
  • the administration is intraparenchymal administration, including delivery to the striatum.
  • identifying a dose for intraparenchymal administration of an AAV9 vector for delivery of a transgene including a transgene operably linked to an hSyn promoter, encoding a therapeutic protein comprising assessing dosage of the AAV9 vector product in a mouse, rat or NHP that achieves therapeutically effective expression of the transgene over 6 months or more (including 1 year, 18 months or 2 years) and correlating that dose with appropriate dosage for intraparenchymal or intracerebroventricular administration to a human (for example, based upon brain weight of a primate being 1 log (10 times) greater than rat and 2 logs (100 times) greater than a mouse brain).
  • Recombinant vectors comprising the capsid proteins also are provided, along with pharmaceutical compositions thereof, nucleic acids encoding the capsid proteins, and methods of making and using the capsid proteins and rA AV vectors having the engineered capsids for targeted delivery, improved transduction and/or treatment of disorders associated with the target tissue.
  • AAV or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses.
  • the AAV can be an AAV derived from a naturally occurring “wildtype” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene.
  • An example of the latter includes a rAAV having a capsid protein comprising a peptide insertion into the amino acid sequence of the naturally-occurring capsid.
  • rAAV refers to a “recombinant AAV.”
  • a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.
  • rep-cap helper plasmid refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.
  • capsid protein refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus.
  • the capsid protein may be VP1, VP2, or VP3.
  • replica gene refers to the nucleic acid sequences that encode the non-structural protein needed for replication and production of virus.
  • nucleic acids and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules.
  • Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases.
  • Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • the nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both singlestranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.
  • a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), or, in certain embodiments, a human.
  • a non-primate e.g., cows, pigs, horses, cats, dogs, rats etc.
  • a primate e.g., monkey and human
  • a “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom.
  • a therapeutically effective amount with respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.
  • prophylactic agent refers to any agent which can be used in the prevention, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder.
  • a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.
  • a prophylactic agent of the invention can be administered to a subject “pre-disposed” to a target disease or disorder.
  • a subject that is “pre-disposed” to a disease or disorder is one that shows symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder.
  • a patient with a family history of a disease associated with a missing gene may qualify as one predisposed thereto.
  • a patient with a dormant tumor that persists after removal of a primary tumor may qualify as one predisposed to recurrence of a tumor.
  • the “central nervous system” refers to neural tissue reaches by a circulating agent after crossing a blood-brain barrier, and includes, for example, the brain, optic nerves, cranial nerves, and spinal cord.
  • the CNS also includes the cerebrospinal fluid, which fdls the central canal of the spinal cord as well as the ventricles of the brain.
  • AAV “serotype” refers to an AAV having an immunologically distinct capsid, a naturally-occurring capsid, or an engineered capsid.
  • rAAVs recombinant adeno-associated viruses
  • capsid proteins engineered relative to a reference capsid protein, such that the rAAV has enhanced desired properties, such as increased tissue targeting, including transduction, genome integration and transgene expression, particularly, preferentially, relative to the reference capsid protein (e.g., the unengineered or wild type capsid), to CNS, including dopaminergic neurons, or to heart and/or skeletal muscle tissue.
  • desired properties such as increased tissue targeting, including transduction, genome integration and transgene expression, particularly, preferentially, relative to the reference capsid protein (e.g., the unengineered or wild type capsid), to CNS, including dopaminergic neurons, or to heart and/or skeletal muscle tissue.
  • the engineered capsid has reduced tropism (i.e., tissue targeting, transduction and integration of the rAAV genome, transcription and/or expression of the transgene) relative to the reference capsid for liver, dorsal root ganglion and/or peripheral nervous tissue to reduce toxicity of the AAV gene therapy.
  • the modifications include amino acid substitutions (including 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions) and/or peptide insertions (4 to 20, or 7 contiguous amino acids, and in embodiments no more than 12 contiguous amino acids from a heterologous protein) as described herein.
  • AAV capsids were modified by introducing selected single to multiple amino acid substitutions which increase effective gene delivery to the CNS or to cardiac or skeletal muscle, detarget the liver and/or dorsal root ganglion to reduce toxicity, and/or reduce immune responses of neutralizing antibodies.
  • the capsids have one or more amino acid substitutions including a W5O3R substitution, a Q474 substitutional a N272A or N266A substitution in AAV9 or the corresponding substitution in another AAV serotype or an A269S substitution in AAV8 or the corresponding substitution in another AAV serotype.
  • rAAV having a capsid with the Q474A substitution may be particularly useful for delivery to skeletal and/or cardiac muscle or CNS tissue and rAAV having a capsid with the W503R substitution may be particularly useful for delivery to CNS tissue, particularly with reduced, compared to reference capsid containing rAAVs, transduction in the liver and/or DRGs.
  • substitutions include S263G/S269R/A273T substitutions in AAV9 or A587D/Q588G in AAV9 or corresponding substitutions in other AAV serotypes.
  • the rAAV capsid can have a R697W substitution.
  • the capsids having these amino acid substitutions and insertions may further have substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) of the AAV9 capsid, or of the NNN (asparagines) at 498 to 500 with AAA (alanines) of the AAV8 capsid corresponding substitutions in other AAV type capsids.
  • AAV serotypes that may be used for the amino acid substitutions and that may be the reference capsid include AAV8, AAV rh.34, AAV4, AAV5, AAV hu.26, AAV rh.31, AAV hu.13, AAV hu.26, AAV hu.56, AAV hu.53, AAV7, rh64Rl, rh46 or rh73.
  • the capsid is rh34, either unmodified or serving as the parental capsid to be modified as detailed herein.
  • the capsid has an insertion of a TFR3 peptide (RTIGPSV, SEQ ID NO: 12), including in variable region IV (see FIG. 3) of a parental capsid, which may be AAV9.
  • the peptide may be inserted between S454 and G455 of the AAV9 capsid sequence or the corresponding position of another capsid (See FIG. 3 for exemplary alignment).
  • Capsids include AAV9S454-TFR3 (SEQ ID NO: 42; see Table 17).
  • Such capsids may have improved transduction efficiency and transcriptional activity (such as 2 fold, 4 fold, 6 fold, 8 fold or 10 fold) in CNS regions of interest relative to parental capsids such as AAV9, including when administered intraparenchymally or by ICV administration.
  • capsids having one or more amino acid substitutions that promote transduction and/or tissue tropism of the rAAV having the modified capsid are provided.
  • capsids having a single mutation at amino acid 269 of the AAV8 capsid replacing alanine with serine (A269S) are provided.
  • capsids having multiple substitutions at amino acids 263, 269, and 273 of the AAV9 capsid resulting in the following substitutions: S263G, S269T, and A273T (herein referred to as AAV9.BBB) or substitutions corresponding to these positions in other AAV types.
  • AAV9.BBB AAV9.BBB
  • Exposure to the AAV capsid can generate an immune response of neutralizing antibodies.
  • One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization.
  • a monoclonal antibody, specific for intact AAV9 capsids, with high neutralizing titer has recently been described (Giles et al, 2018, Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next- Generation Gene Delivery Vectors).
  • the epitope was mapped to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588-QAQAQT-592 of AAV9 (SEQ ID NO:8).
  • Capsid mutagenesis demonstrated that single amino acid substitution within this epitope markedly reduced binding and neutralization.
  • AAVrhlO capsid was modified by substituting three asparagines at amino acid positions 498, 499, and 500 to alanines (AAVrhIO.LD) (Tables 5a-5c).
  • capsids having three asparagines at amino acid positions 496, 497, and 498 of the AAV9 capsid replaced with alanines and also tryptophan at amino acid 503 of the AAV9 capsid with arginine or capsids with substitutions corresponding to these positions in other AAV types.
  • the capsid is an AAV8.BB.LD capsid (A269S,498-NNN/AAA- 500 substitutions in the amino acid sequence of AAV8, SEQ ID NO: 73), an AAV9.BBB.LD capsid (S263G/S269T/A273T, 496-NNN/AAA-498 substitutions in the amino acid sequence of AAV9, SEQ ID NO: 74), an AAV9.496-NNN/AAA-498 capsid (SEQ ID NO: 31), an AAV9.496- NNN/AAA-498.W503R capsid (SEQ ID NO: 32), an AAV9.W503R capsid (SEQ ID NO: 33), or an AAV9.Q474A capsid (SEQ ID NO: 34).
  • AAV8.BB.LD capsid A269S,498-NNN/AAA- 500 substitutions in the amino acid sequence of AAV8, SEQ ID NO: 73
  • the capsid can be an AAV9.N272A.496-NNN-498 capsid (SEQ ID NO:49) or an AAV9.G266A.496-NNN-498 capsid (SEQ ID NO: 50).
  • the rAAVs described herein increase tissue-specific (such as, but not limited to, CNS or skeletal and/or cardiac muscle) cell transduction in a subject (a human, nonhuman-primate, or mouse subject) or in cell culture, compared to the rAAV not comprising the amino acid substitution.
  • the increase in tissue specific cell transduction is at least 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold more than that without the modification.
  • the increase in transduction may be assessed using methods described in the Examples herein and known in the art.
  • the rAAVs described herein increase the incorporation of rAAV genomes into a cell or tissue type in a subject (a human, non-human primate or mouse subject) or in cell culture to the rAAV not comprising the peptide insertion.
  • the increase in genome integration is at least 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold more than an AAV having a capsid without the modification (i.e., the parental capsid).
  • rAAVs having capsid proteins with one or more (generally one or two) peptide insertions wherein the peptide insertion increase effective gene delivery to the CNS or to cardiac or skeletal muscle and to detarget the liver and/or dorsal root ganglion to reduce toxicity relative to the parental capsid protein.
  • the peptides include RTIGPSV (SEQ ID NO: 12), TLAVPFK (SEQ ID NO:20), TLAAPFK (SEQ ID NO: 1), or TILSRSTQTG (SEQ ID NO: 15) (or an at least 4, 5, 6, 7 amino acid portion thereof).
  • the peptides may be inserted into the AAV9 capsid, for example after the positions 138; 262-273; 452-461; 585-593 of AAV9 cap, particularly after position 138, 454 or 588 of AAV9 or a corresponding position in another AAV as detailed herein.
  • the capsid has the peptide TLAVPFK (SEQ ID NO:20) is inserted between G588 and A589 of AAV9, and, in particular, the capsid protein also has amino acid substitutions A587D/Q588G (PHP.eB) and further has the peptide TILSRSTQTG (SEQ ID NO: 15) inserted after position 138 of AAV9 (collectively, AAVPHPeB.VP2Herp; see Table 17), or in the corresponding positions of another AAV.
  • Kidney 1 peptide LPVAS SEQ ID NO: 6
  • Such an engineered capsid may exhibit preferential targeting for heart and skeletal muscle, and reduced targeting (as compared to an AAV having the unengineered capsid) for liver and/or dorsal root ganglion cells and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a muscle disease (such as, but not limited to a muscular dystrophy).
  • the peptide insertion comprises at least 4, 5, 6, 7, 8, 9, or all 10 consecutive amino acids of sequence TTLSRSTQTG (SEQ ID NO: 15), preferably which contains the TQT or STQT (SEQ ID NOV) motif. In some embodiments, the peptide insertion consists of at least 4, 5, 6, 7, 8, 9, or all 10 consecutive amino acids of sequence TILSRSTQTG (SEQ ID NO:15), preferably which contains the TQT or STQT (SEQ ID NOV) motif.
  • the peptide insertion may be a sequence of consecutive amino acids from a domain that targets kidney tissue, or a conformation analog designed to mimic the three-dimensional structure of said domain.
  • the kidney-homing domain comprises the sequence CLPVASC (SEQ ID NOV) (see, e.g., US 5,622,699).
  • the peptide insertion from said kidney-homing domain comprises at least 4, 5, 6, or all 7 amino acids from sequence CLPVASC (SEQ ID NOV).
  • the peptide insertion comprises or consists of the sequence CLPVASC (SEQ ID NOV).
  • a peptide having the sequence LPVAS also can be a kidney-homing peptide.
  • Methods for determining the necessity of a cysteine residue or of amino acid residues N-terminal or C-terminal to a cysteine residue for organ homing activity of a peptide are routine and well known in the art.
  • the peptide insertion comprises at least 4 or all 5 amino acids from sequence LPVAS (SEQ ID NOV).
  • the peptide insertion comprises or consists of the sequence LPVAS (SEQ ID NOV).
  • rAAVs having a capsid that has the peptide TLAAPFK (SEQ ID NO: Q is inserted between Q588 and A589 of AAV9 (AAV9.hDyn; see Table 17), or the corresponding position of another AAV (see, e.g., FIG. 3).
  • Such an engineered capsid may exhibit preferential targeting for CNS tissue, and reduced targeting (as compared to an AAV having the unengineered capsid) for liver and/or dorsal root ganglion cells and may particularly useful for delivery of a transgene encoding a therapeutic protein for treatment of a CNS disease.
  • rAAVs having a capsid with the peptide RTIGPSV (TFR3 peptide; SEQ ID NO: 12).
  • RTIGPSV peptide may be inserted into the VR- IV loop of AAV9 or any other appropriate capsid. For example, it is inserted between S454 and G455 of AAV9 (SEQ ID NO: 74; FIG. 3) and may be AAV9S454-TFR3 (SEQ ID NO: 42).
  • Such capsids have a tropism for dopaminergic neurons (see Example 19), including when administered directly to the CNS, for example, by intraparenchymal administration to the striatum (or ICV administration), resulting in delivery, including by retrograde and/or anterograde transport, to other areas of the brain.
  • Administration to the striatum of such capsids having the TFR3 peptide insert, such as AAV9S454-TFR3 provides delivery to dopaminergic neurons and regions of the brain including the substantia nigra, caudate, putamen, globus pallidus (external), intralaminar thalamus and frontal cortex.
  • the AAV9S454-TFR3 has enhanced transcriptional activity and superior transduction efficiency (including 2 fold, 4 fold, 6 fold, 8 fold or 10 fold) in these CNS regions relative to the parental AAV9 capsid.
  • capsids with peptide insertions at positions amenable to peptide insertions within and near the AAV9 capsid VR-IV loop (see FIG. 2) and corresponding regions on the VR- IV loop of capsids of other AAV types.
  • the rAAV capsid protein comprises a peptide insertion immediately after (z.e., connected by a peptide bond C-terminal to) an amino acid residue corresponding to one of amino acids 451 to 461 of AAV9 capsid protein (amino acid sequence SEQ ID NO: 74 and see FIG.
  • capsid protein amino acid sequence of other AAV serotypes with amino acid sequence of the AAV9 capsid and Table 17 for other capsid sequences
  • said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle.
  • the peptide insertion should not delete any residues of the AAV capsid protein.
  • the peptide insertion occurs in a variable (poorly conserved) region of the capsid protein, compared with other serotypes, and in a surface exposed loop.
  • a peptide insertion described as inserted “at” a given site refers to insertion immediately after, that is having a peptide bond to the carboxy group of, the residue normally found at that site in the wild type virus.
  • insertion at Q588 in AAV9 means that the peptide insertion appears between Q588 and the consecutive amino acid (A589) in the AAV9 wildtype capsid protein sequence (SEQ ID NO: 74).
  • the capsid protein is an AAV9 capsid protein and the insertion occurs immediately after at least one of the amino acid residues 451 to 461.
  • the peptide insertion occurs immediately after amino acid 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid (amino acid sequence SEQ ID NO: 74).
  • the peptide is inserted between residues S454 and G455 of AAV9 capsid protein or between the residues corresponding to S454 and G455 of an AAV capsid protein other than an AAV9 capsid protein (amino acid sequence SEQ ID NO: 74).
  • engineered capsid proteins comprising targeting peptides heterologous to the capsid protein that are inserted into the AAV capsid protein such that, when incorporated into the AAV vector the heterologous peptide is surface exposed.
  • the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh74 (AAVrh74, versions 1 and 2), serotype rh34 (AAVrh34), serotype hu26 (AAVhu26), serotype hu32 (AAVhu32), serotype rh31 (AAVr
  • the peptide insertion occurs immediately after one of the amino acid residues within: 450-459 of AAV1 capsid (SEQ ID NO:63); 449-458 of AAV2 capsid (SEQ ID NO:64); 449-459 of AAV3 capsid (SEQ ID NO:65); 443-453 of AAV4 capsid (SEQ ID NO:68); 442-445 of AAV5 capsid (SEQ ID NO:70); 450-459 of AAV6 capsid (SEQ ID NO:71); 451-461 of AAV7 capsid (SEQ ID NO:72); 451-461 of AAV8 capsid (SEQ ID NO: 73); 451-461 of AAV9 capsid (SEQ ID NO: 74); 452-461 of AAV9e capsid (SEQ ID NO:75); 452-461 of AAVrhlO capsid (SEQ ID NO:76); 452-461 of AAVrh20 capsid (SEQ ID NO:77); 452-4
  • the rAAV capsid protein comprises a peptide insertion immediately after (i.e., C-terminal to) amino acid 588 of AAV9 capsid protein (having the amino acid sequence of SEQ ID NO: 74 and see FIG. 3), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle.
  • the rAAV capsid protein has a peptide insertion that is not immediately after amino acid 588 of AAV9 or corresponding to amino acid 588 of AAV9.
  • the peptide is inserted after 138; 262-272; 450-459; or 585-593 of AAV1 capsid (SEQ ID NO:63); 138; 262-272; 449-458; or 584-592 of AAV2 capsid (SEQ ID NO:64); 138; 262-272; 449-459; or 585-593 of AAV3 capsid (SEQ ID NO:65); 137; 256-262; 443-453; or 583-591 of AAV4 capsid (SEQ ID NO:68); 137; 252-262; 442-445; or 574-582 of AAV5 capsid (SEQ ID NO:70); 138; 262-272; 450-459; 585-593 of AAV6 capsid (SEQ ID NO:71); 138; 263-273; 451-461; 586-594 of AAV7 capsid (SEQ ID NO:72); 138; 263-274; 452- 461; 587-595 of A
  • the peptide insertion is sequence of contiguous amino acids from a heterologous protein or domain thereof.
  • the peptide to be inserted typically is long enough to retain a particular biological function, characteristic, or feature of the protein or domain from which it is derived.
  • the peptide to be inserted typically is short enough to allow the capsid protein to form a coat, similarly or substantially similarly to the native capsid protein without the insertion.
  • the peptide insertion is from about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length.
  • the peptide sequences for insertion are at least 4 amino acids in length and may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide sequences are 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is no more than 7 amino acids, 10 amino acids or 12 amino acids in length.
  • a “peptide insertion from a heterologous protein” in an AAV capsid protein refers to an amino acid sequence that has been introduced into the capsid protein and that is not native to any AAV serotype capsid.
  • Non-limiting examples include a peptide of a human protein in an AAV capsid protein.
  • the rAAVs described herein increase tissue-specific (such as, but not limited to, CNS or skeletal and/or cardiac muscle) cell transduction in a subject (a human, nonhuman-primate, or mouse subject) or in cell culture, compared to the rAAV not comprising the amino acid substitution.
  • the increase in tissue specific cell transduction is at least 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold more than that without the peptide insertion.
  • the rAAVs described herein increase the incorporation of rAAV genomes into a cell or tissue type, particularly CNS or heart and/or skeletal muscle in a subject (a human, non-human primate or mouse subject) or in cell culture to the rAAV not comprising the peptide insertion.
  • the increase in genome integration is at least 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold more than an AAV having a capsid without the peptide insertion.
  • a heterologous peptide insertion library refers to a collection of rAAV vectors that carry the same peptide insertion at different insertion sites in the virus capsid, e.g., at different positions within a given variable region of the capsid or different variant peptides or even one or more amino acid substitutions.
  • the capsid proteins used comprise AAV genomes that contain modified rep and cap sequences to prevent the replication of the virus under conditions in which it could normally replicate (co-infection of a mammalian cell along with a helper virus such as adenovirus).
  • the members of the peptide insertion libraries may then be assayed for functional display of the peptide on the rAAV surface, tissue targeting and/or gene transduction.
  • AAV1 138; 262-272; 450-459; 595-593; and in a particular embodiment, between 453- 454 (SEQ ID NO:63).
  • AAV2 138; 262-272; 449-458; 584-592; and in particular embodiment, between 452-453 (SEQ ID NO:64).
  • AAV3 138; 262-272; 449-459; 585-593; and in particular embodiment, between 452-453 (SEQ ID NO:65).
  • AAV4 137; 256-262; 443-453; 583-591; and in particular embodiment, between 446-447 (SEQ TD NO:68)
  • AAV5 137; 252-262; 442-445; 574-582; and in particular embodiment, between 445-446 (SEQ ID NO:70).
  • AAV6 138; 262-272; 450-459; 585-593; and in particular embodiment, between 452-453 (SEQ ID NO:71).
  • AAV7 138; 263-273; 451-461; 586-594; and in particular embodiment, between 453-454 (SEQ ID NO:72).
  • AAV8 138; 263-274; 451-461, 587-595; and in particular embodiment, between 453-454 (SEQ ID NO: 73).
  • AAV9 138; 262-273; 452-461; 585-593; and in particular embodiment, between 454-455 (SEQ ID NO: 74).
  • AAV9e 138; 262-273; 452-461; 585-593; and in particular embodiment, between 454-455 (SEQ ID NO:75).
  • AAVrhlO 138; 263-274; 452-461; 587-595; and in particular embodiment, between 454- 455 (SEQ ID NO:76).
  • AAVrh20 138; 263-274; 452-461; 587-595; and in particular embodiment, between 454- 455 (SEQ ID NO:70).
  • AAVrh39 138; 263-274; 452-461; 587-595; and in particular embodiment, between 454- 455 (SEQ ID NO:77).
  • AAVrh74 138; 263-274; 452-461; 587-595; and in particular embodiment, between 454- 455 (SEQ ID NO:80 or SEQ ID NO:81).
  • the peptide insertion occurs between amino acid residues 588-589 of the AAV9 capsid, or between corresponding residues of another AAV type capsid as determined by an amino acid sequence alignment (for example, as in FIG. 3).
  • the peptide insertion occurs immediately after amino acid residue 1451 to L461, S268 and Q588 of the AAV9 capsid sequence, or immediately after corresponding residues of another AAV capsid sequence (FIG. 3).
  • one or more peptide insertions can be used in a single system.
  • the capsid is chosen and/or further modified to reduce recognition of the AAV particles by the subject’s immune system, such as avoiding pre-existing antibodies in the subject.
  • the capsid is chosen and/or further modified to enhance desired tropism/targeting.
  • AAV vectors comprising the engineered capsids.
  • the AAV vectors are non-replicating and do not include the nucleotide sequences encoding the rep or cap proteins (these are supplied by the packaging cells in the manufacture of the rAAV vectors).
  • AAV-based vectors comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, A AVI 2, A AVI 3, A AVI 4, A AVI 5, A AVI 6, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC
  • AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV 7m8, AAV.PHP.B, AAV PHP eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HS
  • rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
  • AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV PHP.B, AAV PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, A
  • the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety).
  • the recombinant AAV for use in compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US 9,193,956; US 9,458,517; US 9,587,282; US 2016/0376323, and WO 2018/075798, each of which is incorporated herein by reference in its entirety).
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B.
  • the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., ⁇ ssa etal., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors).
  • the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.
  • rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.R1174 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in US Pat Nos.
  • rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g, SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g, SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs:
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No.
  • WO 2003/052051 see, e.g., SEQ ID NO: 2 of '051 publication
  • WO 2005/033321 see, e.g., SEQ ID NOs: 123 and 88 of '321 publication
  • WO 03/042397 see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication
  • WO 2006/068888 see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication
  • WO 2006/110689 see, e.g., SEQ ID NOs: 5- 38 of '689 publication
  • W02009/104964 see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication
  • W0 2010/127097 see, e.g., SEQ ID NOs: 5-38 of '097 publication
  • WO 2015/191508 see, e.g., SEQ ID NOs: 80-294 of
  • rAAV particles comprise a pseudotyped AAV capsid.
  • the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin etal., Methods 28: 158-167 (2002); and Auricchio etal., Hum. Molec. Genet. 10:3075- 3081, (2001).
  • a single-stranded AAV may be used.
  • a self-complementary vector e.g., scAAV
  • scAAV single-stranded AAV
  • an rAAV particle is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid proteins described herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein.
  • the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein, and retains (or substantially retains) biological function of the capsid protein.
  • the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the one of the capsid proteins described herein, for example, those with sequences in Table 17 or otherwise described herein (see also FIG. 3), while retaining (or substantially retaining) biological function of the capsid protein.
  • the capsid protein, coat, and rAAV particles may be produced by techniques known in the art.
  • the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector.
  • the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene.
  • the cap and rep genes are provided by a packaging cell and not present in the viral genome.
  • the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene.
  • this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat.
  • Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Non-limiting examples include 293 cells or derivatives thereof, HELA cells, or insect cells.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
  • Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below.
  • the rAAV vector also includes regulatory control elements known to one skilled in the art to influence the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulatory control elements and may be tissuespecific, that is, active (or substantially more active or significantly more active) only in the target cell/tissue.
  • the AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues.
  • the promoter may be a constitutive promoter, for example, the CB7 promoter.
  • Additional promoters include: hSyn promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, opsin promoter, the TBG (Thyroxine-binding Globulin) promoter, the APOA2 promoter, SERPINA1 (hAAT) promoter, or MIR122 promoter.
  • an inducible promoter is used, e.g., hypoxia-inducible or rapamycin-inducible promoter.
  • AAV vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the a capsid protein described herein (see Table 17, e.g.), while retaining the biological function of the engineered capsid.
  • the encoded engineered capsid has the sequence of an AAV8.BBB.LD capsid (SEQ ID NO: 27), an AAV9.BBB.LD capsid (SEQ ID NO: 29), an AAV9.496-NNN/AAA-498 capsid (SEQ ID NO: 31), AAV9.496-NNN/AAA-498.W503R capsid (SEQ ID NO: 32), AAV9.W503R capsid (SEQ ID NO: 33), AAV9.Q474A capsid (SEQ ID NO: 34), AAV9.N272A.496-NNN/AAA-498 capsid (SEQ ID NO: 49) or AAV9.N266A.496- NNN/AAA-498 capsid (SEQ ID NO: 50).
  • engineered AAV vectors other than AAV9 vectors such as engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVhu.37, AAVrh39, AAVrh74, AAVrh34, AAVhu26, AAVhu32, AAVrh31, AAVhu56, AAVhu53, AAVrh.46, AAVrh.64.Rl, AAV.rh.73 vectors, including with the amino acid substitutions and/or peptide insert as described herein and 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, or 30 amino acid substitutions relative to the wild type or unengineered sequence for that AAV type and that retains its biological function.
  • the recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene generally is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb.
  • the rAAV vector for delivering the transgene to target tissues, cells, or organs has a tropism for that particular target tissue, cell, or organ. Tissue-specific promoters may also be used.
  • the promoter is an hSyn promoter (SEQ ID NO:95) or in embodiments, the promoter is a CAMKII promoter (SEQ ID NO: 96).
  • the construct further can include expression control elements that enhance expression of the transgene driven by the vector (e.g., introns such as the chicken ⁇ -actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), ⁇ -globin splice donor/immunoglobulin heavy chain spice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron, and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals such as the rabbit ⁇ -globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal.
  • introns such as the chicken ⁇ -actin intron, minute virus
  • nucleic acids sequences disclosed herein may be codon- optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) a promoter and, optionally, enhancer elements to promote expression of the transgene in CNS and/or muscle cells, b) optionally an intron sequence, such as a chicken ⁇ -actin intron, and c) a polyadenylation sequence, such as an SV40 polyA or rabbit ⁇ -globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest.
  • control elements which include a) a promoter and, optionally, enhancer elements to promote expression of the transgene in CNS and/or muscle cells, b) optionally an intron sequence, such as a chicken ⁇ -actin intron, and c) a polyadenylation sequence, such as an SV40 polyA or rabbit ⁇ -globin poly A signal; and (3) transgene providing (e.g., coding
  • constructs and rAAV vectors comprising the constructs in which a transgene encoding a therapeutic protein, including a therapeutic for a neurological disease or disorder, is operably linked to an hSyn promoter (SEQ ID NO: 102) or, in embodiments, a CAMKII promoter (SEQ ID NO: 96).
  • the constructs do not comprise a transgene encoding a tau-specific antibody operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103).
  • the constructs do not comprise a transgene encoding any of the tau-specific antibodies disclosed in WO 2022/147087 operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103). In embodiments, the constructs do not comprise a transgene encoding 4P3, 31B6, or 8H1 operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103).
  • the rAAV vectors do not comprise a construct comprising a transgene encoding a tau-specific antibody operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKII promoter (SEQ ID NO: 103). In embodiments, the rAAV vectors do not comprise a construct comprising a transgene encoding any of the tau-specific antibodies disclosed in WO 2022/147087 operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMK11 promoter (SEQ ID NO: 103).
  • the rAAV vectors do not comprise a construct comprising a transgene encoding 4P3, 31B6, or 8H1 operably linked to an hSyn promoter (SEQ ID NO: 102) or a CAMKTI promoter (SEQ ID NO: 103).
  • rAAV vectors including rAAV9 vectors (and modified version thereof, including those described herein with enhanced CNS targeting properties), including by parenchymal administration, results in durable expression of the transgene in neuronal tissue, for example, in dopaminergic neurons, for 6 months (or more than 6 months, including 8 months, 10 months, 12 months or more).
  • the expression in embodiments, is neuronal specific, including an absence of detectable expression in microglia or astrocytes.
  • Neurological disorders that may be treated include, in embodiments, in human subjects, Parkinson’s Disease, tardive dyskinesia, levodopa-induced dyskinesia (LD), schizophrenia and other neurological diseases and disorders.
  • the viral vectors provided herein may be manufactured using host cells, e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters.
  • host cells e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters.
  • Nonlimiting examples include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and genetic components for producing viruses in the host cells, such as the replication and capsid genes (e.g., the rep and cap genes of AAV).
  • viruses e.g., the rep and cap genes of AAV.
  • the replication and capsid genes e.g., the rep and cap genes of AAV.
  • Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis.
  • Virions may be recovered, for example, by CsCh sedimentation.
  • baculovirus expression systems in insect cells may be used to produce AAV vectors.
  • Aponte-Ubillus etal., 2018, Appl. Microbiol. Biotechnol. 102: 1045-1054 which is incorporated by reference herein in its entirety for manufacturing techniques.
  • In vitro assays e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector.
  • the PER.C6® Cell Line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, e.g., the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression.
  • cell lines derived from liver or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma HT-1080, HKB-11, and CAP cells.
  • characteristics of the expressed product i.e., transgene product
  • characteristics of the expressed product can be determined, including determination of the glycosylation and tyrosine sulfation patterns, using assays known in the art.
  • Another aspect relates to therapies which involve administering a transgene, operably linked an hSyn promoter (SEQ ID NO: 102) via a rAAV vector according to the invention to a subject in need thereof, for delaying, preventing, treating, and/or managing a disease or disorder, and/or ameliorating one or more symptoms associated therewith.
  • a subject in need thereof includes a subject suffering from the disease or disorder, or a subject pre-disposed thereto, e.g., a subject at risk of developing or having a recurrence of the disease or disorder.
  • a rAAV carrying a particular transgene, operably linked an hSyn promoter will find use with respect to a given disease or disorder in a subject where the subject’s native gene, corresponding to the transgene, is defective in providing the correct gene product, or correct amounts of the gene product.
  • the transgene then can provide a copy of a gene that is defective in the subject.
  • Delivery in a recombinant AAV of the transgene operably linked to and under the regulatory control of the hSyn promoter to the CNS results in persistent expression of the transgene of 6 months, 9 months, 12 months, 18 months or at least 6 months, at least 9 months, at least 12 months or at least 18 months, including in neurons within the CNS.
  • the transgene comprises cDNA that restores protein function to a subject having a genetic mutation(s) in the corresponding native gene.
  • the cDNA comprises associated RNA for performing genomic engineering, such as genome editing via homologous recombination.
  • the transgene encodes a therapeutic RNA, such as a shRNA, artificial miRNA, or element that influences splicing.
  • Tables 1A-1B below provides a list of transgenes that may be used in any of the rAAV vectors described herein, in particular, in the novel insertion sites described herein, to treat or prevent the disease with which the transgene is associated, also listed in Tables 1A-1B.
  • the AAV vector may be engineered as described herein to target the appropriate tissue for delivery of the transgene to effect the therapeutic or prophylactic use.
  • the appropriate AAV serotype may be chosen to engineer to optimize the tissue tropism and transduction of the vector.
  • a rAAV vector comprising a transgene operably linked an hSyn promoter (SEQ ID NO: 102) encoding glial derived growth factor (GDGF) finds use treating/preventing/managing Parkinson’s disease.
  • the rAAV vector is administered systemically but may also be administered directly to the CNS, for example to the striatum by intraparenchymal administration or otherwise.
  • the rAAV vector may also be provided by intravenous, intrathecal, intra-nasal, and/or intra-peritoneal administration.
  • the rAAV for delivery of the therapeutic for Parkinson’s disease is a capsid having an insert of the TFR3 peptide (RTIGPSV, SEQ ID NO: 12), including in the VR-IV loop of the capsid (see FIGs. 2 and 3) and, in embodiments, is inserted between S454 and G455 of the AAV9 capsid, or corresponding position in another appropriate capsid protein (see Fig. 3 for alignment), and may be AAV9S454-TFR3 (SEQ ID NO:42).
  • the AAV9S454-TFR3 capsid has enhanced abundance in the putamen ipsilateral and caudate relative to the other capsids in the library when injected in the striatum/putamen of an NHP and is further localized to regions of the brain such as the substantia nigra, intralaminar thalamus (rostral and caudal), frontal cortex, putamen, caudate and globus pallidus.
  • the AAV9S454-TFR3 capsid has superior transduction and enhanced transcriptional activity (3 to 4 fold increase) putamen, caudate, substantia nigra, intralaminar thalamus and frontal cortex after intraparenchymal administration in NHPs, relative to the parental AAV9 capsid. Similar results are seen in mice upon ICV administration, with favorable reduction in biodistribution in liver, heart and DRG in both mice and NHP following ICV administration. Based upon the pattern of localization, the capsids may be transported by retrograde and/or anterograde transport from the site of injection to the substantia nigra.
  • Such capsids may be useful for delivering transgenes encoding a therapeutic protein for diseases associated with dopaminergic neurons, such as Parkinson’s disease and other movement disorders, such as tardive dyskinesia, levodopa-induced dyskinesia (LD), or, alternatively, schizophrenia.
  • Parkinson Parkinson
  • Parkinson's disease and other movement disorders, such as tardive dyskinesia, levodopa-induced dyskinesia (LD), or, alternatively, schizophrenia.
  • LD levodopa-induced dyskinesia
  • a method of treating such CNS disorders associated with dopaminergic neurons by administration, including intraparenchymal administration to the CNS, including the striatum or putamen, of an rAAV having a capsid comprising a TFR3 peptide, including inserted in the VR-IV loop, including between positions S454 and G455 of AAV9 (or other capsid protein, for example based upon the alignment in Fig.
  • rAAV comprises an expression cassette with a transgene encoding a protein therapeutic for the CNS disorder associated with dopaminergic neurons operably linked to regulatory elements, such as the hSyn promoter (SEQ ID NO: 102), for expression in the CNS and, including, in dopaminergic neurons.
  • rAAV vectors comprising a construct in which a transgene encoding a therapeutic protein, including a therapeutic for a neurological disease or disorder, is operably linked to an hSyn promoter (SEQ ID NO: 102) or, in embodiments, a CAMKII promoter (SEQ ID NO: 96).
  • rAAV vectors including rAAV9 vectors (and modified version thereof, including those described herein with enhanced CNS targeting properties), including by parenchymal administration, results in durable expression of the transgene in neuronal tissue, for example, in dopaminergic neurons, for 6 months (or more than 6 months, including 8 months, 10 months, 12 months or more).
  • the expression from the hSyn promoter (SEQ ID NO: 102) in the CNS exhibits slower initial kinetics of expression and then an increase in expression, for example after 1, 2 or 3 months, resulting in a durable level of expression up to 6 months in the CNS.
  • gene therapy vectors in which the transgene encoding the therapeutic protein is operably linked to a hSyn promoter may be used to deliver and express transgenes in the CNS at initially a slower rate of expression for 1, 2 or 3 months and then at a higher durable rate of expression, for at least up to 6 months after administration.
  • expression from the CAG promoter in the CNS is initially rapid and robust, including over the first 5, 10 or 20 days after administration, and then exhibiting a significant reduction in expression.
  • gene therapy vectors in which the transgene encoding the therapeutic protein is operably linked to a CAG promoter may be used to deliver and express a transgene in the CNS at a high rate of expression within 5, 10, or 20 days after administration and then exhibiting a reduction in expression of the transgene.
  • the expression in embodiments, is neuronal specific, including an absence of detectable expression in microglia or astrocytes.
  • Neurological disorders that may be treated include, in embodiments, in human subjects, Parkinson’s Disease, tardive dyskinesia, levodopa-induced dyskinesia (LD), schizophrenia and other neurological diseases and disorders.
  • Example 23 administration of the gene therapy vector in which the hSyn promoter is operably linked to the transgene coding sequence resulted in an increase in the average number of AAV genomes per circular DNA unit increasing over the six months after administration in the striatum, suggesting that more vector genomes are being incorporated into stable episomes in either monomeric or concatermeric forms in the CNS tissue.
  • vectors and methods of delivering vectors to a subject including a human subject, wherein the total number of circular DNAs containing an AAV vector genome per cell in CNS tissue (including the striatum) increases over the first one, two, three, four, five or six months after administration of the rAAV vector, for example, by 1, 2, 3, 4, 5, 6 or more.
  • vectors and methods of delivering vectors to a subject including a human subject, wherein the total number of vector genomes in circular DNA per cell in CNS tissue (including the striatum) increases over the first one, two, three, four, five or six months after administration of the rAAV vector by 5, 10, 15 or 20 or more.
  • vectors and methods of delivering vectors to a subject including a human subject, wherein the average number of AAV genomes per circular DNA unit in CNS tissue, including striatum, increases over the first one, two, three, four, five or six months after administration, for example, by at least 1, 2 or 3 vector genomes.
  • the number of circular DNA units containing AAV genomes, i.e., the gene therapy vector, per cell increases over the first one, two, three, four, five or six months after administration.
  • the total number of vector genomes increases over the first six months after administration of the rAAV vector.
  • the average number of vector genomes per piece of circular DNA increases over the first six months after administration of the rAAV vector.
  • the engineered rAAV capsids and rAAV genomes (including genomes that link a tansgene to, for example, the hSyn promoter) of the present invention find use in delivery to target tissues, or target cell types, including cell matrix associated with the target cell types, associated with the disorder or disease to be treated/prevented.
  • a disease or disorder associated with a particular tissue or cell type is one that largely affects the particular tissue or cell type, in comparison to other tissue of cell types of the body, or one where the effects or symptoms of the disorder appear in the particular tissue or cell type.
  • Methods of delivering a transgene to a target tissue of a subject in need thereof involve administering to the subject tan rAAV where the peptide insertion is a homing peptide.
  • a rAAV vector comprising a peptide insertion that directs the rAAV to neural tissue can be used, in particular, where the peptide insertion facilitates the rAAV in crossing the blood brain barrier to the CNS.
  • an rAAV vector can be used that comprises a peptide insertion from a neural tissue-homing domain, such as any described herein and having an expression cassette in which the transgene is operably linked to the hSyn promoter.
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • Battens disease Batten’s Juvenile NCL form
  • Canavan disease chronic pain
  • Friedreich’s ataxia glioblastoma multiforme
  • Huntington's disease Late Infantile neuronal ceroid lipofuscinosis (LINCL)
  • LINCL Late Infantile neuronal ceroid lipofuscinosis
  • Leber’s congenital amaurosis multiple sclerosis
  • Parkinson's disease Pompe disease
  • Rett syndrome spinal cord injury
  • SMA spinal muscular atrophy
  • stroke and traumatic brain injury.
  • the vector further can contain a transgene for therapeutic/prophylactic benefit to a subject suffering from, or at risk of developing, the disease or disorder (see Tables 1 A-1B).
  • the rAAV vectors of the invention also can facilitate delivery, in particular, targeted delivery, of oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, antibodies to target cells or tissues.
  • the rAAV vectors also can facilitate delivery, in particular, targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.
  • the agents may be provided as pharmaceutically acceptable compositions as known in the art and/or as described herein. Also, the rAAV molecule of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.
  • the dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective.
  • the dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease, the route of administration, as well as age, body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56 th ed., 2002).
  • Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic agents, and whether such agents are administered separately or as an admixture.
  • the amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (/. ⁇ ?., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • Prophylactic and/or therapeutic agents can be tested in suitable animal model systems prior to use in humans.
  • animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. Such model systems are widely used and well known to the skilled artisan.
  • animal model systems for a CNS condition are used that are based on rats, mice, or other small mammal other than a primate.
  • a dosage for a human subject in need of treatment for a neurological disease or disorder for intraparenchymal administration of an AAV9 (or modified AAV9) gene therapy product by comparing the therapeutically effective dosage in a mouse, rat or NHP model for the disease or disorder that achieves durable expression of the transgene encoding the therapeutic protein for at least 6 months.
  • the dosage may be determined based upon the relative size of the brain of the human subject relative to the size of the brain of the animal model subject (for example, a log difference between rat and human or 2 log different between mice and human).
  • prophylactic and/or therapeutic agents of the invention Once the prophylactic and/or therapeutic agents of the invention have been tested in an animal model, they can be tested in clinical trials to establish their efficacy. Establishing clinical trials will be done in accordance with common methodologies known to one skilled in the art, and the optimal dosages and routes of administration as well as toxicity profiles of agents of the invention can be established. For example, a clinical trial can be designed to test a rAAV molecule of the invention for efficacy and toxicity in human patients.
  • Toxicity and efficacy of the prophylactic and/or therapeutic agents of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • a rAAV molecule of the invention generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dosage of an rAAV vector for patients is generally from about 0.1 ml to about 100 ml of solution containing concentrations of from about IxlO 9 to about IxlO 16 genomes rAAV vector, or about IxlO 10 to about IxlO 15 , about IxlO 12 to about IxlO 16 , or about IxlO 14 to about IxlO 16 AAV genomes. Levels of expression of the transgene can be monitored to determine/adjust dosage amounts, frequency, scheduling, and the like.
  • Treatment of a subject with a therapeutically or prophylactically effective amount of the agents of the invention can include a single treatment or can include a series of treatments.
  • pharmaceutical compositions comprising an agent of the invention may be administered once, or may be administered in a series of 2, 3 or 4 or more times, for example, weekly, monthly or every two months, 3 months, 6 months or one year until the series of doses has been administered
  • the rAAV molecules of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.
  • Each prophylactic or therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route.
  • the different prophylactic and/or therapeutic agents are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart.
  • two or more agents are administered within the same patient visit.
  • Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc ⁇ .
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection
  • epidural e.g., epidural
  • epithelial or mucocutaneous or mucosal linings e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc ⁇ .
  • the vector is administered via lumbar puncture or via cisterna magna.
  • the agents of the invention are administered intravenously and may be administered together with other biologically active agents.
  • agents of the invention may be delivered in a sustained release formulation, e.g., where the formulations provide extended release and thus extended half- life of the administered agent.
  • Controlled release systems suitable for use include, without limitation, diffusion-controlled, solvent-controlled, and chemically-controlled systems.
  • Diffusion controlled systems include, for example reservoir devices, in which the molecules of the invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier.
  • Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers.
  • Monolithic (matrix) device are a second type of diffusion controlled system, wherein the dual antigen-binding molecules are dispersed or dissolved in an rate-controlling matrix (e.g., a polymer matrix).
  • an rate-controlling matrix e.g., a polymer matrix
  • Agents of the invention can be homogeneously dispersed throughout a rate-controlling matrix and the rate of release is controlled by diffusion through the matrix.
  • Polymers suitable for use in the monolithic matrix device include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers, as well as polymer derivatives.
  • any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents described herein. See, e.g. U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al., “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology, 39: 179 189, 1996; Song et al., “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology, 50:372 397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro.
  • a pump may be used in a controlled release system (see Langer, supra, Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J.
  • polymeric materials can be used to achieve controlled release of agents comprising dual antigen-binding molecule, or antigen-binding fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science, 228:190, 1985; During et al., Ann.
  • a controlled release system can be placed in proximity of the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)).
  • Other controlled release systems are discussed in the review by Langer, Science, 249: 1527 1533, 1990.
  • rAAVs can be used for in vivo delivery of transgenes for scientific studies such as optogenetics, gene knock-down with miRNAs, recombinase delivery for conditional gene deletion, gene editing with CRISPRs, and the like.
  • the invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent of the invention, said agent comprising a rAAV molecule of the invention.
  • the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like
  • Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbi
  • the pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol
  • compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising a therapeutically and/or prophylactically effective amount of an agent of the invention along with a pharmaceutically acceptable carrier.
  • the agent of the invention is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects).
  • the host or subject is an animal, e.g., a mammal such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as, a cynomolgus monkey and a human).
  • the host is a human.
  • kits that can be used in the above methods.
  • a kit comprises one or more agents of the invention, e.g., in one or more containers.
  • a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a condition, in one or more containers.
  • the invention also provides agents of the invention packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent or active agent.
  • the agent is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject.
  • the agent is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.
  • the lyophilized agent should be stored at between 2 and 8°C in its original container and the agent should be administered within 12 hours, usually within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • an agent of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of agent or active agent Typically, the liquid form of the agent is supplied in a hermetically sealed container at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml.
  • compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) as well as pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient).
  • Bulk drug compositions can be used in the preparation of unit dosage forms, e.g., comprising a prophylactically or therapeutically effective amount of an agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier.
  • the invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of the target disease or disorder can also be included in the pharmaceutical pack or kit.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
  • compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of agent or active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of agent or active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. 6.
  • the following examples report an analysis of surface-exposed loops on the AAV9 capsid to identify candidates for capsid engineering via insertional mutagenesis.
  • the invention is illustrated by way of examples, describing the construction of rAAV9 capsids engineered to contain 7-mer peptides designed on the basis of the human axonemal dynein heavy chain tail. Briefly, three criteria were used for selecting surface loops that might be amenable to short peptide insertions: 1) minimal side chain interactions with adjacent loops; 2) variable sequence and structure between serotypes (lack of conserved sequences); and 3) the potential for interrupting commonly targeted neutralizing antibody epitopes.
  • a panel of peptide insertion mutants was constructed and the individual mutants were screened for viable capsid assembly, peptide surface exposure, and potency. The top candidates were then used as templates for insertion of homing peptides to test if these peptide insertion points could be used to re-target rAAV vectors to tissues of interest. Further examples, demonstrate the increased transduction and tissue tropism for certain of the modified AAV capsids described herein.
  • FIGs. 1 and 2 depict analysis of variable region four of the adeno-associated virus type 9 (AAV9 VR-IV) by amino acid sequence comparison to other AAVs VR-IV (FIG. 1) and protein model (FIG. 2). As seen, AAV9 VR-IV is exposed on the surface at the tip or outer surface of the 3-fold spike. Further analysis indicated that there are few side chain interactions between VR-IV and VR-V and that the sequence and structure of VR-IV is variable amongst AAV serotypes, and further that there is potential for interrupting a commonly-targeted neutralizing antibody epitope and thus, reducing immunogenicity of the modified capsid.
  • AAV9 VR-IV adeno-associated virus type 9
  • AAV9 mutants were constructed, to each include a heterologous peptide but at different insertion points in the VR-IV loop.
  • the heterologous peptide was a FLAG tag that was inserted immediately following the following residues in vectors identified as pRGNXl 090- 1097, as shown in Table 2.
  • All candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097) were packaged with high efficiency.
  • Each candidate vector contains a FLAG insert at different sites within AAV9’s VR-IV. Briefly, each candidate vector was packaged with a luciferase transgene in lOmL culture. The titer was measured in genome copies per mL (GC/mL) (results not shown).
  • transduced cells were lysed and centrifuged. 500 pL of cell culture supernatant was loaded on 20 pL agarose-FLAG beads and eluted with SDS-PAGE loading buffer also loaded directly on the gel. All 8 capsid candidates were pulled down with anti-FLAG beads, indicating that the FLAG insert is on the surface of the capsid (results not shown).
  • Transduction efficiency of each of the eight candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097) was measured in Lec2 cells by measuring relative light units per microgram of protein.
  • CHO-derived Lec2 cells were grown in aMEM and 10% FBS.
  • the Lec2 cells were transduced at a multiplicity of infection (MOI) of about 2xl0 8 GC vector (a MOI of about 10,000) and were treated with ViraDuctin reagent (similar results were observed on transducing Lec2 cells at a MOI of about 10,000 GC/cell but treated with 40 pg/mL zinc chloride (ZnCh); results not shown).
  • MOI multiplicity of infection
  • ZnCh 40 pg/mL zinc chloride
  • AAV9 vectors having a capsid protein containing a homing peptide of the following peptide sequences (Table 3) at the S454 insertion site were studied.
  • Suspension-adapted HEK293 cells were seeded at 1x10° cells/mL one day before transduction in lOmL of media.
  • Triple plasmid DNA transfections were done with PEIpro® (Polypus transfection) at a DNA:PEI ratio of E 1.75.
  • qPCR was performed on harvested supernatant of transfected suspension HEK293 cells five days post-transfection. Samples were subjected to DNase I treatment to remove residual plasmid or cellular DNA and then heat treated to inactivate DNase I and denature capsids. Samples were titered via qPCR using TaqMan Universal PCR Master Mix, No AmpEraseUNG (ThermoFisherScientific) and primer/probe against the polyA sequence packaged in the transgene construct. Standard curves were established using RGX-501 vector BDS.
  • Example 7 Homing peptides alter the transduction properties of AAV9 in vitro when inserted after S454.
  • AAV9 wild type and S454 insertion homing peptide capsids of Table 3 containing GFP transgene were used to transduce a Lec2 cell line (sialic acid-deficient epithelial cell line), a HT- 22 cell line (neuronal cell line), a hCMEC/D3 cell line (brain endothelial cell line), and a C2C12 cell line (muscle cell line).
  • Lec2 cell line sialic acid-deficient epithelial cell line
  • HT- 22 cell line neurovascular endothelial cell line
  • C2C12 cell line muscle cell line
  • Cell lines were plated at 5-20xl0 3 cells/well (depending on the cell line) in 96-well 24 hours before transduction.
  • Cells were transduced with AAV9-GFP vectors (with or without insertions) at IxlO 10 particles/well and analyzed via Cytation5 (BioTek) 48-96 hours after transduction, depending on the difference in expression rate in each cell line.
  • Lec2 cells were cultured as in Example 5
  • blood-brain barrier hCMEC/D3 (EMD Millipore) cells were cultured according to manufacturer’s protocol
  • HT-22 and HUH7 cells were cultured in DMEM and 10% FBS
  • C2C12 myoblasts were plated in DMEM and 10% FBS and differentiated for three days pre-transfection in DMEM supplemented with 2% horse serum and 0.1% insulin.
  • AAV9.S454.FLAG showed low transduction levels in every cell type tested.
  • Homing peptides can alter the transduction properties of AAV9 in vitro when inserted after S454 in the AAV9 capsid protein, as compared to unmodified AAV9 capsid (data not shown).
  • P7 TfRl peptide, HAIYPRH (SEQ ID NO: 10)
  • P9 TIR3 peptide, RTIGPSV (SEQ ID NO: 12)
  • P4 Kidney 1 peptide, LPVAS (SEQ ID NO:6)
  • FIG. 3 depicts alignment of AAVs l-9e, rhlO, rh20, rh39, rh74, hul2, hu21, hu26, hu37, hu51 and hu53 capsid sequences within insertion sites for capsid sequences within insertion sites for human peptides within or near the initiation codon of VP2, variable region 1 (VR-I), variable region 4 (VR-IV), and variable region 8 (VR-VIII) highlighted in grey; a particular insertion site within variable region eight (VR-VIII) of each capsid protein is shown by the symbol “#” (after amino acid residue 588 according to the amino acid numbering of AAV9).
  • GFP green fluorescent protein transgene expression was measured in mouse brain cells, following administration of the AAV vectors: AAV9; AAV.PHP.eB; AAV.hDyn (AAV9 with TLAAPFK (SEQ ID NO: 1) between 588-589 with no other amino acid modifications to the capsid sequence); AAV.PHP.S; and AAV.PHP.SH (see Table 17).
  • AAV.PHP.B is a capsid having a TLAVPFK (SEQ ID NO:20) insertion in AAV9 capsid, with no other amino acid modifications to the capsid sequence.
  • AAV.PHP.eB is a capsid having a TLAVPFK (SEQ ID NO:20) insertion in AAV9 capsid, with two amino acid modifications of the capsid sequence upstream of the PHP.B insertion (see also Table 17).
  • Table 4A summarizes the capsids utilized in the study.
  • mice were 8-12 weeks of age at the start date. At day 15 post administration, the animals were euthanized, and peripheral tissues were collected, including brain tissue, liver, forelimb biceps, heart, kidney, lung, ovaries, and the sciatic nerve. Table 4B
  • Quantitative PCR was used to determine the number of vector genomes per pg of brain genomic DNA. Brain samples from injected mice were processed and genomic DNA was isolated using Blood and Tissue Genomic DNA kit from Qiagen. The qPCR assay was run on a QuantStudio 5 instrument (Life Technologies Inc) using primer-probe combination specific for eGFP following a standard curve method.
  • AAV vector genome copies per pg of brain genomic DNA was at least a log higher in mice that were administered AAV.hDyn compared to all other AAV serotypes: AAV9, AAV.PHPeB, PHP.S, and PHP.SH (data not shown).
  • AAV.hDyn which is AAV9 capsid containing the “TLAAPFK” (SEQ ID NO:1) peptide insert (a peptide from human axonemal dynein) between residues 588-589 of the AAV9 capsid.
  • Other modified AAV9 capsids, however, including the vector AAV.PHPeB, which contains the “TLAVPFK” (SEQ ID NO:20) sequence demonstrated transduction in mouse brain at less than 1E03 GC/pg transgene upon systemic treatment.
  • AAV capsid sequences were modified either by peptide insertions or guided mutagenesis and pooled to give a bar-coded library packaged with a GFP expression cassette.
  • the modified vectors were then evaluated in an in vitro assay, as well as for in vivo bio-distribution in mice using next generation sequencing (NGS) and quantitative PCR.
  • NGS next generation sequencing
  • AAV.hDyn was identified as a high brain transduction vector from this pool and was further evaluated in individual delivery studies in mice to characterize its transduction profile. Additionally, immunohistochemistry analysis of brain sections was performed to understand the cellular tropism of this vector.
  • Example llA-/n vitro testing of transduction an crossing blood brain barrier [00258] The ability of the modified capsids to cross the blood brain barrier was tested in an in vitro transwell assay using hCMEC/D3 BBB cells (SCC066, Millipore- Sigma). More specifically, the assay was essentially adapted from Sade, H. et al. (2014 PLoS ONE 9(4): e96340) A human Blood-Brain Barrier transcytosis assay reveals Antibody Transcytosis influenced by pH-dependent Receptor Binding, April 2014, Vol.
  • Capsid modifications were performed on widely used AAV capsids including AAV8, AAV9, and AAVrh.10 by inserting various peptide sequences after the position S454 of the VR- IV (Tables 5a-5c) or after position Q588 of the VR-VIII surface exposed loop of the AAV capsid, as well as insertions after the initiation codon of VP2, which begins at amino acid 137 (AAV4, AAV4-4, and AAV5) or at amino acid 138 (AAV1, AAV2, AAV3, AAV3-3, AAV6, AAV7, AAV8, AAV9, AAV9e, rh.10, rh.20, rh.39, rh.74, and hu.37) (FIG.
  • rAAVs with certain modified capsids were tested for transduction in vitro in Lec2 cells as described above in Example 5.
  • Modified AAVs tested for transduction in Lec2 cells as follows: eB 588 Ad, eB 588 Hep, eB 588 p79, eB 588 Rab, AAV9 588 Ad, AAV9 588 Hep, AAV9 588 p79, AAV9 588 Rab, eB VP2 Ad, eB VP2 Hep, eB VP2 p79, eB VP2 Rab, AAV9 VP2 Ad, AAV9 VP2 Hep, AAV9 VP2 p79, AAV9 VP2 Rab as compared to AAV9. See Table 5B below for identity of AAV capsids.
  • modified AAVs were packaged with an eGFP transgene cassette containing specific barcodes corresponding to each individual capsid. Novel barcoded vectors were pooled and injected into mice in order to increase the efficiency of screening.
  • mice were randomized into treatment groups based on Day 1 bodyweight and their age at start date was 8-12 weeks. At day 15 post administration, the animals were euthanized and peripheral tissues were collected, including brain, kidney, liver, sciatic nerve, lung, heart, and muscle tissue. In the studies where selected capsids from the pool were injected individually, the same protocol was followed.
  • Genomic DNA was isolated from tissue samples using DNeasy Blood and Tissue kit (69506) from Qiagen. Each vector’s barcode region was amplified with primers containing overlaps for NGS and unique dual indexing (UDI) and multiplex sequencing strategies, as recommended by the manufacturer (Illumina). Illumina MiSeq using reagent nano and micro kits v2 (MS- 103 -1001/1002) were used to determine the relative abundance of each barcoded AAV vector per sample collected from the mice. Accordingly, each vector sample in Tables 5A-C below was barcoded as noted above to allow for each read to be identified and sorted before the final data analysis. The data was normalized based on the composition of AAVs in the originally injected pool and quantified using the total genome copy number obtained from qPCR analysis with a primer-probe combination specific to the barcoded sample.
  • mice injected with AAV.hDyn were sectioned using a Vibratome (Leica, VT-1000) and the GFP expression was evaluated using an anti-GFP antibody (AB3080, Millipore Sigma), Vectastain ABC kit (PK-6100, Vector Labs) and DAB Peroxidase kit (SK-4100, Vector Labs). Broad distribution of GFP expressing cells were present throughout the brain in mice injected with AAV.hDyn, including distribution in the cortex, striatum, and hippocampus of the brain (data not shown).
  • AAV9 588 Hep AAV9 with the peptide TILSRSTQTG (SEQ ID NO: 15) 5 inserted after position 588) exhibited significantly greater transduction (4-fold) than wild type AAV9
  • AAV9 VP2 Ad AAV9 with the peptide SITLVKSTQTV (SEQ ID NO: 14) inserted after position 138
  • AAV9 VP2 Hep AAV9 with the peptide TILSRSTQTG (SEQ ID NO: 15) inserted after position 138
  • AAV9 VP2 Rab AAV9 with the peptide RSSEEDKSTQTT (SEQ ID NO: 19) inserted after position 138
  • NGS Next Generation Sequencing
  • brain gDNA reveals relative abundances (percent composition) of the capsid pool delivered to mouse brains following intravenous injection.
  • the data was normalized based on the composition of AAVs in the originally injected pool and quantified using the total genome copy number obtained from qPCR analysis with a primer-probe combination specific to the eGFP sequence. Data was collected from three different experiments. Parental AAV9 was used as standard and included in each pool.
  • AAV.hDyn shows increased brain bio-distribution compared to AAV9.
  • the AAV vector genome copies per pg of brain genomic DNA was at least a log higher in mice that were administered AAV.hDyn compared to the parental AAV9 vector (data not shown).
  • AAV capsid modifications performed either by insertions in surface exposed loops of VR-IV and VR-VIII or by specific amino acid mutations did not affect their packaging efficiency and were able to produce similar titers in the production system described herein.
  • Intravenous administration of AAV9 S454 Kidney 1 and AAV9 S454 Kidney 1C to mice resulted in higher relative abundance of the viral genome and greater kidney cell transduction than other modified AAV9 vectors and the parental AAV9 vector tested.
  • Intravenous administration of the AAV9 S454 Kidneyl or AAV9 S454 Musclel vector to mice resulted also in lower liver cell transduction.
  • FIG. 14 depicts the amino acid sequence for a recombinant AAV9 vector capsid including a peptide insertion of amino acid sequence TLAVPFK (SEQ ID NO:20) between S454 and G455 of VR-IV.
  • the administration, in vivo and post-mortem observations, and biodistribution of a pool of recombinant AAVs having engineered capsids and a GFP transgene will be evaluated following a single intravenous, intracerebroventricular or intravitreal injection in cynomolgus monkeys (Table 7).
  • the pool contains multiple capsids each of which contains a unique barcode identification allowing identification using next generation sequencing (NGS) analysis following administration to cynomolgus monkeys.
  • NGS next generation sequencing
  • the cynomolgus monkey is chosen as the test system because of its established usefulness and acceptance as a model for AAV biodistribution studies in a large animal species and for further translation to human. All animals on this study are naive with respect to prior treatment.
  • the pool may comprise at least the following recombinant AAVs having the engineered capsids listed in Table 7.
  • the IV infusion will be administered at a rate of 3 mL/min followed by 0.2 mL of vehicle to flush the dose from the IV catheter.
  • the three intravenous animals will receive a single dose of the pooled recombinant AAVs at a volume of 4 mL/kg.
  • the total dose (vg) and dose volume (mL/kg) will be recorded in the raw data.
  • the IV dose of IxlO 13 GC/kg body weight was determined to be required to have the desired distribution in the CNS from a systemic delivery as well as the peripheral tissues including skeletal muscle.
  • the ICV implanted animals will receive a single bolus dose at a volume of 1 mL of AAV- NAV-GFPbc (by slow infusion, approximately 0.1 mL/min) followed by 0.1 mL of vehicle to flush the dose from the catheter system.
  • the ICV dose is based on distribution data from a previous non-human primate study to support current clinical programs.
  • IVT intravitreal
  • Clinical signs will be recorded at least once daily beginning approximately two weeks prior to initiation of dosing and continuing throughout the study period. The animals will be observed for signs of clinical effects, illness, and/or death. Additional observations may be recorded based upon the condition of the animal at the discretion of the Study Director and/or technicians.
  • Ophthalmological examinations will be performed on Group 3 animals prior to dose administration, and on Days 2, 8, 15 and 22. All animals will be sedated with ketamine hydrochloride IM for the ophthalmologic examinations performed following Day 1.
  • the animals will be sedated with injectable anesthesia (refer to Section 15.3.3).
  • the eyes will be dilated with 1% tropicamide prior to the examination.
  • the examination will include slit-lamp biomicroscopy and indirect ophthalmoscopy. Additionally, applanation tonometry will be performed on Group 3 animals prior to dosing, immediately following dose administration (-10 to 15 minutes) and on Days 2 and 22.
  • Blood samples ( ⁇ 3 mL) will be collected from a peripheral vein for neutralizing antibodies analysis approximately 2 to 3 weeks prior to dose administration.
  • Blood samples (-5 mL) will be collected from fasted animals from a peripheral vein for PBMC analysis prior to dose administration (Day 1), on Days 8 and 15 and prior to necropsy (Day 22). The samples will be obtained using lithium heparin tubes and the times recorded.
  • Blood samples will be collected from a peripheral vein for bioanalytical analysis prior to dose administration (Day 1, 2 mL) and necropsy (Day 22, 5 mL). The samples will be collected in clot tubes and the times recorded. The tubes will be maintained at room temperature until fully clotted, then centrifuged at approximately 2400 rpm at room temperature for 15 minutes. The serum will be harvested, placed in labeled vials (necropsy sample split into 1 mL aliquots), frozen in liquid nitrogen, and stored at -60°C or below.
  • CSF (-1.5 mL) will be collected prior to dose administration from a cistema magna spinal tap from animals in Group 1 only.
  • CSF (-2 mL) will be collected immediately prior to necropsy from a cisterna magna spinal tap from all animals (Groups 1 to 3). An attempt to collect CSF will be made but due to unsuccessful spinal taps, samples may not be collected at all intervals from an animal(s). Upon collection, the samples will be stored on ice until processing.
  • a gross necropsy will be performed on any animal found dead or sacrificed moribund, and at the scheduled necropsy, following at least 21 days of treatment (Day 22). All animals, except those found dead, will be sedated with 8 mg/kg of ketamine HC1 IM, maintained on an isoflurane/oxygen mixture and provided with an intravenous bolus of heparin sodium, 200 lU/kg. The animals will be perfused via the left cardiac ventricle with 0.001% sodium nitrite in saline. Animals found dead will be necropsied but will not be perfused.
  • PBMC samples collected from all animals will be evaluated by flow cytometry and enzyme-linked immune absorbent spot (ELISpot), if required.
  • ELISpot enzyme-linked immune absorbent spot
  • FIG. 5 depicts the relative abundance (RA) of the viral genomes (normalized to input) in the frontal cortex of the cynomolgus monkey model, and Table 8 lists the RA values for those capsids with the highest RA shown in FIG. 5.
  • FIGS. 6 and 7 depict the RA of the viral genomes (normalized to input) in the hippocampus and the cerebellum of the cynomolgus monkey model, respectively.
  • the RA of AAV.rh34 is shown by the shaded column on the left side of the graphs and the RA of AAV9 reference is showed by the shaded column in the middle or the graphs.
  • FIGS. 6 and 7 show that AAV.rh34 is a top performing capsid in the intravenous administration pool
  • AAV.rh34 displayed a favorable profile with respect to CNS toxicity as well.
  • the rh34 capsid displayed decreased transduction in dorsal root ganglion (DRG) while exhibiting a high frontal cortex tropism (transduction efficiency).
  • AAVrh34 exhibits an increased RA to AAV9 in CNS regions as follows: 1.8-fold in Hippocampus, 7.4-fold in frontal cortex, 1.9-fold in amygdala, 6.0-fold in medulla, 3.1-fold in midbrain, 1.2-fold in hypothalamus, 8.8-fold in thalamus, 13-fold in globus pallidus, 5.7-fold in SNc, 3.5-fold in dorsal raphe, 2.0-fold in claustrum, 13-fold in putamen, 9-fold in occipital cortex, and 9.6-fold in cerebellum. Additionally, AAVrh34 exhibits a decreased RA in: DRGs: 90-99.5%, Liver: -99%, Biceps: -30%, Sciatic nerve: 83%, and Optic nerve: 17%.
  • the AAVrh34 capsid is the only capsid present in the top 45 performers in FC (highest RA to AAV9), and bottom 45 performers in the cervical, thoracic, and lumbar DRGs (lowest RA to AAV9) .
  • AAV capsids with a combination of these recited characteristics are considered “DRG- friendly” capsids, such that their low rate of transduction in DRG should have minimal neurotoxicity and/or reduced or negligible axonopathy symptoms in a subject administered the AAV capsid.
  • a procedure like that described in Example 13 with ICV administration was used to study the biodistribution of a pool of rAAV capsids to cynomolgus monkey model.
  • Several capsids exhibited good spread in CNS with high relative abundance (RA, compared to AAV9 reference capsid) in most brain regions, notably AAV4, AAV5, rh 34, hu26, rh31, and hul3.
  • Favorable capsids exhibiting CNS-tropism have DNA relative abundance values resulting in greater than 1.1- fold increase in DNA values in at least one CNS region, except dorsal root ganglion (DRG).
  • DRG dorsal root ganglion
  • FIG. 8 depicts the RA of the viral genomes (normalized to input) in the frontal cortex of the cynomolgus monkey model, and Table 9 lists the RA values for those capsids with the highest RA shown in FIG. 8.
  • FIG. 9 depicts the relative abundance of the viral genomes (normalized to input) in the hippocampus of the cynomolgus monkey model, and Table 10 lists the RA values for those capsids with the highest RA shown in FIG. 9.
  • FIG. 10 depicts the relative abundance (RA) of the viral genomes (normalized to input) in the midbrain of the cynomolgus monkey model, and Table 11 lists the RA values for those capsids with the highest RA shown in FIG. 10.
  • FIG. 11 depicts the RA of the viral genomes (normalized to input) in the cerebellum of the cynomolgus monkey model, and Table 12 lists the RA values forthose capsids with the highest RA shown in FIG. 11.
  • FIG. 12 depicts the RA of the viral genomes (normalized to input) in the cervical DRGs of the cynomolgus monkey model, and Table 13 lists the RA values for those capsids with the highest RA shown in FIG. 12.
  • FIG. 13 depicts the RA of the viral genomes (normalized to input) in the lumbar DRGs of the cynomolgus monkey model, and Table 14 lists the RA values for those capsids with the highest RA shown in FIG. 13.
  • FIG. 14 depicts a Venn diagram of the top performing 15 capsids transducing the frontal cortex, hippocampus, midbrain and cerebellum following ICV administration. As indicated in the diagram, AAV6, AAV8.BBB, AAV.rh.46, and AAV1 were the only AAVs represented in each of the top performing groups.
  • FIG. 15 depicts a Venn diagram of the top performing 45 capsids transducing the hippocampus and the 45 capsids with the lowest transduction values for DRG, to identify hippocampus-targeting DRG friendly capsids.
  • AAV.hu.60, AAV.rh.21, AAV.PHP.hB, AAV.rh.15, AAV.rh.24, AAV9.W503R, hu.5, AAV9.Q474A, and AAV.hu.10 were the only AAVs represented in each of the groups.
  • FIG. 16 depicts a Venn diagram of the top performing 40 capsids transducing the heart, biceps, and gastrocnemius and the 40 capsids with the lowest transduction values for the liver, to identify muscle-targeting liver-friendly capsids.
  • AAV.PHPeB.VP2Herp was the only AAVs represented in each of the groups.
  • FIG. 17 depicts a Venn diagram of the top performing 15 capsids transducing the heart, biceps, and gastrocnemius and Table 15 provides a list of the top performing capsids in three different cells of the diagram. Table 15.
  • FIGS. 18A and B depict the RA of the viral genomes (normalized to input) in the gastrocnemius and the liver of the cynomolgus monkey model, respectively.
  • Table 16 provides the rank of each capsid by RA values for the cynomolgus monkey model and the MDX mouse model. Capsids were ranked relative to one another in each animal to decrease variability across animals. Gastrocnemius, TA, heart, bicep, and triceps contributed 70% to the ranking for the MDX Mouse Model and the gastrocnemius, heart, and biceps contributed 70% to the ranking for the cynomolgus monkey model. Liver RA contributed 30% to rankings for each animal. The overall ranking was determined by weighting the ranking for each animal 50%.
  • pooled barcoded vectors were administered toNHPs by IV injection.
  • the pooled mixture consists of 118 different AAV capsids, including natural isolates and engineered AAVs, as described herein, expressing the GFP reporter gene from the universal CAG promoter.
  • the intravenous study followed the protocol described in Examples 13 and 14, infra.
  • Several capsids exhibited tropism that “detargeted” the liver, as such, mutated capsids exhibited lower abundance in liver tissue than the parental capsid (AAV9), e.g.
  • AAV8.BBB.LD (A269S, 498-NNN/AAA- 500), AAV9.BBB.LD (S263G/S269T/A273T, 496-NNN/AAA-498), AAV9.496-NNN-498, AAV9.496-NNN-498.W503R, AAV9.W503R, and AAV9.Q474A.
  • AAV8 capsids having the NNN/AAA mutation exhibit overall approximately an 11 -fold reduction in transduction in liver, and 42-fold reduction in expression of transcript in liver.
  • AAV9 capsids having the NNN/AAA and W5O3R mutation exhibits approximately a 400-fold reduction in transduction in liver, and results in zero expression of transcript in liver. In some instances, brain distribution of these modified vectors was also diminished.
  • AAV8.BBB.LD additionally exhibits a high level of transduction in gastrocnemius muscle.
  • FIGs. 19A and 19B depict the number of genome copies of DNA (A) or RNA (B) of select “liver-detargeting” (LD) vectors as detected in the liver of NHPs following IV administration of the capsid library.
  • FIG. 20 depicts the biodistribution of select “liver- detargeting” (LD) vectors compared to their parental AAV9 capsid in various tissues, in NHPs following IV administration of the capsid library.
  • FIG. 21 depicts the biodistribution of select LD vectors compared to their parental AAV8 capsid in various tissues, in NHPs following IV administration of the capsid library.
  • a fold change >1 indicates that the capsid makes up a lower percentage of the total capsid “pool” present in the blood at 24hr compared to 3hr after dosing (i.e. faster blood clearance).
  • a fold change ⁇ 1 indicates that the capsid makes up a greater percentage of the total capsid “pool” present in the blood at 24hr compared to 3hr after dosing (i.e. slower clearance).
  • slower clearance correlates with lower liver transduction/liver detargeting.
  • FIG. 23A As represented by increase in blood retention, a depiction of the change in abundance for a given capsid in a given animal was plotted. Allowing for the calculation of the fold increase in blood retention over the baseline retention of AAV9, for example, the representations (FIGs 23A and 23B) compare that change in abundance value of the select capsid to the change in abundance of the parental capsid (setting the parental capsid to 1). Thus, various mutations to the AAV9 capsid increase retention in the circulation by 3 to 5 fold (see. e.g. FIG. 23A).
  • pooled barcoded vectors were administered to mdx mice by IV (tail vein) injection.
  • the pooled mixture consists of 118 different AAV capsids, including natural isolates and engineered AAVs, as described herein, expressing the GFP reporter gene from the universal CAG promoter.
  • the IV study followed a protocol analogous to that described in Examples 12 and 16, infra.
  • FIG. 24 depicts the biodistribution of select AAV vectors in muscle tissues, including cardiac muscle, as well as cerebrum, liver and pancreas in wild-type B6 mice following IV administration of the capsid library.
  • IP intraparenchymal
  • the pooled mixture consists of 118 different AAV capsids, including natural isolates and engineered AAVs, as described herein, expressing the GFP reporter gene from the universal CAG promoter.
  • the targeted CNS tissue was the putamen in this study, and capsids were identified that have distinct expression patterns locally and that demonstrate retrograde or anterograde transport.
  • the IP striatum study followed the protocol as described in Examples 13 and 14, infra, except that the vector material was administered to the anterior striatum by MRI-guided stereotactic injection.
  • Peripheral tissues analyzed were from liver, spleen, and heart. Cerebrospinal fluid (CSF) and brain sections were extracted, e.g. putamen (bilateral) - three tissue samples at different rostro- caudal levels; Caudate (bilateral); Cortex (mPFC, dlPFC, Cg, Frontal - Ml, Parietal - SI, Occipital, Insular, Temporal (bilateral)); White matter of extreme capsule (bilateral); Globus pallidus (internal/external) (external — GPe); Basolateral amygdala (bilateral); Intralaminar thalamus (CM/Pf) (rostral and caudal); Thalamus (pulvinar); STN; Substantia nigra (SNr/SNc) (bilateral); Pedunculopontine tegmentum (PPT); Hippocampus; Cerebellum; Spin
  • Dosing MRIs were obtained as T1 images taken following injection in each of the monkeys to visualize the location of the injection (Prohance contrast agent was added to injected test article).
  • FIG. 26 depicts qPCR analysis which revealed high copy number (RNA cp/ug) of total vector transcripts localized to putamen, caudate, GPe and interestingly, SNc, as well as other tissues (RNA copy number/ug, adjusted to log scale). This distribution is consistent with other known inputs to the putamen (Smith et al., Journal of Neurophsyiology, 2012; Weiss et al., Scientific Reports, 2020).
  • FIG. 27 depicts on overview of the relationship between vector spread from posterior compartments to anterior compartments, as an absolute measure of RNA copy number vs. log scale. Localization was observed in the putamen, as well as the caudate regions. Vector expression was strongest in injection site (e g. putamen anterior).
  • FIGs. 28A and 28B depict absolute RNA copy numbcr/pg distribution by qPCR (A) and results adjusted to log scale (B), including peripheral tissues. Expression in the periphery and in unrelated brain regions was negligible, which comports with known lack of anatomical connectivity from cerebellum or hippocampus to putamen.
  • FIG 29 depicts NGS analysis to identify vector DNA from the capsid pool in putamen.
  • the BC029 barcode indicates the mutated AAV9 capsid, AAV9.S454.Tfr3 is the highest transduced capsid identified in the putamen ipsilateral (relative to the location of injection) of this particular injected subject.
  • FIG. 30 depicts RNA abundance adjusted to input (normalized to 1 per pg). This is a representation of transcripts containing the barcode of interest per pg calculated using the barcode relative abundance adjusted for input normalized to 1 and total number of library-derived transcripts per pg in that tissue.
  • qPCR was performed to identify vector transcripts relative to high expressing capsids in the selected tissues: putamen ipsilateral (sample “punch” 1 or 2), caudate ipsilateral (punch 1 or 2), extreme capsule ipsilateral, GPe (globus pallidus external) ipsilateral, amygdala ipsilateral, substantia nigra ipsilateral.
  • AAV9.S454.Tfr3 demonstrates improved transduction relative to AAV9 following intraparenchymal delivery, for example a 4 to 5 fold increase RNA expression compared to AAV9 is observed in most brain regions analyzed.
  • AAV9.454.Tfr3 is biodistributed to peripheral tissues similarly to AAV9 yet AAV9.454.Tfr3 is diminished in brain regions compared to AAV9. The same trend is seen in mouse brains following IV administration. The data suggests that AAV9.454.Tfr3 is less efficient at transport across the blood-brain-barrier than AAV9 following an IV administration of pooled capsids.
  • FIGs. 31A and 31B show biodistribution of AAV9 and AAV9.454.Tfr3 vector RNA and DNA following IV administration of pooled capsids intravenously in NHPs.
  • FIGs. 32A and 32B illustrate the biodistribution of AAV9 and AAV9.454.Tfr3 vector RNA and DNA in selected tissues following IV administration of pooled capsids intravenously in mice.
  • FIGs. 33A and 33B illustrate the correlation between abundances of transgene DNA and transcribed transgene RNA in NHP and mouse in the putamen (NHP) or striatum (mouse) following intraparenchymal delivery to striatum (mouse) or putamen (NHP).
  • AAV9.454.TFR3 identified as top hit in both NHP and mouse experiments and findings translated well between mouse and primate at both the DNA and RNA levels.
  • FIGs. 34A and 34B illustrate that rAAVs having an AAV9.S454.Tfr3 capsid produces 2-10 fold more transgene RNA than an rAAV with an AAV9 capsid with similar biodistribution following intraparenchymal administration in NHPs.
  • a difference was observed in RNA relative abundance (RA) for AAV9.S454.Tfr3 compared to AAV9 (AAV9.S454.Tfr3>AAV9 by 2-10 fold) which was not seen at the DNA level.
  • FIGs. 35A and 35B illustrate that rAAV having an AAV9.S454.Tfr3 capsid produces 2- 10 fold more RNA than AAV9 with similar biodistribution following intraparenchymal administration in mouse.
  • FIGs. 36A and 36B Further analysis of the intraparenchymal administration of the PAVE118 library (2.4el lGC in lOOpl) to NHP putamen is depicted in FIGs. 36A and 36B.
  • GC/pg DNA was calculated from the relative abundance of each library member and the qPCR total biodistribution data in 3 tissue punches each from putamen (FIG. 36A) and caudate (FIG. 36B) from the ipsilateral hemisphere.
  • BC029 AAV9S454-TFR3 genome copy (GC) levels were determined to be equivalent to those of AAV9, AAV5 and AAV1.
  • the fold change in genome copy (GC/pg DNA) for each capsid, normalized to AAV9, is depicted in white bars on the right y-axis.
  • BC029 (AAV9S454-TFR3) results in higher RNA expression levels than does AAV9 at or near the injection site in putamen (FIG. 37A) and caudate (FIG. 37B) RNA expression levels (transcripts/pg RNA) produced from each capsid were calculated (left Y-axis).
  • the fold change in expression level (transcripts/pg RNA) for each capsid, normalized to AAV9, is depicted in white bars on the right y-axis.
  • a 4-fold improvement in putamen and nearly 6-fold improvement in caudate of BC029 was observed over transcription from AAV9, which in resulted in further improvement of BC029 over AAV2, AAV5, and AAV8 as compared to AAV9.
  • the superior transduction efficiency for AAV9S454-TFR3 can be attributed to an improved RNA:DNA ratio. Taking the DNA and RNA analysis together (transcripts/pg RNA per copies/pg DNA), AAV9S454-TFR3(BC029) produces approximately 4 fold more RNA per genome copy than does AAV9 in both putamen (solid bars) and caudate (hatched bars) (FIG. 38A). The ratio of RNA expression levels to DNA genome copy number, normalized to AAV9, in putamen (solid bars) and caudate (hatched bars) is shown in FIG. 38B.
  • FIG. 39A shows the genome copies (GC/ug DNA) for AAV9S454-TFR3 (BC029) relative to AAV9 in the CNS regions sampled.
  • AAV9S454-TFR3 (BC029) has, on average, 40% of the number of genome copies of AAV9 and 10% of the number of genome copies of AAV5.
  • FIG 39B shows RNA expression levels of AAV9S454-TFR3 (BC029) relative to AAV9 in the CNS regions sampled.
  • AAV9S454-TFR3 (BC029) expresses 3-25 times more RNA than AAV9. The average fold change across these regions is depicted by the white bars Extensive sampling across the entire brain was performed and BC029 consistently outperformed AAV9, driven by an increase in RNA production (data not shown).
  • BC029 produces 3-4 fold more RNA transcripts than AAV9 in the mouse striatum (FIG. 41A), thalamus (FIG. 41B) and frontal cortex (FIG. 41C). RNA expression levels (transcripts/pg RNA) produced from each capsid were calculated (left Y-axis). The fold change in expression level (transcripts/pg RNA) for each capsid, normalized to AAV9, is depicted in white bars on the right y-axis. BC029 produces significantly higher transcript levels than reference AAV serotypes by approximately 1-2 logs.
  • BC029 (AAV9S454-TFR3) has approximately two-fold higher transcriptional activity than AAV9 in striatum and thalamus relative to AAV9 in mice administered AAV particles to the striatum.
  • RNA DNA ratios, absolute (FTG. 42A) and relative to AAV9 (FTG. 42B) in striatum (solid bars) thalamus (hatched bars) and frontal cortex (checked bars) were calculated as described above.
  • the RNA:DNA ratio of BC029 was improved by only 2 fold in striatum and thalamus relative to AAV9 due to increase in BC029 GC/cell (this increase was not observed in these regions in NHP as shown above). This difference trended higher in the cortex, but this was largely driven by a single animal.
  • BC029 AAV9S454-TFR3
  • AAV9S454-TFR3 displays improved transduction over AAV9 in multiple species via multiple routes of administration, largely doing so by producing more transcript copies than does AAV9 from equivalent genome copy levels in regions of interest in the CNS.
  • Example 20 Comparative analysis of transgene expression durability via different promoters in the striatum of mice delivered by intraparenchymal (IPa) injection of rAAV9
  • rAAV9 having a recombinant genome encoding GFP as transgene under the control of one of the neuronal-specific promoters hSyn (SEQ ID NO.: 95) or CamKII (SEQ ID NO.: 96), or under the control of the ubiquitous pan-cellular promoter CAG (SEQ ID NO.: 101) was injected bilaterally (lelO GC in 2pl each hemisphere) into the dorsal striatum of C57B16 female mice at 6- 8 weeks old. The sequences of the promoters are shown in Table 18, below. Brains were harvested three weeks, three months, and six months after the AAV9 administration.
  • the right hemisphere was processed for histological analyses (H&E Stain, DAPI, GFP, NeuN, GFAP (multi- fluorescent)).
  • the left hemisphere was used to extract DNA and RNA from the striatum for ddPCR analyses.
  • FIGs. 43A-43C Bar graphs show GFP gene copy number per cell (FIG. 43A), GFP RNA expression (FIG. 43B) and GFP RNAZDNA (FIG. 43C) at 3 weeks, 3 months and 6 months after injection of GFP under control of the CAG promoter (circles), the hSyn promoter (square) or the CamKll promoter (triangle). Expression under the hSyn promoter increases with time in the striatum samples.
  • FIGs. 44A-44C show quantitative luminance analysis of GFP expression.
  • FIG. 44A is a plot of GFP luminance in striatum samples under the control of the CAG promoter (circles), the hSyn promoter (squares), and the CamKII promoter (triangles) at 3 weeks, 3 months and 6 months.
  • FIG. 44B is a line graph showing transgene expression over time in the harvested samples.
  • FIG. 44C is a dot plot showing the quantitative analysis of GFP luminance under control of the CAG promoter (circles), hSyn promoter (squares), and CamKII promoter (triangles) at 6 months.
  • FIGs. 45A-45C show the percentage of neurons (FIG. 45A), the percentage of astrocytes (FIG. 45B) and the percentage of microglia (FIG. 45C) expressing GFP under the CAG promoter (circles), the hSyn promoter (squares) and CAMKII promoter (triangles). The data show that both the hSyn promoter and CAMKII promoter effect neuronal specific expression with no detected expression in astrocytes or microglia.
  • GFP under control of the CAG promoter is expressed in oligodendrocytes (data not shown).
  • GFP under control of the CAG promoter, the hSyn promoter, and the CamKII promoter co-localizes with parvalbumin (PV) (including, dopaminergic neurons), but not choline acetyltansf erase (ChAT) or cholinergic neurons (data not shown) at 6 months post-injection (data also not shown).
  • PV parvalbumin
  • ChAT choline acetyltansf erase
  • cholinergic neurons data not shown
  • Luminance analysis of native GFP fluorescence demonstrated that GFP expression driven by the CAG promoter was highest at all three time points, CamKII weakly expressing, and hSyn in the middle.
  • the RNA-to-DNA ratios from ddPCR analyses showed similar trends to the luminance quantification for all three promoters in terms of relative strength of expression.
  • all three promoters showed increasing levels of transgene expression all the way to six months.
  • a vector genome expressing a unique fluorescent reporter either GFP, tdTomato, or iRFP670, under the control of the universal CAG reporter and including a unique 20bp barcode between the fluorescent reporter coding sequence and the polyadenylation signal, was packaged into AAV9 (GFP), AAV9 TfR3 (tdTomato), or AAV9.Ref (iRFP) capsids.
  • a vector genome expressing a codon-optimized, human ApoE transgene, under the control of the universal CAG reporter and containing a unique 20bp barcode between the ApoE coding sequence and the polyadenylation signal (a different set of barcodes than those used in the fluorescent reporter cassettes) was packaged into AAV9, AAV9.TfR3, or AAV9.Ref such that ApoE transcripts produced from one of these capsids could be attributed to a transduction event with that capsid.
  • each fluorescent reporter prep was used to calculate the volume of each prep required to formulate a test article with equimolar concentrations of AAV9.CAG.GFP, A AV9.TfR3. CAG tdTomato, and AAV9.Ref.CAG.iRFP, and vector was formulated accordingly.
  • each ApoE reporter prep was used to calculate the volume of each prep required to formulate a test article with equimolar concentrations of AAV9.CAG.hcoApoE.BCl, AAV9.TfR3.hcoApoE.BC4, and AAV9.Ref.hcoApoE.BC5, and vector was formulated accordingly.
  • these 2 vector pools were pooled such that 90% of total GC in the final preparation were derived from fluorescent reporter preps, with the 10% of contributing GC being from ApoE reporter preps.
  • final “Tricolor-2” formulated vector pool had a titer of 2el3 GC/mL, with each fluorescent reporter prep having an effective concentration of 6el2 GC/mL and each ApoE reporter prep having an effective concentration of 6.6el l GC/mL.
  • the pooled Tricolor-2 test article was administered intraparenchymally to adult, cynomolgus macaques by MRT-guided delivery, following a bilateral dosing scheme into both the hippocampus (60pl of 2el3 GC/mL test article, 1.2el2 GC total) and the putamen (75pL of 2el3 GC/mL test article, 1.5el2 GC total).
  • a custom 16 gauge, 10ft. SmartFlow Neuro Ventricular Cannula was used, and Prohance was added prior to cannula fill in order to allow for repeated Tl- weighted MRI monitoring of dosing solution delivery.
  • the cannula was first primed with dosing solution, followed by dosing solution hold in the cannula for at least 5m to control for any vector adsorption to the device. Dosing solution was then eluted, followed by adjustment of flow rate to 2-5pL/min, cannula was inserted into target region, flow rate reduced to IpL/min until complete dose was delivered. Animals were sacrificed 3 weeks post-test article delivery, and brain was collected in 3mm thick coronal sections; alternating sections were fixed for histological analysis and direct visualization of fluorescent reporter expression or were sampled using a 4mm punch for nucleic acid analysis. Additional tissues were also collected for nucleic acid analysis.
  • DNA and RNA were extracted using a custom workflow such that paired DNA-RNA data could be obtained from the same tissue sample.
  • the DNA and RNA (following conversion to cDNA) were then subjected to amplicon sequencing using the Illumina MiSeq platform after amplification of barcode-containing regions from nucleic acids derived both from the fluorescent and ApoE reporters.
  • barcode counts were adjusted for test article input concentrations and normalized to 1 , such that the fraction of total fluorescent reporter or total ApoE reporter DNA or RNA that was derived from one of the 3 capsids of interest could be determined.
  • the pooled vector cocktail was administered to the striatum of mice (1.2elOGC total in putamen in 2pl) and mice were sacrificed 3 weeks after administration and tissue samples analyzed.
  • BC029 and AAV display similar tropism for hippocampal regions of interest (ROIs) (dentate gyrus, CA3, CA2 and subiculum regions of the hippocampus) (data not shown).
  • ROIs hippocampal regions of interest
  • BC029 and AAV9 similarly transduce the polymorphic and granule cell layers (data not shown).
  • PAVE118 library was administered by ICV infusion to the left ventricle in mice (1.44el0 GC/brain) and NHP (3el0 GC/g brain).
  • Genome copy (FIG. 46A) and RNA transcript abundance (FIG. 46B) were measured in CNS ROI (frontal cortex, striatum and hippocampus). Genome copy was equivalent between BC029 and AAV9, however, the relative abundance of RNA transcripts was up to 6.4 fold higher in BC029 compared to AAV9.
  • Off-target biodistribution (GC/pg DNA) was analyzed in mouse liver, NHP liver, NHP heart, NHP lumbar DRG and NHP thoracic DRG (FIG. 46C).
  • BC029 genome copies were approximately 5 fold lower than AAV9 genome copies. There was approximately a 30% decline in BC029 genome copies in lumbar DRG compared to AAV genome copies. BC029 and AAV9 GC levels were equivalent in thoracic DRG. In conclusion, improved transduction profile of BC029 relative to AAV9 is also seen in ICV administration. Finally, due to a more favorable transduction profile in off-target tissues, use of BC029 has the potential to ameliorate concerns surrounding undesired peripheral tissue transduction.
  • CAG circles
  • hSyn squares
  • CamKII triangles
  • the transgene modulates a neural circuit
  • the lower increment of transgene expression may allow for a larger capacity of plasticity of the neural circuits being modulated.
  • the comparatively rapid and robust transgene expression driven by CAG may then limit the achievable effect on neural circuits by trying to modulate the circuit too quickly. While this is speculative, understanding these dynamics would be critical for gene therapy development
  • transgene expression and kinetics are not only vital in the longterm for gene therapies, but it is also vital for the design and analysis of basic neuroscience studies. Our results demonstrate that transgene expression continues to change until at least three months depending on promoter. Therefore, differences in transgene expression could confound results if a response is recorded over a long period of time making the inclusion of experimental controls paramount.
  • Extracted DNA for each sample was diluted to 20 ng per pl.
  • a total of 100 ng of DNA for each sample was digested with PS-DNase (Lucigen, Middleton, WI, USA) to isolate circular DNA by removing any linear DNA within the sample.
  • the reaction mix consisted of 100 ng of DNA, 10 units of PS-DNase, 33 mM Tris-acetate (pH 7.5), 66 mM potassium acetate, 10 mM of magnesium acetate, 0.5 mM DTT, and 1 mM ATP.
  • DNA samples were incubated with PS-DNase at 37°C for 16 h, and then heat inactivated for 30 min at 70°C.
  • an additional restriction enzyme digestion using ECORT was added to the ddPCR workflow.
  • ECORI digestion breaks up concatamers containing multiple copies of the AAV genome into individual vector genomes by only cleaving once in each genome, allowing for accurate quantification of AAV genomes regardless of concatemer size.
  • Five pl of the PS-DNase digested samples were added to BioRad ddPCR reaction mixture containing an additional 5 units of ECORI enzyme. Reactions were incubated at 37°C for 30 min before continuing to droplet generation and PCR. Copies of AAV genomes were again quantified using GFP primers and probes, then normalized by copies of mouse glucagon.
  • Table 17 provides the amino acid sequences of certain engineered capsid proteins described and/or used in studies described herein. Heterologous peptides and amino acid substitutions are indicated in gray shading.

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Abstract

La présente invention concerne des virus adéno-associés recombinants (VAAr) ayant une cassette d'expression dans laquelle le transgène est lié de manière fonctionnelle à un promoteur hSyn qui favorise l'expression neuronale persistante du transgène lors de la transduction du VAAr. L'invention concerne également des protéines de capside modifiées pour comprendre des séquences d'acides aminés et/ou des substitutions d'acides aminés qui confèrent et/ou améliorent des propriétés souhaitées, en particulier une transduction accrue dans le SNC lors d'une administration intraparenchymateuse relative à un VAAr ayant une capside de référence.
PCT/US2023/065692 2022-04-14 2023-04-12 Virus adéno-associés recombinants pour administration tropique au snc WO2023201277A1 (fr)

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