WO2021226167A1 - Variants de vaa issus de bibliothèques de second tour présentant un tropisme pour des tissus du système nerveux central - Google Patents

Variants de vaa issus de bibliothèques de second tour présentant un tropisme pour des tissus du système nerveux central Download PDF

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WO2021226167A1
WO2021226167A1 PCT/US2021/030779 US2021030779W WO2021226167A1 WO 2021226167 A1 WO2021226167 A1 WO 2021226167A1 US 2021030779 W US2021030779 W US 2021030779W WO 2021226167 A1 WO2021226167 A1 WO 2021226167A1
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aav
sequence
capsid
viral genome
disease
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PCT/US2021/030779
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Jinzhao Hou
Shaoyong LI
Wei Wang
Xiaodong Lu
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Voyager Therapeutics, Inc.
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Publication of WO2021226167A1 publication Critical patent/WO2021226167A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—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
    • C12N2750/14123—Virus like particles [VLP]
    • 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
    • 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
    • C12N2750/14171—Demonstrated in vivo effect

Definitions

  • AAV adeno-associated viral vectors
  • AAV-based therapies are currently being tested for the treatment of a wide-range of human disease.
  • the AAV-based therapy may be brought easily into contact with the system in need, such as for treating diseases of the blood (e.g, lipoprotein lipase deficiency or hemophilias), the eye (e.g., Leber congenital amaurosis or inherited retinal diseases), or muscle (e.g., Duchenne muscular dystrophy).
  • diseases of the blood e.g, lipoprotein lipase deficiency or hemophilias
  • the eye e.g., Leber congenital amaurosis or inherited retinal diseases
  • muscle e.g., Duchenne muscular dystrophy
  • delivery of the AAV-based therapy becomes more complicated.
  • Direct administration generally involves invasive surgeries, while the blood brain barrier impedes access of the AAV-based therapy to the CNS if administered systemically. Further, high doses of AAV- based therapies are necessary to yield sufficient transduction of target CNS tissue, giving rise to enhanced risk of side effects and/or production difficulties given the high volumes needed.
  • PNS peripheral nervous system
  • some PNS tissues such as dorsal root ganglia remain difficult to target.
  • libraries of novel capsids have been created and screened.
  • capsid engineering methods including DNA barcoding, directed evolution, random peptide insertions, and capsid shuffling and/or chimeras.
  • CREATE a series of targeting peptides was identified that enhanced the AAV capsid protein tropism for CNS tissues. The majority of this work has been carried out in mouse models, meaning that there still remains an issue of translatability to human subjects and conditions. [0008] Even though such ongoing efforts to generate AAV capsids with enhanced tropism to CNS tissues and/or blood brain barrier permeability have yielded improvements, a need still exists for AAV particles with enhanced tropism for CNS or PNS tissue for the use of treating human disease.
  • AAV particles with enhanced tropism for CNS or PNS tissues which may be delivered intravenously to a subject in need, at a low dose, and yet still provide sufficient transduction of the target cell-type, tissue and/or organ.
  • the present disclosure addresses this need by providing novel targeting peptides and associated AAV capsid proteins, capsids, and AAV particles for enhanced tropism to the human CNS or PNS following intravenous delivery.
  • the viral genomes of these AAV particles may be manipulated to suit the needs of a wide variety of CNS or PNS- associated disorders, or neurological disease.
  • the present disclosure provides AAV particles with enhanced central nervous system (CNS) transduction.
  • CNS central nervous system
  • the AAV particle may comprise a capsid having a nucleotide sequence comprising SEQ ID NO: 659. In some embodiments, the AAV particle may comprise a capsid having an amino acid sequence given by SEQ ID NO: 660.
  • the disclosure provides AAV particles with enhanced peripheral nervous system (PNS) transduction (e.g., dorsal root ganglia).
  • PNS peripheral nervous system
  • the disclosure provides AAV particles comprising an AAV capsid protein with a targeting sequence insert and a viral genome.
  • the viral genome comprises a nucleic acid sequence encoding a payload, wherein the payload may be an RNAi agent or a polypeptide.
  • the AAV particles as described herein comprise a viral genome encoding an RNAi agent payload.
  • the RNAi agent may be, but is not limited to, a dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA.
  • the RNAi agent When the RNAi agent is expressed, it inhibits or suppresses the expression of a gene of interest in a cell, wherein the gene of interest may be, but is not limited to, SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A- SCN5A, or SCN8A-SCN11A.
  • the AAV particles described herein comprise a viral genome encoding a polypeptide payload.
  • the polypeptide may be, but is not limited to, an antibody, survival motor neuron 1 (SMN1), ApoE2, GBA1, GRN, ASPA, ARSA, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, or GAN.
  • the AAV particles described herein may be formulated with a pharmaceutically acceptable excipient to prepare a pharmaceutical composition.
  • the pharmaceutical composition may be administered to a subject in need in order to treat a disease in said subject.
  • the disease may be, but is not limited to, Huntington’s Disease, Amyotrophic Lateral Sclerosis, Parkinson’s Disease, Alzheimer’s Disease, a tauopathy or neuropathic pain, Metachromatic Leukodystrophy, and Niemann-Pick Type C.
  • the disclosure provides peptides (targeting peptides) with amino acid sequences set forth as any of SEQ ID NO: 4-221.
  • the targeting peptide may comprise the amino acid sequence given by SEQ ID NO: 4.
  • Nucleic acid sequences encoding these peptides are also provided and are set forth as any of SEQ ID NO: 222-658.
  • the targeting peptide may be encoded by any one of SEQ ID NO: 222, 223, or 441.
  • the targeting peptide is encoded by SEQ ID NO: 223.
  • targeting sequences may be inserted into a parent sequence
  • a targeting peptide may be inserted into a parent AAV capsid protein comprising a VP1 amino acid sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 3.
  • the parent Vp1 amino acid sequence may comprise a VP2 region and/or a VP3 region.
  • Targeting peptides may be inserted into the parent sequence in any of the VP1, VP2, or VP3 regions.
  • a targeting peptide may be inserted at any amino acid position between amino acids 586-592, inclusive, of the parent VP1 amino acid sequence.
  • an AAV particle described herein comprises a capsid with targeting peptide SMPREPG (SEQ ID NO: 4) inserted between amino acids at positions 588 and 589 of a parent AAV capsid protein (SEQ ID NO: 2), resulting in capsid AAV-G (SEQ ID NO: 660).
  • SMPREPG SEQ ID NO: 4
  • SEQ ID NO: 2 AAV capsid protein
  • capsid AAV-G SEQ ID NO: 660
  • an AAV particle described herein comprises a capsid encoded by a nucleic acid sequence comprising a targeting sequence given by SEQ ID NO: 223.
  • the nucleic acid sequence of an AAV particle capsid described herein is given by SEQ ID NO: 659.
  • FIG.1A and FIG.1B show native GFP imaging of HEK293T and Lec2 cells, respectively, transduced with AAV9-GFP or AAV-G-GFP particles.
  • FIG.2A, FIG.2B and FIG.2C show native GFP imaging of mouse, monkey and human brain microvascular endothelial cells (BMVEC), respectively, transduced with AAV9-GFP or AAV-G-GFP particles.
  • FIG.3A and FIG.3B show native GFP imaging of undifferentiated and differentiated SH-SY5Y cells, respectively, transduced with AAV9-GFP or AAV-G-GFP particles.
  • DETAILED DESCRIPTION [0023] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described.
  • AAV particles with enhanced tropism for a target tissue e.g., CNS
  • target tissue e.g., CNS
  • Targeting peptides and nucleic acid sequences encoding the targeting peptides are provided.
  • an “AAV particle” or “AAV vector” comprises a capsid protein and a viral genome, wherein the viral genome comprises at least one payload region and at least one inverted terminal repeat (ITR).
  • ITR inverted terminal repeat
  • the AAV particle and/or its component capsid and viral genome may be engineered to alter tropism to a particular cell-type, tissue, organ or organism.
  • viral genome or “vector genome” refers to the nucleic acid sequence(s) encapsulated in an AAV particle.
  • a viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.
  • a “payload region” is any nucleic acid molecule which encodes one or more “payloads” as described herein.
  • a payload region may be a nucleic acid sequence encoding a payload comprising an RNAi agent or a polypeptide.
  • a “targeting peptide” refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein.
  • the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism.
  • the AAV particles and payloads as described herein may be delivered to one or more target cells, tissues, organs, or organisms.
  • the AAV particles described herein demonstrate enhanced tropism for a target cell type, tissue or organ.
  • the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively).
  • the AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ. II.
  • Adeno-associated viruses are small non-enveloped icosahedral capsid viruses of the Parvoviridae family characterized by a single stranded DNA viral genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates.
  • the Parvoviridae family comprises the Dependovirus genus which includes AAV, capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.
  • AAV AAV have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile.
  • the genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload.
  • the wild-type AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length.
  • Inverted terminal repeats (ITRs) traditionally cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome. While not wishing to be bound by theory, an AAV viral genome typically comprises two ITR sequences.
  • ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region.
  • the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.
  • the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes).
  • the Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid.
  • Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame.
  • VP1 refers to amino acids 1-736
  • VP2 refers to amino acids 138-736
  • VP3 refers to amino acids 203-736.
  • VP1 is the full length capsid sequence
  • VP2 and VP3 are shorter components of the whole.
  • changes in the sequence in the VP3 region are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three.
  • the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3. As used herein, an “AAV serotype” is defined primarily by the AAV capsid. In some instances, the ITRs are also specifically described by the AAV serotype (e.g., AAV2/9).
  • the wild-type AAV viral genome can be modified to replace the rep/cap sequences with a nucleic acid sequence comprising a payload region with at least one ITR region.
  • a nucleic acid sequence comprising a payload region with at least one ITR region.
  • the rep/cap sequences can be provided in trans during production to generate AAV particles.
  • AAV vectors may comprise the viral genome, in whole or in part, of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant.
  • AAV variants may have sequences of significant homology at the nucleic acid (genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms.
  • AAV particles are recombinant AAV viral vectors which are replication defective and lacking sequences encoding functional Rep and Cap proteins within their viral genome. These defective AAV vectors may lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, a tissue, an organ, or an organism.
  • the viral genome of the AAV particles comprise at least one control element which provides for the replication, transcription, and translation of a coding sequence encoded therein.
  • Non-limiting examples of expression control elements include sequences for transcription initiation and/or termination, promoter and/or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (e.g., Kozak consensus sequence), sequences that enhance protein stability, and/or sequences that enhance protein processing and/or secretion.
  • AAV particles for use in therapeutics and/or diagnostics comprise a virus that has been distilled or reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest.
  • AAV particles are engineered as vehicles for specific delivery while lacking the deleterious replication and/or integration features found in wild-type viruses.
  • AAV vectors as described herein may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences.
  • AAV a “vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.
  • scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell.
  • the AAV particle is an scAAV.
  • the AAV particle is an ssAAV.
  • Methods for producing and/or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos.
  • AAV particles may be modified to enhance the efficiency of delivery. Such modified AAV particles can be packaged efficiently and be used to successfully infect the target cells at high frequency and with minimal toxicity.
  • the capsids of the AAV particles are engineered according to the methods described in US Publication Number US20130195801, the contents of which are incorporated herein by reference in their entirety.
  • the AAV particles described herein comprising a capsid with an inserted targeting peptide and a viral genome, may have enhanced tropism for a cell- type or tissue of the human CNS.
  • AAV Capsids [0046] AAV particles described herein may comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profiles. While not wishing to be bound by theory, the AAV capsid protein is often considered to be the driver of AAV particle tropism to a particular tissue.
  • an AAV particle may have a capsid protein and ITR sequences derived from the same parent serotype (e.g., AAV2 capsid and AAV2 ITRs).
  • the AAV particle may be a pseudo-typed AAV particle, wherein the capsid protein and ITR sequences are derived from different parent serotypes (e.g., AAV9 capsid and AAV2 ITRs; AAV2/9).
  • the AAV particles described herein may comprise an AAV capsid protein with a targeting peptide inserted into the parent sequence.
  • the parent capsid or serotype may comprise or be derived from any natural or recombinant AAV serotype.
  • a “parent” sequence is a nucleotide or amino acid sequence into which a targeting sequence is inserted (i.e., nucleotide insertion into nucleic acid sequence or amino acid sequence insertion into amino acid sequence).
  • the parent AAV capsid nucleotide sequence is as set forth in SEQ ID NO: 1.
  • the parent AAV capsid nucleotide sequence is a K449R variant of SEQ ID NO: 1, wherein the codon encoding a lysine (e.g., AAA or AAG) at position 449 in the amino acid sequence (nucleotides 1345-1347) is exchanged for one encoding an arginine (CGT, CGC, CGA, CGG, AGA, AGG).
  • the K449R variant has the same function as wild-type AAV9.
  • the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 2.
  • the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 3.
  • the parent AAV capsid sequence is any of those shown in Table 1. Table 1.
  • AAV Capsid Sequences [0054] Each of the patents, applications and or publications listed in Table 1 are hereby incorporated by reference in their entirety. [0055] The parent AAV serotype and associated capsid sequence may be any of those known in the art.
  • Non-limiting examples of such AAV serotypes include, AAV9, AAV9 K449R (or K449R AAV9), AAV1, AAVrh10, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1- 35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.
  • the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887- 5911 (2008), US Publication US20140359799 and US Patent No.7,588,772, each of which is herein incorporated by reference in its entirety).
  • the amino acid sequence of AAVDJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD).
  • HBD heparin binding domain
  • the AAV-DJ sequence is as described by SEQ ID NO: 1 in U.S.
  • Patent No.7,588,772 the contents of which are herein incorporated by reference in their entirety, and the AAVDJ8 sequence may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • the AAVDJ8 sequence may comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • the parent AAV capsid sequence comprises an AAV9 sequence.
  • the parent AAV capsid sequence comprises an K449R AAV9 sequence.
  • the parent AAV capsid sequence comprises an AAVDJ sequence. [0060] In some embodiments, the parent AAV capsid sequence comprises an AAVDJ8 sequence. [0061] In some embodiments, the parent AAV capsid sequence comprises an AAVrh10 sequence. [0062] In some embodiments, the parent AAV capsid sequence comprises an AAV1 sequence. [0063] In some embodiments, the parent AAV capsid sequence comprises an AAV5 sequence. [0064] While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region.
  • a parent AAV capsid sequence comprises a VP1, VP2 and/or VP3 region, or any combination thereof.
  • a parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.
  • the parent AAV capsid sequence may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above.
  • the parent AAV capsid sequence may be encoded by a nucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of those described above.
  • the parent sequence is not an AAV capsid sequence and is instead a different vector (e.g., lentivirus, plasmid, etc).
  • the parent sequence is a delivery vehicle (e.g., a nanoparticle) and the targeting peptide is attached thereto.
  • Capsid Engineering [0068] Recombinant or engineered AAV vectors have shown promise for use in therapy for the treatment of human disease. However, a need still exists for AAV particles with more specific and/or enhanced tropism for target tissues. Capsid engineering methods have been used to try to identify capsids with enhanced transduction of target tissues (e.g., brain, spinal cord, DRG).
  • Rational engineering and mutational methods have been used to direct AAV to a target tissue.
  • structure-function relationships are used to determine regions in which changes to the capsid sequence may be made.
  • surface loop structures, receptor binding sites, and/or heparin binding sites may be mutated, or otherwise altered, for rational design of recombinant AAV capsids for enhanced targeting to a target tissue.
  • Rational design also encompasses the addition of targeting peptides to a parent AAV capsid sequence, wherein the targeting peptide may have an affinity for a receptor of interest within a target tissue.
  • AAV capsids and/or targeting peptides having enhanced transduction of a target tissue e.g., CNS or PNS.
  • Capsid shuffling, and/or chimeras describe a method in which fragments of at least two parent AAV capsids are combined to generate a new recombinant capsid protein. The number of parent AAV capsids used may be 2-20, or more than 20.
  • capsid shuffling is used to identify AAV capsids and/or targeting peptides having enhanced transduction of a target tissue (e.g., CNS or PNS).
  • Directed evolution involves the generation of AAV capsid libraries ( ⁇ 10 4 -10 8 ) by any of a variety of mutagenesis techniques and selection of lead candidates based on response to selective pressure by properties of interest (e.g., tropism). Directed evolution of AAV capsids allows for positive selection from a pool of diverse mutants without necessitating extensive prior characterization of the mutant library. Directed evolution libraries may be generated by any molecular biology technique known in the art, and may include, DNA shuffling, random point mutagenesis, insertional mutagenesis (e.g., targeting peptides), random peptide insertions, or ancestral reconstructions.
  • AAV capsid libraries may be subjected to more than one round of selection using directed evolution for further optimization.
  • Directed evolution methods are most commonly used to identify AAV capsid proteins with enhanced transduction of a target tissue. Capsids with enhanced transduction of a target tissue have been identified for targeting human airway epithelium, neural stem cells, human pluripotent stem cells, retinal cells, and other in vitro and in vivo cells.
  • directed evolution methods are used to identify AAV capsids and/or targeting peptides having enhanced transduction of a target tissue (e.g., CNS or PNS).
  • AAV Barcode-Seq (Adachi K et al, Nature Communications 5:3075 (2014), the contents of which are herein incorporated by reference in their entirety).
  • NGS next-generation sequence
  • AAV libraries are created comprising DNA barcode tags, which can be assessed by multi-plexed Illumina barcode sequencing. Barcode design confers the ability to detect AAV presence and expression via DNA (biodistribution) and RNA (transduction) barcodes, respectively.
  • AAV capsid libraries were generated, wherein each mutant carried a wild-type AAV2 rep gene and an AAV cap gene derived from a series of variants or mutants, and a pair of left and right 12-nucleotide long DNA bar-codes downstream of an AAV2 polyadenylation signal (pA). In this manner, 7 different DNA barcode AAV capsid libraries were generated. Capsid libraries were then provided to mice.
  • samples were collected, DNA extracted and PCR-amplified using AAV-clone specific virus bar codes and sample-specific bar code attached PCR primers. All the virus barcode PCR amplicons were Illumina sequenced and converted to raw sequence read number data by a computational algorithm.
  • the core of the Barcode-Seq approach is a 96-nucleotide cassette comprising the two DNA bar-codes (left and right) described above, three PCR primer binding sites and two restriction enzyme sites.
  • an AAV rep-cap genome was used, but the system can be applied to any AAV viral genome, including one devoid of rep and cap genes.
  • the advantage of the Barcode Seq method is the collection of a large data set and correlation to desirable phenotype with few replicates and in a short period of time.
  • the DNA Barcode Seq method can be similarly applied to RNA.
  • the Barcode Seq method is used to identify AAV capsids and/or targeting peptides having enhanced transduction of a target tissue (e.g., CNS or PNS).
  • barcoded AAV capsid libraries are generated, wherein a representative viral genome comprises a 5’ and 3’ ITR, AAV rep and cap sequences, regulatory sequences (e.g., polyadenylation, termination or promoter sequences) and one viral barcode (VBC) sequence.
  • VBC viral barcode
  • a representative viral genome in a barcoded AAV capsid library when read in the 5’ to 3’ direction, comprises an ITR region, an AAV rep sequence, an AAV cap sequence, a polyadenylation sequence, an H1 alternative promoter, a viral barcode (VBC) sequence, a termination signal and an ITR region.
  • a viral genome of a barcoded library may comprise more than one barcode sequence.
  • a viral genome of a barcoded library may comprise six barcode sequences.
  • the viral barcode (VBC) sequence is 5-100 nucleotides in length.
  • the VBC sequence may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides.
  • the VBC sequence is about 50 nucleotides in length. In some embodiments, the VBC sequence is 50 nucleotides in length.
  • Targeting peptides Disclosed herein are targeting peptides and associated AAV particles comprising a capsid protein with one or more targeting peptide inserts, for enhanced or improved transduction of a target tissue (e.g., cells of the CNS or PNS).
  • a target tissue e.g., cells of the CNS or PNS.
  • the targeting peptide may direct an AAV particle to a cell or tissue of the CNS.
  • the cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or supporting cells of the brain such as immune cells (e.g., T cells).
  • neurons e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc
  • glial cells e.g., microglia, astrocytes, oligodendrocytes
  • immune cells e.g., T cells
  • the tissue of the CNS may be, but is not limited to, the cortex (e.g, frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei.
  • the targeting peptide may direct an AAV particle to a cell or tissue of the PNS.
  • the cell or tissue of the PNS may be, but is not limited to, a dorsal root ganglion (DRG).
  • DRG dorsal root ganglion
  • the targeting peptide may direct an AAV particle to the CNS (e.g., the cortex) after intravenous administration.
  • the targeting peptide may direct and AAV particle to the PNS (e.g., DRG) after intravenous administration.
  • a targeting peptide may vary in length. In some embodiments, the targeting peptide is 3-20 amino acids in length. As non-limiting examples, the targeting peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length.
  • Targeting peptides as described herein may be identified and/or designed by any method known in the art.
  • the CREATE system as described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and in International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are herein incorporated by reference in their entirety, may be used as a means of identifying targeting peptides, in either mice or other research animals, such as, but not limited to, non-human primates.
  • Targeting peptides and associated AAV particles may be identified from libraries of AAV capsids comprised of targeting peptide variants.
  • the targeting peptides may be 7 amino acid sequences (7-mers).
  • the targeting peptides may be 9 amino acid sequences (9-mers).
  • the targeting peptides may also differ in their method of creation or design, with non-limiting examples including, random peptide selection, site saturation mutagenesis, and/or optimization of a particular region of the peptide (e.g., flanking regions or central core).
  • a targeting peptide library comprises targeting peptides of 7 amino acids (7-mer) in length randomly generated by PCR.
  • a targeting peptide library comprises targeting peptides with 3 mutated amino acids. In some embodiments, these 3 mutated amino acids are consecutive amino acids. In another embodiment, these 3 mutated amino acids are not consecutive amino acids.
  • the parent targeting peptide is a 7-mer.
  • a targeting peptide library comprises 7-mer targeting peptides, wherein the amino acids of the targeting peptide and/or the flanking sequences are evolved through site saturation mutagenesis of 3 consecutive amino acids.
  • the AAV particles as described herein are prepared via the CREATE system, as described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and in International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are herein incorporated by reference in their entirety.
  • CREATE or “Cre- recombinant-based AAV targeted evolution” refers to an AAV capsid selection strategy that selects for capsids that transduce target tissues (e.g, CNS or PNS) following intravenous injection.
  • AAV capsids with one or more targeting peptide inserts are developed and administered intravenously to transgenic mice. These transgenic cre-expressing mice may be developed for specific targeting, for example, GFAP-Cre mice may be used for targeting to astrocytes.
  • Variation of the targeting sequence as well as the transgenic animal model enables the selection of AAV variants with desired transduction profiles, for example, tropism to neurons or astrocytes, as compared to other AAV serotypes, including the parent AAV particle and capsid.
  • the CREATE method involves the generation of a library of targeting peptides which are then assembled into a viral genome backbone comprising a parent AAV capsid sequence.
  • An AAV capsid library (AAV particles) is then generated, purified and administered to a transgenic animal (e.g., mouse).
  • Target tissue is collected and AAV sequences selectively recovered from Cre expressing cells. These sequences are assessed and characterized for the identification of targeting peptides that lead to enrichment in a target tissue (i.e., enhanced transduction or tropism).
  • Targeting peptides and associated AAV particles can then be generated for further testing and characterization. This process is considered one round of evolution or selection. In some embodiments, more than one round of evolution is conducted.
  • the CREATE system uses a rAAV-Cap-in-cis-lox viral genome comprising AAV cap and regulator elements of the AAV rep genes and a Cre-invertible switch. Since this viral genome lacks a fully function rep gene necessary for AAV particle production, the rep is provided in trans.
  • a modified AAV2/9 Rep-Cap plasmid may be provided, wherein stop-codons are provided in-frame to prevent the expression of VP1-VP3 proteins.
  • Capsid libraries are generated using the rAAV-Cap-in-cis-lox viral genome as a backbone.
  • Targeting peptides are inserted into the parent AAV capsid protein (e.g., AAV9) at any position that results in the generation of a fully functional AAV capsid protein and AAV particle.
  • AAV capsid protein e.g., AAV9
  • targeting peptides comprising 7 random amino acids were inserted between amino acids 588 and 589 of the VP1 sequence of K449 AAV9. Regions flanking the targeting peptide insert may or may not be stay the same as the parent capsid sequence.
  • Targeting peptides may be designed by any method known in the art. In some embodiments, targeting peptides are generated using polymerase chain reaction (PCR).
  • AAV particles comprising capsid proteins with targeting peptide inserts are generated and viral genomes encoding a reporter (e.g., GFP) encapsulated within. These AAV particles (or AAV capsid library) are then administered to a transgenic mouse by intravenous delivery to the tail vein. Administration of these capsid libraries to cre- expressing mice results in expression of the reporter payload in the target tissue, due to the expression of Cre. [0101] AAV particles and/or viral genomes may be recovered from the target tissue for identification of targeting peptides and associated AAV particles that are enriched, indicating enhanced transduction of target tissue.
  • a reporter e.g., GFP
  • a target tissue may be any cell, tissue or organ of a subject.
  • samples may be collected from brain, spinal cord, dorsal root ganglia and associated roots, liver, heart, gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal glands, stomach, sciatic nerve, saphenous nerve, thyroid gland, eyes (with or without optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, skin of the leg, superior cervical ganglia, urinary bladder, ovaries, uterus, prostate gland, testes, and/or any sites identified as having a lesion, or being of interest.
  • Targeting peptides and associated AAV capsid proteins and AAV particles identified using a CREATE system include AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B- SNP, AAVPHP.B-QGT, AAVP
  • the targeting peptides identified using a CREATE system are described in International Patent Application WO2015038958, the contents of which are herein incorporated by reference in their entirety, such as, but not limited to PHP.B (SEQ ID NO: 1 of WO2015038958), PHP.B2 (SEQ ID NO: 28 of WO2015038958), PHP.B3 (SEQ ID NO: 29 of WO2015038958), or PHP.A (SEQ ID NO: 60 of WO2015038958).
  • the targeting peptides identified using a CREATE system are described in International Patent Application WO2017100671, the contents of which are herein incorporated by reference in their entirety, such as, but not limited to PHP.N (SEQ ID NO: 4 of WO2017100671), PHP.B (SEQ ID NO: 1 of WO2017100671), PHP.B2 (SEQ ID NO: 2 of WO2017100671), PHP.B3 (SEQ ID NO: 3 of WO2017100671), G2B4/G2B-A7 (SEQ ID NO: 43 of WO2017100671), G2B5/G2B5-G9 (SEQ ID NO: 44 of WO2017100671), PHP.B-EST (SEQ ID NO: 5 of WO2017100671), PHP.B-GGT (SEQ ID NO: 6 of WO2017100671), PHP.B-ATP (SEQ ID NO: 7 of WO2017100671), PHP.B-ATT-T (SEQ ID NO: 4 of WO2017
  • CREATE in mice is used to identify AAV capsids and/or targeting peptides having enhanced transduction of a target tissue (e.g., CNS or PNS).
  • a method may be used as described in Challis et al (Nature Protocols 14(2):379-414 (2019), the contents of which are herein incorporated by reference in their entirety).
  • CREATE system in NHP [0108] The CREATE system has proven efficacious in identifying targeting peptides for enhanced transduction to the CNS of mice after intravenous administration. However, translation of findings from mouse to human is not always straightforward.
  • Modifying the CREATE system for non-transgenic animals or model systems that more closely resemble humans may help identify targeting peptides and associated AAV capsids and particles useful for the treatment of human disease.
  • AAV Cre-vectors may be used to transduce cells and induce subsequent Cre expression.
  • these AAV Cre-vectors may be AAV1-Cre and/or AAV- Syn-Cre vectors.
  • These AAV Cre-vectors may comprise viral genomes with a cell-type specific promoter.
  • these cell-type specific promotors may be, but are not limited to, CAG, UBC, EF1 ⁇ , synapsin, GFAP, MBP, VGLUT, VGAT, Nav1.8, parvalbumin, TH, ChaT, and/or any promoter known in the art.
  • these AAV-Cre vectors are delivered to a target tissue by intracranial infusion.
  • these AAV-Cre vectors are delivered to a target tissue by intraparenchymal administration.
  • the intraparenchymal administration is directly to the putamen of the subject.
  • the intraparenchymal administration is directly to the thalamus of a subject.
  • the intraparenchymal administration is directly to the cortex of a subject. In some embodiments, the intraparenchymal administration is indirectly to the cortex of a subject. In some embodiments, the intraparenchymal administration is simultaneously to one or more of the putamen, the thalamus and or the cortex of a subject, and may be bi-lateral administrations. [0112] In some embodiments, the subject is a non-human primate. [0113] As for the CREATE method developed in mice, the AAV capsid libraries may be administered intravenously. In another embodiment, the AAV capsid libraries may be administered by intraparenchymal delivery. In some embodiments, the AAV capsid library is administered prior to the delivery of the AAV-Cre vectors.
  • the AAV capsid library is administered after the delivery of the AAV-Cre vectors.
  • the length of time between the administration of the AAV-Cre vectors and the AAV capsid libraries may be seconds, minutes, hours, days, weeks, or years.
  • the AAV capsid library may comprise AAV particles comprising a viral genome encoding a reporter (e.g., GFP). Only those cells of the target tissue (e.g., CNS or DRG) also expressing Cre (co-transduced by a Cre-vector administered intraparenchymally) will express the reporter.
  • Target tissues may be collected and analyzed for the identification of AAV particles and targeting peptides that lead to enrichment in the target tissue, i.e., enhanced transduction. Standard methods in the art may be used to assess, analyze, or characterize sample tissues and AAV sequences, including but not limited to, next generation sequencing, viral genome quantification, biochemical assays, immunohistochemistry and/or imaging.
  • CREATE in NHP is used to identify AAV capsids and/or targeting peptides having enhanced transduction of a target tissue (e.g., CNS or PNS).
  • Targeting peptide sequences [0117]
  • the targeting peptide may comprise a sequence as set forth in SEQ ID NO: 4-221.
  • targeting peptides may be encoded by a sequence as set forth in SEQ ID NO: 222-658. These sequences are shown in Table 2.
  • the targeting peptides may be inserted in a parent capsid sequence. Exemplary full length capsid sequences comprising the targeting sequence(s) are provided in Table 3. [0118]
  • the targeting nucleic acid sequences may undergo further mutagenesis. In certain embodiments, this further mutagenesis enhances the tropism of the capsid for a target tissue (e.g., the CNS).
  • the targeting peptide may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 2.
  • the targeting peptide may be encoded by a nucleic acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 2.
  • the full length capsid sequence comprising a targeting peptide may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 3.
  • the full length capsid sequence comprising a sequence encoding a targeting peptide may comprise a nucleic acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 3.
  • a targeting peptide may comprise 4 or more contiguous amino acids of any of the targeting peptides disclosed herein. In some embodiments the targeting peptide may comprise 4 contiguous amino acids of any of the sequences as set forth in SEQ ID NO: 4-221. In some embodiments the targeting peptide may comprise 5 contiguous amino acids of any of the sequences as set forth in SEQ ID NO: 4-221. In some embodiments the targeting peptide may comprise 6 contiguous amino acids of any of the sequences as set forth in SEQ ID NO: 4-221.
  • the AAV particle comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence as set forth in any of SEQ ID NO: 4-221.
  • the AAV particle comprises an AAV capsid polynucleotide with a targeting nucleic acid insert, wherein the targeting nucleic acid insert has a nucleotide sequence as set forth in any of SEQ ID NO: 222-658.
  • the AAV particle comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence comprising at least 4 contiguous amino acids of any of the sequences as set forth in any of SEQ ID NO: 4-221.
  • the AAV particle comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence substantially comprising any of the sequences as set forth in any of SEQ ID NO: 4-221.
  • the AAV particle comprises an AAV capsid polynucleotide with a targeting nucleic acid insert, wherein the targeting nucleic acid insert has a nucleotide sequence substantially comprising any of those set forth as SEQ ID NO: 222-658.
  • the AAV particle comprising a targeting nucleic acid insert may have a polynucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence.
  • the AAV particle comprising a targeting peptide insert may have an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence. Table 2. Targeting peptides (7-mers)
  • Exemplary full length capsid sequences [0131]
  • the single letter symbol has the following description: A for adenine; C for cytosine; G for guanine; T for thymine; U for Uracil; W for weak bases such as adenine or thymine; S for strong nucleotides such as cytosine and guanine; M for amino nucleotides such as adenine and cytosine; K for keto nucleotides such as guanine and thymine; R for purines adenine and guanine; Y for pyrimidine cytosine and thymine; B for any base that is not A (e.g., cytosine, guanine, and thymine); D for any base that is not C (e.g., adenine, guanine, and thymine); H for any base that is not
  • G (Gly) for Glycine A (Ala) for Alanine; L (Leu) for Leucine; M (Met) for Methionine; F (Phe) for Phenylalanine; W (Trp) for Tryptophan; K (Lys) for Lysine; Q (Gln) for Glutamine; E (Glu) for Glutamic Acid; S (Ser) for Serine; P (Pro) for Proline; V (Val) for Valine; I (Ile) for Isoleucine; C (Cys) for Cysteine; Y (Tyr) for Tyrosine; H (His) for Histidine; R (Arg) for Arginine; N (Asn) for Asparagine; D (Asp) for Aspartic Acid; T (Thr) for Threonine; B (Asx) for Aspartic acid or Asparag
  • Targeting peptides may be stand-alone peptides or may be inserted into or conjugated to a parent sequence. In some embodiments, the targeting peptides are inserted into the capsid protein of an AAV particle. [0134] One or more targeting peptides may be inserted into a parent AAV capsid sequence to generate the AAV particles described herein. [0135] Targeting peptides may be inserted into a parent AAV capsid sequence in any location that results in fully functional AAV particles. The targeting peptide may be inserted in VP1, VP2 and/or VP3.
  • the targeting peptides are inserted in a hypervariable region of the AAV capsid sequence.
  • hypervariable regions include Loop IV and Loop VIII of the parent AAV capsid. While not wishing to be bound by theory, these surface exposed loops are unstructured and poorly conserved, making them ideal regions for insertion of targeting peptides.
  • the targeting peptide is inserted into Loop IV.
  • the targeting peptide is used to replace a portion, or all of Loop IV.
  • addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.
  • the targeting peptide is inserted into Loop VIII.
  • the targeting peptide is used to replace a portion, or all of Loop VIII.
  • addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.
  • more than one targeting peptide is inserted into a parent AAV capsid sequence.
  • targeting peptides may be inserted at both Loop IV and Loop VIII in the same parent AAV capsid sequence.
  • Targeting peptides may be inserted at any amino acid position of the parent AAV capsid sequence, such as, but not limited to, between amino acids at positions 586-592, 588- 589, 586-589, 452-458, 262-269, 464-473, 491-495, 546-557 and/or 659-668.
  • the targeting peptides are inserted into a parent AAV capsid sequence between amino acids at positions 588 and 589 (Loop VIII).
  • the parent AAV capsid is AAV9 (SEQ ID NO: 2).
  • the parent AAV capsid is K449R AAV9 (SEQ ID NO: 3).
  • the targeting peptides described herein may increase the transduction of the AAV particles described herein to a target tissue as compared to the parent AAV particle lacking a targeting peptide insert.
  • the targeting peptide increases the transduction of an AAV particle to a target tissue by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.
  • the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the CNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.
  • the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the PNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.
  • the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the DRG by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.
  • the initiation codon for translation of the AAV VP1 capsid protein may be CTG, TTG, or GTG as described in US Patent No. US8163543, the contents of which are herein incorporated by reference in its entirety.
  • the present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e. capsid) of a viral vector such as AAV.
  • VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Met1), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence.
  • a first-methionine (Met1) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases.
  • This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.).
  • Met- clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.
  • Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Met1/AA1 amino acid (Met+/AA+) and some of which may lack a Met1/AA1 amino acid as a result of Met/AA-clipping (Met-/AA-).
  • Met/AA-clipping in capsid proteins see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods.2017 Oct.28(5):255-267; Hwang, et al.
  • references to capsid proteins is not limited to either clipped (Met-/AA-) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure.
  • a direct reference to a “capsid protein” or “capsid polypeptide” may also comprise VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA-clipping (Met-/AA-).
  • a reference to a specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Met1/AA1 amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Met1/AA1).
  • VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Met1” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Met1” amino acid (Met-) of the 736 amino acid Met+ sequence.
  • VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.
  • references to viral capsids formed from VP capsid proteins can incorporate VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA1-clipping (Met-/AA1-), and combinations thereof (Met+/AA1+ and Met-/AA1-).
  • an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met-/AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met- /AA1-).
  • An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met-/AA1-).
  • Viral Genome of the AAV particle AAV particles as described herein, comprising targeting peptides, may be used for the delivery of any viral genome to a target tissue (e.g., CNS and/or DRG).
  • the viral genome may encode any payload, such as but not limited to a polypeptide, an antibody, an enzyme, an RNAi agent and/or components of a gene editing system.
  • a viral genome of an AAV particle as described herein comprises a nucleic acid sequence with at least one payload region encoding a payload, and at least one ITR.
  • a viral genome typically comprises two ITR sequences, one at each of the 5’ and 3’ ends.
  • a viral genome of the AAV particles described herein may comprise nucleic acid sequences for additional components, such as, but not limited to, a regulatory element (e.g., promoter), untranslated regions (UTR), a polyadenylation sequence (polyA), a filler or stuffer sequence, an intron, and/or a linker sequence for enhanced expression.
  • a regulatory element e.g., promoter
  • UTR untranslated regions
  • polyA polyadenylation sequence
  • filler or stuffer sequence e.g., an intron
  • linker sequence for enhanced expression.
  • These viral genome components can be selected and/or engineered to further tailor the specificity and efficiency of expression of a given payload in a target tissue (e.g., CNS or DRG).
  • Viral Genome Component Inverted Terminal Repeats (ITRs)
  • the AAV particles described herein comprise a viral genome with at least one ITR and a payload region.
  • the viral genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends.
  • the ITRs function as origins of replication comprising recognition sites for replication.
  • ITRs comprise sequence regions which can be complementary and symmetrically arranged.
  • ITRs incorporated into viral genomes as described herein may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.
  • the ITRs may be derived from the same serotype as the capsid, selected from any of the known serotypes, or a derivative thereof.
  • the ITR may be of a different serotype than the capsid.
  • the AAV particle has more than one ITR.
  • the AAV particle has a viral genome comprising two ITRs.
  • the ITRs are of the same serotype as one another.
  • the ITRs are of different serotypes.
  • Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid.
  • both ITRs of the viral genome of the AAV particle are AAV2 ITRs.
  • each ITR may be about 100 to about 150 nucleotides in length.
  • An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length.
  • the ITRs are 140-142 nucleotides in length.
  • Non-limiting examples of ITR length are 102, 105, 130, 140, 141, 142, 145 nucleotides in length.
  • ITRs encompassed by the present disclosure include those with at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to a known AAV serotype ITR sequence.
  • Viral Genome Component Promoters [0160]
  • the payload region of the viral genome comprises at least one element to enhance the payload target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety).
  • Non-limiting examples of elements to enhance payload target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.
  • PREs post-transcriptional regulatory elements
  • PolyA polyadenylation
  • USEs upstream enhancers
  • CMV enhancers and introns CMV enhancers and introns.
  • a person skilled in the art may recognize that expression of a payload in a target cell may require a specific promoter, including but not limited to, a promoter that is species specific, inducible, tissue-specific, or cell cycle-specific (Parr et al., Nat. Med.3:1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).
  • the promoter is deemed to be efficient when it drives expression of the payload encoded by the viral genome of the AAV particle. [0163] In some embodiments, the promoter is a promoter deemed to be efficient when it drives expression in a cell being targeted. [0164] In some embodiments, the promoter is a promoter having a tropism for a cell being targeted. [0165] In some embodiments, the promoter drives expression of the payload for a period of time in targeted tissues.
  • Expression driven by a promoter may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years,
  • the promoter is a selected for sustained expression of a payload in tissues and/or cells of the central or peripheral nervous system.
  • Promoters may be naturally occurring or non-naturally occurring.
  • Non-limiting examples of promoters include those derived from viruses, plants, mammals, or humans. In some embodiments, the promoters may be those derived from human cells or systems.
  • the promoter may be truncated or mutated.
  • Promoters which drive or promote expression in most tissues include, but are not limited to, the human elongation factor 1 ⁇ -subunit (EF1 ⁇ ) promoter, the cytomegalovirus (CMV) immediate-early enhancer and/or promoter, the chicken ⁇ -actin (CBA) promoter and its derivative CAG, ⁇ glucuronidase (GUSB) promoter, or ubiquitin C (UBC) promoter.
  • EF1 ⁇ human elongation factor 1 ⁇ -subunit
  • CMV cytomegalovirus
  • CBA chicken ⁇ -actin
  • GUSB ⁇ glucuronidase
  • UBC ubiquitin C
  • Tissue-specific promoters can be used to restrict expression to certain cell types such as, but not limited to, cells of the central or peripheral nervous systems, targeted regions within (e.g., frontal cortex), and/or sub-sets of cells therein (e.g., excitatory neurons).
  • cell-type specific promoters may be used to restrict expression of a payload to excitatory neurons (e.g., glutamatergic), inhibitory neurons (e.g., GABA-ergic), neurons of the sympathetic or parasympathetic nervous system, sensory neurons, neurons of the dorsal root ganglia, motor neurons, or supportive cells of the nervous systems such as microglia, astrocytes, oligodendrocytes, and/or Schwann cells.
  • Cell-type specific promoters also exist for other tissues of the body, with non- limiting examples including, liver promoters (e.g., hAAT, TBG), skeletal muscle specific promoters (e.g., desmin, MCK, C512), B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, and/or cardiac or cardiovascular promoters (e.g., ⁇ MHC, cTnT, and CMV-MLC2k) .
  • liver promoters e.g., hAAT, TBG
  • skeletal muscle specific promoters e.g., desmin, MCK, C512
  • B cell promoters e.g., monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue
  • Non-limiting examples of tissue-specific promoters for targeting payload expression to central nervous system tissues and cells include synapsin (Syn), glutamate vesicular transporter (VGLUT), vesicular GABA transporter (VGAT), parvalbumin (PV), sodium channel Na v 1.8, tyrosine hydroxylase (TH), choline acetyltransferase (ChaT), methyl-CpG binding protein 2 (MeCP2), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), neuron-specific enolase (NSE), ⁇ -globin minigene n ⁇ 2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters.
  • Synapsin Sesynapsin
  • VGLUT gluta
  • tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters.
  • GFAP glial fibrillary acidic protein
  • EAAT2 EAAT2 promoters
  • a non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.
  • MBP myelin basic protein
  • the promoter may be less than 1 kb.
  • the promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides.
  • the promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300- 400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800 nucleotides.
  • the promoter may be a combination of two or more components of the same or different starting or parental promoters such as, but not limited to, CMV and CBA.
  • Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides.
  • Each component may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300- 400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800 nucleotides.
  • the promoter is a combination of a 382 nucleotide CMV-enhancer sequence and a 260 nucleotide CBA-promoter sequence.
  • the viral genome comprises a ubiquitous promoter.
  • Non- limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1 ⁇ , PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1- CBX3).
  • Yu et al. (Molecular Pain 2011, 7:63; the contents of which are herein incorporated by reference in their entirety) evaluated the expression of eGFP under the CAG, EFI ⁇ , PGK and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors and found that UBC showed weaker expression than the other 3 promoters and only 10-12% glial expression was seen for all promoters. Soderblom et al. (E.
  • NFL is a 650-nucleotide promoter and NFH is a 920 nucleotide promoter which are both absent in the liver but NFH is abundant in the sensory proprioceptive neurons, brain and spinal cord and NFH is present in the heart.
  • SCN8A Na v 1.6
  • SCN8A is a 470 nucleotide promoter which expresses throughout the DRG, spinal cord and brain with particularly high expression seen in the hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus and hypothalamus (See e.g., Drews et al.
  • the promoter is not cell specific.
  • the promoter is a RNA pol III promoter.
  • the RNA pol III promoter is U6.
  • the RNA pol III promoter is H1.
  • the viral genome comprises an H1 promoter.
  • the viral genome comprises an H1 alternative promoter.
  • the viral genome comprises an enhancer element.
  • the viral genome comprises an engineered promoter.
  • the viral genome comprises a promoter from a naturally expressed protein.
  • Viral Genome Component Untranslated Regions (UTRs) [0181] By definition, wild type untranslated regions (UTRs) of a gene are transcribed but not translated.
  • the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.
  • a 5’ UTR from mRNA normally expressed in the brain e.g., huntingtin
  • wild-type 5 ⁇ untranslated regions include features which play roles in translation initiation.
  • Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G'.
  • R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G'.
  • the 5’UTR in the viral genome includes a Kozak sequence.
  • the 5’UTR in the viral genome does not include a Kozak sequence.
  • AU rich elements can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in its entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions.
  • Class II AREs such as, but not limited to, GM- CSF and TNF-a, possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers.
  • Class III ARES such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif.
  • Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3 ⁇ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • AREs 3 ⁇ UTR AU rich elements
  • the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • the viral genome may include at least one miRNA seed, binding site or full sequence.
  • microRNAs or miRNA or miR are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
  • a microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick sequence complementarity to the miRNA target sequence of the nucleic acid.
  • the viral genome may be engineered to include, alter or remove at least one miRNA binding site, full sequence or seed region.
  • any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location.
  • the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, made with one or more other 5 ⁇ UTRs or 3 ⁇ UTRs known in the art.
  • the term “altered” as it relates to a UTR means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3 ⁇ or 5 ⁇ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • the viral genome of the AAV particle comprises at least one artificial UTR which is not a variant of a wild type UTR.
  • the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • the viral genome of the AAV particles described herein may comprise at least one polyadenylation sequence.
  • the viral genome of the AAV particle comprises a polyadenylation sequence between the 3’ end of the payload encoding region and the 5’ end of the 3’ITR.
  • the polyadenylation sequence or “polyA sequence” may range from absent to about 500 nucleotides in length.
  • the polyadenylation sequence may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • the polyadenylation sequence is 50-100 nucleotides in length. [0197] In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length. [0198] In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length. [0199] In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length. [0200] In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length. [0201] In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length.
  • the polyadenylation sequence is 60-160 nucleotides in length. [0203] In some embodiments, the polyadenylation sequence is 60-200 nucleotides in length. [0204] In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length. [0205] In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length. [0206] In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length. [0207] In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length.
  • the polyadenylation sequence is 80-100 nucleotides in length. [0209] In some embodiments, the polyadenylation sequence is 80-150 nucleotides in length. [0210] In some embodiments, the polyadenylation sequence is 80-160 nucleotides in length. [0211] In some embodiments, the polyadenylation sequence is 80-200 nucleotides in length. [0212] In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length. [0213] In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length.
  • the polyadenylation sequence is 90-160 nucleotides in length.
  • the polyadenylation sequence is 90-200 nucleotides in length.
  • Viral Genome Component Introns
  • the viral genome of the AAV particles as described herein comprises at least one element to enhance the payload target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, Discov. Med, 2015, 19(102): 49-57; the contents of which are herein incorporated by reference in their entirety) such as an intron.
  • Non- limiting examples of introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), ⁇ - globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/immunoglobin splice acceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S) (180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
  • the intron or intron portion may be 100-500 nucleotides in length.
  • the intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nucleotides.
  • the intron may have a length between 80-100, 80- 120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200- 300, 200-400, 200-500, 300-400, 300-500, or 400-500 nucleotides.
  • Viral Genome Component Stuffer sequences [0218]
  • the viral genome of the AAV particles described herein comprises at least one element to improve packaging efficiency and expression, such as a stuffer or filler sequence.
  • stuffer sequences include albumin and/or alpha-1 antitrypsin. Any known viral, mammalian, or plant sequence may be manipulated for use as a stuffer sequence.
  • the stuffer or filler sequence may be from about 100-3500 nucleotides in length.
  • the stuffer sequence may have a length of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 nucleotides.
  • Viral Genome Component miRNA
  • the viral genome comprises at least one sequence encoding a miRNA to reduce the expression of the payload in an “off-target” tissue.
  • off-target indicates a tissue or cell-type unintentionally targeted by the AAV particles described herein.
  • an “off-target” tissue or cell when targeting the DRG may be neurons of other ganglia, such as those of the sympathetic or parasympathetic nervous system.
  • miRNAs and their targeted tissues are well known in the art.
  • a miR-122 miRNA may be encoded in the viral genome to reduce the expression of the viral genome in the liver.
  • Viral Genome Component Selectable marker [0221]
  • the viral genome of the AAV particles described herein optionally encodes a selectable marker.
  • the selectable marker may comprise a cell-surface marker, such as any protein expressed on the surface of the cell including, but not limited to receptors, CD markers, lectins, integrins, or truncated versions thereof.
  • selectable marker reporter genes are described in International Publication Nos. WO 1996023810 and WO 1996030540; Heim et al., Current Biology 2:178-182 (1996); Heim et al., Proc. Natl. Acad. Sci. USA (1995); or Heim et al., Science 373:663-664 (1995), the contents of each of which are incorporated herein by reference in their entirety.
  • the viral genome comprises a reporter sequence.
  • the viral genome may comprise a sequence encoding a fluorescent reporter sequence.
  • the fluorescent reporter may be a green fluorescent protein (GFP) or enhanced GFP (eGFP).
  • the viral genome may comprise a CAG-NLSeGFP-wpre sequence.
  • the CAG-NLSeGFP viral genome read in 5’ to 3’ order, comprises a 5’ AAV2 inverted terminal repeat (ITR), a CAG promoter, an intron, a nuclear localization signal (NLS), a sequence encoding eGFP, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a polyadenylation sequence (PolyA) and an AAV23’ ITR.
  • the viral genome may comprise SEQ ID NO: 661. Genome Size
  • the AAV particles described herein may comprise a single- stranded or double-stranded viral genome.
  • the size of the viral genome may be small, medium, large or the maximum size.
  • the viral genome may comprise a promoter and a polyA tail.
  • the viral genome may be a small single stranded viral genome.
  • a small single stranded viral genome may be 2.1 to 3.5 kb in size such as, but not limited to, about 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size.
  • the viral genome may be a small double stranded viral genome.
  • a small double stranded viral genome may be 1.3 to 1.7 kb in size such as, but not limited to, about 1.3, 1.4, 1.5, 1.6, and 1.7 kb in size.
  • the viral genome may be a medium single stranded viral genome.
  • a medium single stranded viral genome may be 3.6 to 4.3 kb in size such as, but not limited to, about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size.
  • the viral genome may be a medium double stranded viral genome.
  • a medium double stranded viral genome may be 1.8 to 2.1 kb in size such as, but not limited to, about 1.8, 1.9, 2.0, and 2.1 kb in size.
  • the viral genome may be a large single stranded viral genome.
  • a large single stranded viral genome may be 4.4 to 6.0 kb in size such as, but not limited to, about 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb in size.
  • the viral genome may be a large double stranded viral genome.
  • a large double stranded viral genome may be 2.2 to 3.0 kb in size such as, but not limited to, about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size.
  • Payloads [0232]
  • the AAV particles of the present disclosure comprise a viral genome with at least one payload region.
  • a “payload region” is any nucleic acid sequence (e.g., within the viral genome) which encodes one or more “payloads” described herein.
  • a payload region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is an RNAi agent or a polypeptide.
  • Payloads of the present disclosure may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc.
  • the payload region may comprise a combination of coding and non-coding nucleic acid sequences.
  • the payload region may encode a coding or non-coding RNA.
  • the AAV particle comprises a viral genome with a payload region encoding more than one payload of interest. In such an embodiment, a viral genome encoding more than one payload may be replicated and packaged into a viral particle. A target cell transduced with a viral particle comprising more than one payload may express each of the payloads in a single cell.
  • the polypeptide may be a peptide or protein.
  • the payload region may encode at least one allele of apolipoprotein E (APOE) such as, but not limited to ApoE2, ApoE3 and/or ApoE4.
  • APOE apolipoprotein E
  • the payload region encodes ApoE2 (cys112, cys158).
  • the payload region encodes ApoE3 (cys112, arg158).
  • the payload region may encode an antibody, or a fragment thereof.
  • the payload region may encode human survival of motor neuron (SMN) 1 or SMN2, or fragments or variants thereof.
  • the payload region may encode APOE (APOE2, APOE3, APOE4), or fragments or variants thereof.
  • the payload region may encode glucocerebrosidase (GBA1), or a fragment or variant thereof.
  • the payload region may encode granulin precursor or progranulin (GRN), or a fragment or variant thereof.
  • the payload region may encode aspartoacylase (ASPA), or a fragment or variant thereof.
  • the payload region may encode arylsulfatase A (ARSA).
  • the payload region may encode tripeptidyl peptidase I (CLN2), or a fragment or variant thereof.
  • the payload region may encode beta-galactosidase (GLB1), or a fragment or variant thereof.
  • the payload region may encode N-sulphoglucosamine sulphohydrolase (SGSH), or a fragment or variant thereof.
  • the payload region may encode N-acetyl-alpha-glucosaminidase (NAGLU), or a fragment or variant thereof.
  • the payload region may encode iduronate 2-sulfatase (IDS), or a fragment or variant thereof.
  • the payload region may encode Intracellular cholesterol transporter (NPC1), or a fragment or variant thereof.
  • the payload region may encode gigaxonin (GAN), or a fragment or variant thereof.
  • the AAV viral genomes encoding polypeptides described herein may be useful in the fields of human disease, viruses, infections veterinary applications and a variety of in vivo and in vitro settings.
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long.
  • polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked.
  • the term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
  • Antibodies may be an antibody, a fragment, or any derivative thereof, which may contribute to the formation of a “functional antibody”, exhibiting the desired biological activity.
  • an antibody may be a native antibody (e.g., with two heavy and two light chains), a heavy chain variable region, a light chain variable region, a heavy chain constant region, a light chain constant region, Fab, Fab', F(ab')2, Fv, or scFv fragments, a diabody, a linear antibody, a single-chain antibody, a multi-specific antibody, an intrabody, one or more heavy chain complementarity determining regions (CDR), one or more light chain CDRs, a bi-specific antibody, a monoclonal antibody, a polyclonal antibody, a humanized antibody, an antibody mimetic, an antibody variant, a miniaturized antibody, a unibody, a maxibody, and/or a chimeric antigen receptor.
  • a native antibody e.g., with two heavy and two light chains
  • a heavy chain variable region e.g., with two heavy and two light chains
  • a heavy chain variable region e.g., with
  • antibody-based or “antibody-derived” compositions are monomeric or multi-meric polypeptides which comprise at least one amino-acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence, e.g., mammalian protein.
  • Payload regions may encode polypeptides that form or function as any antibody, including antibodies that are known in the art and/or antibodies that are commercially available.
  • the encoded antibodies may be therapeutic, diagnostic, or for research purposes.
  • the encoded antibodies may be useful in the treatment of neurological disease or any disorders associated with the central and/or peripheral nervous systems.
  • the viral genome of the AAV particle may comprise nucleic acids which have been engineered to enable or enhance the expression of antibodies, antibody fragments, or components thereof.
  • Antibodies encoded in payload regions of the AAV particles described herein may be, but are not limited to, antibodies targeting ⁇ -amyloid, APOE, tau, SOD1, TDP-43, huntingtin, and/or synuclein.
  • RNAi agents [0244] RNAi (also known as post-transcriptional gene silencing (PTGS), quelling, or co- suppression) is a post-transcriptional gene silencing process in which RNA molecules, in a sequence specific manner, inhibit gene expression, typically by causing the destruction of specific mRNA molecules.
  • RNAi mediated gene silencing can specifically inhibit targeted gene expression.
  • the payload region of the viral genome of the AAV particles described herein encodes an RNAi agent
  • the RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA.
  • Non-limiting examples of a target gene of an RNAi agent include, SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A- SCN5A, or SCN8A-SCN11A.
  • the AAV particles described herein may comprise viral genomes encoding RNAi agents, wherein the RNAi agent targets the mRNA of a gene of interest to interfere with gene expression and/or protein production. Such AAV particles may be used as a therapeutic, a diagnostic, or for research purposes.
  • the RNAi agent may target the gene of interest along any segment of their respective nucleotide sequence.
  • the RNAi agent may target the gene of interest at the location of a single-nucleotide polymorphism (SNP) or variant within the nucleotide sequence.
  • SNP single-nucleotide polymorphism
  • RNAi agent a nucleic acid sequence encoding an RNAi agent, or a single strand of an RNAi agent, is inserted into the viral genome of the AAV particle and introduced into cells, specifically cells in the central nervous system or cells of the DRG.
  • the RNAi agent may be an siRNA duplex, wherein the siRNA duplex contains an antisense strand (guide strand) and a sense strand (passenger strand) hybridized together forming a duplex structure, wherein the antisense strand is complementary to the nucleic acid sequence of the targeted gene, and wherein the sense strand is homologous to the nucleic acid sequence of the targeted gene.
  • each strand of an siRNA duplex targeting a gene of interest may be about 19 to 25, 19 to 24 or 19 to 21 nucleotides in length, preferably about 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.
  • an siRNA or dsRNA includes at least two sequences that are complementary to each other.
  • the dsRNA includes a sense strand having a first sequence and an antisense strand having a second sequence.
  • the antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding the target gene, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length.
  • the dsRNA is 19 to 25, 19 to 24 or 19 to 21 nucleotides in length.
  • the dsRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the dsRNA is from about 25 to about 30 nucleotides in length. In some embodiments, the dsRNA is about 15 nucleotides in length, 16 nucleotides in length,17 nucleotides in length, 18 nucleotides in length, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, or 30 nucleotides in length.
  • the dsRNA upon contacting with a cell expressing the target protein, inhibits the expression of the protein by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method known in the art.
  • the RNAi agent may be used to reduce the expression of target protein by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the expression of target protein expression may be reduced 50-90%.
  • the RNAi agent may be used to reduce the expression of target mRNA by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%
  • RNAi agent may be used to reduce the expression of target protein and/or mRNA in at least one region of the CNS.
  • the expression of target protein and/or mRNA is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-90%, 70-9
  • the expression of target protein and mRNA in neurons is reduced by 50-90%.
  • the expression of target protein and mRNA in neurons is reduced by 40- 50%.
  • the AAV particle described herein, comprising a viral genome encoding at least one RNAi agent targeting a gene of interest is administered to a subject in need for treating and/or ameliorating a disease, e.g., a neurological disorder of any disease associated with the central or peripheral nervous systems.
  • the RNAi agent is an siRNA.
  • AAV particles described herein may comprise a viral genome encoding one or more siRNA molecules (e.g., siRNA duplexes or encoded dsRNA) that are specifically designed to target a gene of interest and suppress target gene expression and protein production.
  • the siRNA molecules are designed and used to selectively “knock out” target gene variants in cells, i.e., transcripts that are identified in neurological disease.
  • the siRNA molecules are designed and used to selectively “knock down” target gene variants in cells.
  • siRNA molecules targeting a gene of interest may be designed using any available design tools.
  • siRNAs for insertion into a viral genome of the AAV particles described herein. These guidelines generally recommend generating a 19-nucleotide duplexed region, symmetric 2-3 nucleotide 3’overhangs, 5-phosphate and 3-hydroxyl groups targeting a region in the gene to be silenced.
  • siRNA sequence preference include, but are not limited to, (i) A/U at the 5 ⁇ end of the antisense strand; (ii) G/C at the 5 ⁇ end of the sense strand; (iii) at least five A/U residues in the 5 ⁇ terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nucleotides in length.
  • highly effective siRNA molecules essential for suppressing mammalian target gene expression may be readily designed.
  • the sense and/or antisense strand is designed based on the method and rules outlined in European Patent Publication No.
  • the 3’-terminal base of the sequence is adenine, thymine or uracil.
  • the 5’-terminal base of the sequence is guanine or cytosine.
  • the 3’- terminal sequence comprises seven bases rich in one or more bases of adenine, thymine and uracil.
  • an siRNA molecule comprises a sense strand and a complementary antisense strand in which both strands are hybridized together to form a duplex structure.
  • the antisense strand has sufficient complementarity to the target mRNA sequence to direct target-specific RNAi, i.e., the siRNA molecule has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • the antisense strand and target mRNA sequences have 100% complementarity.
  • the antisense strand may be complementary to any part of the target mRNA sequence. Neither the identity of the sense sequence nor the homology of the antisense sequence need be 100% complementary to the target.
  • the antisense strand and target mRNA sequences comprise at least one mismatch.
  • the antisense strand and the target mRNA sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30- 50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40- 80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-
  • the siRNA molecule may have a length from about 10-50 or more nucleotides, i.e., each strand comprising 10-50 nucleotides (or nucleotide analogs).
  • the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region.
  • the siRNA molecule has a length from about 19 to 25, 19 to 24 or 19 to 21 nucleotides.
  • the siRNA molecule can be a synthetic RNA duplex comprising about 19 nucleotides to about 25 nucleotides, and two overhanging nucleotides at the 3'-end.
  • the siRNA molecule may comprise an antisense sequence and a sense sequence, or a fragment or variant thereof.
  • the antisense sequence and the sense sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80-
  • the sense and antisense sequences may be completely complementary across a substantial portion of their length. In other embodiments, the sense sequence and antisense sequence may be at least 70, 80, 90, 95 or 99% complementary across independently at least 50, 60, 70, 80, 85, 90, 95, or 99% of the length of the strands. [0269] In some embodiments, the sense and antisense strands of a siRNA duplex are linked by a short spacer sequence leading to the expression of a stem-loop structure termed short hairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer, thus generating mature siRNA molecules.
  • shRNA short hairpin RNA
  • the siRNA molecules can be encoded by the viral genome of the AAV particles described herein, for delivery to a cell.
  • the siRNA molecules may be encoded in a modulatory polynucleotide which also comprises a molecular scaffold.
  • a “molecular scaffold” is a framework or starting molecule that forms the sequence or structural basis against which to design or make a subsequent molecule.
  • the modulatory polynucleotide which comprises the payload includes a molecular scaffold which comprises at least one 5’ flanking sequence which may be of any length and may be derived in whole or in part from wild type microRNA sequence or be completely artificial.
  • a 3’ flanking sequence may mirror the 5’ flanking sequence in size and origin. Either flanking sequence may be absent. In some embodiments, both the 5’ and 3’ flanking sequences are absent.
  • the 3’ flanking sequence may optionally contain one or more CNNC motifs, where “N” represents any nucleotide.
  • the 5’ and 3’ flanking sequences are the same length.
  • the 5’ flanking sequence is from 1-10 nucleotides in length, from 5-15 nucleotides in length, from 10-30 nucleotides in length, from 20-50 nucleotides in length, greater than 40 nucleotides in length, greater than 50 nucleotides in length, greater than 100 nucleotides in length or greater than 200 nucleotides in length.
  • the 5’ flanking sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • the molecular scaffold comprises at least one 3’ flanking region.
  • the 3’ flanking region may comprise a 3’ flanking sequence which may be of any length and may be derived in whole or in part from wild type microRNA sequence or be a completely artificial sequence.
  • the 3’ flanking sequence is from 1-10 nucleotides in length, from 5-15 nucleotides in length, from 10-30 nucleotides in length, from 20-50 nucleotides in length, greater than 40 nucleotides in length, greater than 50 nucleotides in length, greater than 100 nucleotides in length or greater than 200 nucleotides in length.
  • the 3’ flanking sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • the 5’ and 3’ flanking sequences are the same sequence. In some embodiments they differ by 2%, 3%, 4%, 5%, 10%, 20% or more than 30% when aligned to each other.
  • Forming the stem of a stem loop structure is a minimum of at least one payload sequence.
  • the payload sequence comprises at least one nucleic acid sequence which is in part complementary or will hybridize to the target sequence.
  • the payload is an siRNA molecule or fragment of an siRNA molecule.
  • the 5’ arm of the stem loop comprises a sense sequence.
  • the 3’ arm of the stem loop comprises an antisense sequence.
  • the antisense sequence in some instances, comprises a “G” nucleotide at the 5’ most end.
  • the sense sequence may reside on the 3’ arm while the antisense sequence resides on the 5’ arm of the stem of the stem loop structure.
  • Separating the sense and antisense sequence of the stem loop structure is a loop (also known as a loop motif).
  • the loop may be of any length, between 4-30 nucleotides, between 4-20 nucleotides, between 4-15 nucleotides, between 5-15 nucleotides, between 6-12 nucleotides, 6 nucleotides, 7, nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, and/or 12 nucleotides.
  • the loop comprises at least one UGUG motif. In some embodiments, the UGUG motif is located at the 5’ terminus of the loop.
  • Spacer regions may be present in the modulatory polynucleotide to separate one or more modules from one another. There may be one or more such spacer regions present.
  • a spacer region of between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the sense sequence and a flanking sequence.
  • the spacer is 13 nucleotides and is located between the 5’ terminus of the sense sequence and a flanking sequence.
  • a spacer is of sufficient length to form approximately one helical turn of the sequence.
  • a spacer region of between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the antisense sequence and a flanking sequence.
  • the spacer sequence is between 10-13, i.e., 10, 11, 12 or 13 nucleotides and is located between the 3’ terminus of the antisense sequence and a flanking sequence. In some embodiments, a spacer is of sufficient length to form approximately one helical turn of the sequence.
  • the modulatory polynucleotide comprises in the 5’ to 3’ direction, a 5’ flanking sequence, a 5’ arm, a loop motif, a 3’ arm and a 3’ flanking sequence.
  • the 5’ arm may comprise a sense sequence and the 3’ arm comprises the antisense sequence.
  • the 5’ arm comprises the antisense sequence and the 3’ arm comprises the sense sequence.
  • the 5’ arm, payload (e.g., sense and/or antisense sequence), loop motif and/or 3’ arm sequence may be altered (e.g., substituting 1 or more nucleotides, adding nucleotides and/or deleting nucleotides). The alteration may cause a beneficial change in the function of the construct (e.g., increase knock-down of the target sequence, reduce degradation of the construct, reduce off target effect, increase efficiency of the payload, and reduce degradation of the payload).
  • the molecular scaffold of the modulatory polynucleotides is aligned in order to have the rate of excision of the guide or antisense strand be greater than the rate of excision of the passenger or sense strand.
  • the rate of excision of the guide or passenger strand may be, independently, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%.
  • the rate of excision of the guide strand is at least 80%.
  • the rate of excision of the guide strand is at least 90%.
  • the rate of excision of the guide strand is greater than the rate of excision of the passenger strand.
  • the rate of excision of the guide strand may be at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% greater than the passenger strand.
  • the efficiency of excision of the guide strand is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%. As a non-limiting example, the efficiency of the excision of the guide strand is greater than 80%.
  • the efficiency of the excision of the guide strand is greater than the excision of the passenger strand from the molecular scaffold.
  • the excision of the guide strand may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times more efficient than the excision of the passenger strand from the molecular scaffold.
  • the molecular scaffold comprises a dual-function targeting modulatory polynucleotide.
  • a “dual-function targeting” modulatory polynucleotide is a polynucleotide where both the guide and passenger strands knock down the same target or the guide and passenger strands knock down different targets.
  • the molecular scaffold may comprise one or more linkers known in the art. The linkers may separate regions or one molecular scaffold from another. As a non-limiting example, the molecular scaffold may be polycistronic.
  • the modulatory polynucleotide is designed using at least one of the following properties: loop variant, seed mismatch/bulge/wobble variant, stem mismatch, loop variant and basal stem mismatch variant, seed mismatch and basal stem mismatch variant, stem mismatch and basal stem mismatch variant, seed wobble and basal stem wobble variant, or a stem sequence variant.
  • AAV Production describes processes and methods for producing AAV particles (with enhanced, improved and/or increased tropism for a target tissue) that may be used to contact a target cell to deliver a payload.
  • the present disclosure provides methods for the generation of AAV particles comprising targeting peptides.
  • the AAV particles are prepared by viral genome replication in a viral replication cell. Any method known in the art may be used for the preparation of AAV particles.
  • AAV particles are produced in mammalian cells (e.g., HEK293).
  • AAV particles are produced in insect cells (e.g., Sf9). [0302] Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos.
  • the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety.
  • the viral replication cell may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells.
  • Viral replication cells commonly used for production of recombinant AAV viral particles include, but are not limited to, HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines as described in U.S. Patent. Nos. US6156303, US5387484, US5741683, US5691176, and US5688676; U.S.
  • the HEK293 cells may be HEK-293T cells.
  • Viral replication cells may comprise other mammalian cells such as A549, WEH1, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals.
  • Viral replication cells may comprise cells derived from mammalian species including, but not limited to, human, monkey, mouse, rat, rabbit, and hamster. Viral replication cells may comprise cells derived from a cell type, including but not limited to fibroblast, hepatocyte, tumor cell, cell line transformed cell, etc.
  • the present disclosure provides a method for producing an AAV particle in mammalian cells, comprising the steps of 1) simultaneously co-transfecting mammalian cells, such as, but not limited to HEK293 cells, with a viral genome comprising a payload region (payload construct), a viral genome comprising polynucleotide sequences for rep and cap genes (rep/cap construct) and a viral genome comprising polynucleotide sequences encoding helper components (helper construct), 2) harvesting and purifying the AAV particles comprising a viral genome.
  • This triple transfection method of AAV particle production may be utilized to produce small lots of virus.
  • the AAV particles may be produced in a viral replication cell that comprises an insect cell.
  • a viral replication cell that comprises an insect cell.
  • Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art, see U.S. Pat. No.6,204,059, the contents of which are herein incorporated by reference in their entirety.
  • Any insect cell which allows for replication of parvovirus and which can be maintained in culture can be used in accordance with the present disclosure.
  • Cell lines may be used from Spodoptera frugiperda, including, but not limited to the Sf9 or Sf21 cell lines, Drosophila cell lines, or mosquito cell lines, such as Aedes albopictus derived cell lines.
  • nucleic acids such as vectors, e.g., insect-cell compatible vectors
  • methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors into such cells and methods of maintaining such cells in culture. See, for example, Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir.63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir.
  • the present disclosure provides a method for producing an AAV particle in a baculovirus/Sf9 system, comprising the steps of: 1) co-transfecting competent bacterial cells with a bacmid vector and either a viral construct vector and/or AAV payload construct vector, 2) isolating the resultant viral construct expression vector and AAV payload construct expression vector and separately transfecting viral replication cells, 3) isolating and purifying resultant payload and viral construct particles comprising viral construct expression vector or AAV payload construct expression vector, 4) co-infecting a viral replication cell with both the AAV payload and viral construct particles comprising viral construct expression vector or AAV payload construct expression vector, and 5) harvesting and purifying AAV particles comprising a viral genome.
  • the viral construct vector and the AAV payload construct vector are each incorporated by a transposon donor/acceptor system into a bacmid, also known as a baculovirus plasmid, by standard molecular biology techniques known and performed by a person skilled in the art.
  • Transfection of separate viral replication cell populations produces two baculoviruses, one that comprises the viral construct expression vector, and another that comprises the AAV payload construct expression vector.
  • the two baculoviruses may be used to infect a single viral replication cell population for production of AAV particles.
  • Baculovirus expression vectors for producing viral particles in insect cells including but not limited to Spodoptera frugiperda (Sf9) cells, provide high titers of viral particle product.
  • Recombinant baculovirus encoding the viral construct expression vector and AAV payload construct expression vector initiates a productive infection of viral replicating cells.
  • Infectious baculovirus particles released from the primary infection secondarily infect additional cells in the culture, exponentially infecting the entire cell culture population in a number of infection cycles that is a function of the initial multiplicity of infection, see Urabe, M. et al., J Virol.2006 Feb; 80 (4):1874-85, the contents of which are herein incorporated by reference in their entirety.
  • Production of AAV particles with baculovirus in an insect cell system may address known baculovirus genetic and physical instability.
  • the production system addresses baculovirus instability over multiple passages by utilizing a titerless infected-cells preservation and scale-up system.
  • Small scale seed cultures of viral producing cells are transfected with viral expression constructs encoding the structural, non-structural, components of the viral particle.
  • Baculovirus-infected viral producing cells are harvested into aliquots that may be cryopreserved in liquid nitrogen; the aliquots retain viability and infectivity for infection of large scale viral producing cell culture Wasilko DJ et al., Protein Expr Purif.2009 Jun; 65(2):122-32, the contents of which are herein incorporated by reference in their entirety.
  • a genetically stable baculovirus may be used as the source of one or more of the components for producing AAV particles in invertebrate cells.
  • defective baculovirus expression vectors may be maintained episomally in insect cells.
  • the bacmid vector is engineered with replication control elements, including but not limited to promoters, enhancers, and/or cell-cycle regulated replication elements.
  • replication control elements including but not limited to promoters, enhancers, and/or cell-cycle regulated replication elements.
  • stable viral replication cells permissive for baculovirus infection are engineered with at least one stable integrated copy of any of the elements necessary for AAV replication and viral particle production including, but not limited to, the entire AAV genome, Rep and Cap genes, Rep genes, Cap genes, each Rep protein as a separate transcription cassette, each VP protein as a separate transcription cassette, the AAP (assembly activation protein), or at least one of the baculovirus helper genes with native or non-native promoters.
  • AAV particles described herein may be produced by triple transfection or baculovirus mediated virus production, or any other method known in the art. Any suitable permissive or packaging cell known in the art may be employed to produce the particles. Mammalian cells are often preferred. Also preferred are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other E1a trans-complementing cells. A packaging cell line may be used that is stably transformed to express cap and/or rep genes. Alternatively, a packaging cell line may be used that is stably transformed to express helper constructs necessary for AAV particle assembly.
  • AAV virus particles are, in some cases, produced and purified from culture supernatants according to the procedure as described in US20160032254, the contents of which are incorporated by reference.
  • AAV particles are produced wherein all three VP proteins are expressed at a stoichiometry around 1:1:10 (VP1:VP2:VP3). While not wishing to be bound by theory, the regulatory mechanisms that allow this controlled level of expression include the production of two mRNAs, one for VP1, and the other for VP2 and VP3, produced by differential splicing.
  • 293T cells are transfected with polyethyleneimine (PEI) with plasmids required for production of AAV, i.e., AAV2 rep, an adenoviral helper construct and a ITR flanked payload cassette.
  • AAV2 rep plasmid also contains the cap sequence of the particular virus being studied. Twenty-four hours after transfection (no medium changes for suspension), which occurs in DMEM/F17 with/without serum, the medium is replaced with fresh medium with or without serum. Three (3) days after transfection, a sample is taken from the culture medium of the 293 adherent cells.
  • AAV particle titers are measured according to genome copy number (genome particles per milliliter).
  • Genome particle concentrations are based on DNA qPCR of the vector DNA as previously reported (Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278).
  • Large-scale production [0319] In some embodiments, AAV particle production may be modified to increase the scale of production.
  • Large scale viral production methods may include any of those taught in US Patent Nos.5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526, 7,291,498 and 7,491,508 or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353 and WO2001023597, the contents of each of which are herein incorporated by reference in their entirety.
  • viral replication cells comprise adherent cells.
  • adherent cells To increase the scale of viral particle production by adherent viral replication cells, larger cell culture surfaces are required.
  • large-scale production methods comprise the use of roller bottles to increase cell culture surfaces.
  • Other cell culture substrates with increased surface areas are known in the art. Examples of additional adherent cell culture products with increased surface areas include, but are not limited to CELLSTACK ® , CELLCUBE ® (Corning Corp., Corning, NY) and NUNC TM CELL FACTORY TM (Thermo Scientific, Waltham, MA).
  • large- scale adherent cell surfaces may comprise from about 1,000 cm 2 to about 100,000 cm 2 .
  • large-scale adherent cell cultures may comprise from about 10 7 to about 10 9 cells, from about 10 8 to about 10 10 cells, from about 10 9 to about 10 12 cells or at least 10 12 cells. In some cases, large-scale adherent cultures may produce from about 10 9 to about 10 12 , from about 10 10 to about 10 13 , from about 10 11 to about 10 14 , from about 10 12 to about 10 15 or at least 10 15 viral particles. [0320] In some embodiments, large-scale viral production methods of the present disclosure may comprise the use of suspension cell cultures. Suspension cell culture allows for significantly increased numbers of cells. Typically, the number of adherent cells that can be grown on about 10-50 cm 2 of surface area can be grown in about 1 cm 3 volume in suspension.
  • Transfection of replication cells in large-scale culture formats may be carried out according to any methods known in the art.
  • transfection methods may include, but are not limited to the use of inorganic compounds (e.g. calcium phosphate), organic compounds [e.g. polyethyleneimine (PEI)] or the use of non-chemical methods (e.g. electroporation.)
  • inorganic compounds e.g. calcium phosphate
  • organic compounds e.g. polyethyleneimine (PEI)
  • non-chemical methods e.g. electroporation.
  • transfection methods may include, but are not limited to the use of calcium phosphate and the use of PEI.
  • transfection of large scale suspension cultures may be carried out according to the section entitled “Transfection Procedure” described in Feng, L. et al., 2008. Biotechnol Appl.
  • PEI-DNA complexes may be formed for introduction of plasmids to be transfected.
  • cells being transfected with PEI- DNA complexes may be ‘shocked’ prior to transfection. This comprises lowering cell culture temperatures to 4°C for a period of about 1 hour. In some cases, cell cultures may be shocked for a period of from about 10 minutes to about 5 hours. In some cases, cell cultures may be shocked at a temperature of from about 0°C to about 20°C.
  • transfections may include one or more vectors for expression of an RNA effector molecule to reduce expression of nucleic acids from one or more AAV payload constructs.
  • Such methods may enhance the production of viral particles by reducing cellular resources wasted on expressing payload constructs.
  • such methods may be carried out according to those methods taught in US Publication No. US2014/0099666, the contents of which are herein incorporated by reference in their entirety.
  • III. METHODS AND USES OF THE COMPOSITIONS [0323]
  • the AAV particles described herein, having enhanced tropism for a desired target tissue, may be used for the delivery of a payload to the target tissue.
  • the payload is a polypeptide.
  • the payload is an antibody.
  • the payload is an RNAi agent and/or modulatory polynucleotide.
  • the AAV particles described herein may be delivered by intravenous injection or infusion for the targeting of CNS and/or PNS tissues (e.g., neurons, DRG). [0324] The AAV particles described herein may be used for regulating expression of a gene of interest (or its protein product) in a cell, tissue, organ or subject.
  • methods for increasing expression of a target protein in a cell, tissue, organ or subject comprising administering to the cell, tissue, organ or subject an effective amount of the AAV particles described herein, comprising a viral genome with a payload region encoding the target protein.
  • the target protein may be increased by at least about 10%, preferably by at least about 10%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40- 70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50- 100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the AAV particles described herein may be used to increase the expression of a target protein in a cell of the CNS, such as a neuron, astrocyte, microglial cell, and/or oligodendrocyte.
  • the gene may encode a protein including, but not limited to, an antibody, survival motor neuron 1 (SMN1), APOE (APOE2, APOE3, or APOE4), GBA1, GRN, ASPA, ARSA, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, or GAN.
  • RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA specific for a target gene of interest.
  • Non-limiting examples of a target gene of interest include, SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, or SCN8A-SCN11A.
  • the present disclosure provides methods for inhibiting/silencing target gene expression in a cell.
  • the RNAi agent can be used to substantially inhibit target gene expression in a cell, such as but not limited to, in astrocytes, microglia, or cortical, hippocampal, entorhinal, thalamic, motor or sensory neurons.
  • the inhibition of target gene expression refers to an inhibition by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the protein product of the targeted gene may be inhibited by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30- 50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40- 80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the gene to be inhibited may include but is not limited to SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, or SCN8A-SCN11A.
  • Therapeutic Applications [0332] The present disclosure provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject any of the AAV particles described herein or administering to the subject any of the described compositions, including pharmaceutical compositions, described herein.
  • the AAV particles described herein are administered to a subject prophylactically, to prevent on-set of disease.
  • the AAV particles described herein are administered to treat (lessen the effects of) a disease or symptoms thereof.
  • the AAV particles of the described herein are administered to cure (eliminate) a disease.
  • the AAV particles described herein are administered to prevent or slow progression of disease.
  • the AAV particles as described herein are used to reverse the deleterious effects of a disease. Disease status and/or progression may be determined or monitored by standard methods known in the art.
  • the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of neurological diseases and/or disorders. [0335] In some embodiments, the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of tauopathy. [0336] In some embodiments, the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Alzheimer’s Disease. [0337] In some embodiments, the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Parkinson’s Disease.
  • the AAV particles described herein useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Amyotrophic lateral sclerosis.
  • the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Huntington’s Disease.
  • the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of chronic or neuropathic pain.
  • the AAV particles described herein are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the central nervous system. [0342] In some embodiments, the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Metachromatic Leukodystrophy. [0343] In some embodiments, the AAV particles described herein are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Niemann-Pick Type C.
  • the AAV particles described herein are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the peripheral nervous system.
  • the AAV particles described herein are administered to a subject having at least one of the diseases or symptoms described herein.
  • any disease associated with the central or peripheral nervous system and components thereof e.g., neurons
  • Any neurological disease may be treated with the AAV particles described herein, or pharmaceutical compositions thereof, including but not limited to, Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS - Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-
  • the present disclosure are methods for introducing the AAV particles described herein into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for an increase in the production of target mRNA and protein to occur.
  • the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.
  • a target protein e.g., ApoE
  • the method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising AAV particles described herein.
  • the AAV particles can increase target gene expression, increase target protein production, and thus reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.
  • the composition comprising the AAV particles described herein is administered to the central nervous system of the subject via systemic administration.
  • the systemic administration is intravenous injection.
  • the composition comprising the AAV particles described herein is administered to the central nervous system of the subject.
  • the composition comprising the AAV particles described herein is administered to a CNS tissue of a subject (e.g., putamen, thalamus or cortex of the subject).
  • the composition comprising the AAV particles described herein is administered to the central nervous system of the subject via intraparenchymal injection.
  • intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.
  • the composition comprising the AAV particles as described herein is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.
  • the AAV particles as described herein may be delivered into specific types of targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.
  • the AAV particles described herein may be delivered to neurons in the putamen, thalamus and/or cortex.
  • the AAV particles described herein may be used as a therapy for neurological disease. [0357] In some embodiments, the AAV particles described herein may be used as a therapy for tauopathies. [0358] In some embodiments, the AAV particles described herein may be used as a therapy for Alzheimer’s Disease. [0359] In some embodiments, the AAV particles described herein may be used as a therapy for Amyotrophic Lateral Sclerosis. [0360] In some embodiments, the AAV particles described herein may be used as a therapy for Huntington’s Disease. [0361] In some embodiments, the AAV particles described herein may be used as a therapy for Parkinson’s Disease.
  • the AAV particles described herein may be used as a therapy for chronic or neuropathic pain.
  • the AAV particles described herein may be used as a therapy for Metachromatic Leukodystrophy.
  • the AAV particles described herein may be used as a therapy for Niemann-Pick Type C.
  • administration of the AAV particles described herein to a subject may increase target protein levels in a subject.
  • the target protein levels may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject.
  • the AAV particles may increase the protein levels of a target protein by at least 50%.
  • the AAV particles may increase the proteins levels of a target protein by at least 40%.
  • a subject may have an increase of 10% of target protein.
  • the AAV particles may increase the protein levels of a target protein by fold increases over baseline. In some embodiments, AAV particles lead to 5-6 times higher levels of a target protein. [0366] In some embodiments, administration of the AAV particles described herein to a subject may increase the expression of a target protein in a subject.
  • the expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject.
  • the AAV particles may increase the expression of a target protein by at least 50%.
  • the AAV particles may increase the expression of a target protein by at least 40%.
  • intravenous administration of the AAV particles described herein to a subject may increase the CNS expression of a target protein in a subject.
  • the expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject.
  • the AAV particles may increase the expression of a target protein in the CNS by at least 50%. As a non-limiting example, the AAV particles may increase the expression of a target protein in the CNS by at least 40%. [0368] In some embodiments, the AAV particles of the present disclosure may be used to increase target protein expression in astrocytes in order to treat a neurological disease.
  • Target protein in astrocytes may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5- 80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20
  • the AAV particles may be used to increase target protein in microglia.
  • the increase of target protein in microglia may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10- 25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10- 75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15- 50%, 15-55%, 15-60%, 15-65%, 15-7
  • the AAV particles may be used to increase target protein in cortical neurons.
  • the increase of target protein in the cortical neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5- 40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%
  • the AAV particles may be used to increase target protein in hippocampal neurons.
  • the increase of target protein in the hippocampal neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5- 30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10- 60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15- 35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15
  • the AAV particles may be used to increase target protein in DRG and/or sympathetic neurons.
  • the increase of target protein in the DRG and/or sympathetic neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-55%, 15-5
  • the AAV particles described herein may be used to increase target protein in sensory neurons in order to treat neurological disease.
  • Target protein in sensory neurons may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5- 80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%
  • the AAV particles described herein may be used to increase target protein and reduce symptoms of neurological disease in a subject.
  • the increase of target protein and/or the reduction of symptoms of neurological disease may be, independently, altered (increased for the production of target protein and reduced for the symptoms of neurological disease) by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5- 80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-2
  • the AAV particles described herein may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.
  • TFC total functional capacity
  • the AAV particles described herein may be used to improve performance on any assessment used to measure symptoms of neurological disease.
  • Such assessments include, but are not limited to ADAS-cog (Alzheimer Disease Assessment Scale – cognitive), MMSE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activities Questionnaire), ADL (Activities of Daily Living), GPCOG (General Practitioner Assessment of Cognition), Mini-Cog, AMTS (Abbreviated Mental Test Score), Clock-drawing test, 6-CIT (6-item Cognitive Impairment Test), TYM (Test Your Memory), MoCa (Montreal Cognitive Assessment), ACE-R (Addenbrookes Cognitive Assessment), MIS (Memory Impairment Screen), BADLS (Bristol Activities of Daily Living Scale), Barthel Index, Functional Independence Measure, Instrumental Activities of Daily Living, IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly), Neuropsychiatric Inventory, The Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and/or MBR Caregiver Stra
  • the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.
  • the AAV particles encoding the target protein may be used in combination with one or more other therapeutic agents.
  • combination with it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
  • Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • Therapeutic agents that may be used in combination with the AAV particles described herein can be small molecule compounds which are antioxidants, anti- inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation.
  • the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.
  • Compounds tested for treating neurological disease which may be used in combination with the AAV particles described herein include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3 ⁇ (lithium) or PP2A, immunization with A ⁇ peptides or tau
  • Neurotrophic factors may be used in combination therapy with the AAV particles described herein for treating neurological disease.
  • a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron.
  • the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment.
  • Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.
  • the AAV particle described herein may be co-administered with AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the contents of which are incorporated herein by reference in their entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).
  • AAV-IGF-I See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the contents of which are incorporated herein by reference in their entirety
  • AAV-GDNF See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).
  • administration of the AAV particles to a subject will increase the expression of a target protein in a subject and the increase of the expression of the target protein will reduce the effects and/or symptoms of neurological disease in a subject.
  • the target protein may be an antibody, or fragment thereof.
  • AAV Particles Comprising RNAi agents or Modulatory Polynucleotides
  • Provided in the present disclosure are methods for introducing the AAV particles described herein, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for degradation of a target mRNA to occur, thereby activating target-specific RNAi in the cells.
  • the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.
  • the method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules.
  • the siRNA molecules can silence target gene expression, inhibit target protein production, and reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.
  • the composition comprising the AAV particles described herein, comprising a viral genome encoding one or more siRNA molecules comprise an AAV capsid that allows for enhanced transduction of CNS and/or PNS cells after intravenous administration.
  • the composition comprising the AAV particles described herein, with a viral genome encoding at least one siRNA molecule is administered to the central nervous system of the subject.
  • the composition comprising the AAV particles is administered to a tissue of a subject (e.g., putamen, thalamus or cortex of the subject).
  • the composition comprising the AAV particles described herein, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via systemic administration.
  • the systemic administration is intravenous injection.
  • the composition comprising the AAV particles described herein, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection.
  • Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.
  • the composition comprising the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered into specific types or targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered to neurons in the putamen, thalamus, and/or cortex.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for neurological disease.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for tauopathies.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Alzheimer’s Disease.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Amyotrophic Lateral Sclerosis.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Huntington’s Disease.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Parkinson’s Disease.
  • the administration of AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower target protein levels in a subject.
  • the target protein levels may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30- 60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40- 90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • the AAV particles may lower the protein levels of a target protein by at least 50%. As a non-limiting example, the AAV particles may lower the proteins levels of a target protein by at least 40%. [0401] In some embodiments, the administration of AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in a subject.
  • the expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40- 70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50- 100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject.
  • the AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the AAV particles may lower the expression of a target protein by at least 40%. [0402] In some embodiments, the administration of AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in the CNS of a subject.
  • the expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a
  • the AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the AAV particles may lower the expression of a target protein by at least 40%.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in astrocytes in order to treat neurological disease.
  • Target protein in astrocytes may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5- 95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10- 65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15- 40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15- 90%, 15-95%, 20-30%
  • Target protein in astrocytes may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5- 35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10- 65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15- 40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15- 90%, 15-95%, 20-30%,
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in microglia.
  • the suppression of the target protein in microglia may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5- 30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10- 60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15- 3
  • the reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5- 45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%,
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress target protein in cortical neurons.
  • the suppression of a target protein in cortical neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5- 30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10- 60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-
  • the reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5- 45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%,
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in hippocampal neurons.
  • the suppression of a target protein in the hippocampal neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5- 20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10- 50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15- 25%, 15
  • the reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5- 35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10- 65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15- 40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15- 90%, 15-95%, 20-30%, 20-35%, 20-40%,
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in DRG and/or sympathetic neurons.
  • the suppression of a target protein in the DRG and/or sympathetic neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5- 60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10- 35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10- 85%, 10-90%, 10-95%, 10-95%, 10
  • the reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5- 15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in sensory neurons in order to treat neurological disease.
  • Target protein in sensory neurons may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5- 30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10- 60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15- 35%, 15
  • Target protein in the sensory neurons may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5- 80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein and reduce symptoms of neurological disease in a subject.
  • the suppression of target protein and/or the reduction of symptoms of neurological disease may be, independently, reduced or suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5- 30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10- 60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-90%, 10-90%,
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.
  • TFC total functional capacity
  • the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.
  • the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used in combination with one or more other therapeutic agents.
  • compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • Therapeutic agents that may be used in combination with the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation.
  • Compounds tested for treating neurological disease which may be used in combination with the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3 ⁇ (lithium)
  • Neurotrophic factors may be used in combination therapy with the AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules for treating neurological disease.
  • a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron.
  • the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment.
  • Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.
  • the AAV particle encoding the nucleic acid sequence for the at least one siRNA duplex targeting the gene of interest may be co-administered with AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).
  • AAV-IGF-I See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety
  • AAV-GDNF See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).
  • the AAV particles may be prepared as pharmaceutical compositions.
  • pharmaceutical composition refers to compositions comprising at least one active ingredient and optionally, one or more pharmaceutically acceptable excipients.
  • Relative amounts of the active ingredient e.g. AAV particle
  • a pharmaceutically acceptable excipient e.g. any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary.
  • compositions may depend upon the identity, size, and/or condition of the subject being treated, the route by which the composition is to be administered, the nature of the AAV particle composition or the excipient, and/or any other factor.
  • the composition may comprise between 0.0001% and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.0001% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
  • the pharmaceutical composition comprises AAV particles having one type of AAV capsid protein.
  • the pharmaceutical composition comprises a mixture of AAV particles, having different AAV capsid proteins.
  • the pharmaceutical compositions described herein may comprise at AAV particles comprising a viral genome encoding at least one payload.
  • the pharmaceutical compositions may contain an AAV particle with 1, 2, 3, 4, 5, or more payloads.
  • the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • pharmaceutical compositions are administered to humans, human patients or subjects.
  • the pharmaceutical composition is administered to the human, human patient, and/or subject by intravenous delivery.
  • the pharmaceutical composition may be prepared in a manner optimized for administration by intravenous delivery.
  • Formulations of the present disclosure can include, without limitation, one or more AAV particles, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • Such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • active ingredient generally refers either to an AAV particle with a viral genome encoding a payload or to the end product, or payload itself.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the AAV formulations described herein may contain sufficient AAV particles for expression of at least one expressed functional payload.
  • the AAV particles may contain viral genomes encoding 1, 2, 3, 4 or 5 functional payloads.
  • the formulations described herein may contain at least one AAV particle comprising a viral genome with a nucleic acid sequence encoding a protein of interest.
  • the protein of interest may include but is not limited to an antibody, survival motor neuron 1 (SMN1), APOE (APOE2, APOE3, or APOE4), GBA1, GRN, ASPA, ARSA, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, or GAN.
  • the formulations described herein may contain at least one AAV particle comprising a viral genome with a nucleic acid sequence encoding the siRNA molecules described herein.
  • the siRNA molecules may target a gene of interest at one target site.
  • the formulation comprises a plurality of AAV particles, each AAV particle comprising a viral genome with a nucleic acid sequence encoding a siRNA molecule targeting the gene of interest at a different target site.
  • the target gene may be targeted at 1, 2, 3, 4, 5 or more than 5 sites.
  • the target gene may include, but is not limited to, SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, or SCN8A-SCN11A.
  • AAV particles may be formulated for systemic delivery.
  • AAV particles may be formulated for CNS delivery.
  • the formulation of AAV particles may be optimized for delivery to any target tissue by any administration route described herein.
  • the formulation of AAV particles may be optimized for CNS as the target tissue and intravenous administration as the method of delivery.
  • the formulation of AAV particles may be optimized for DRG as the target tissue and intravenous administration as the method of delivery.
  • the AAV particles described herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed expression of the payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein; and/or (7) allow for regulatable expression of the payload.
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia. [0435] Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • the use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • AAV particle formulations may comprise at least one inactive ingredient.
  • the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations.
  • composition formulations of AAV particles disclosed herein may include cations or anions.
  • the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mn2+, Mg+ and combinations thereof.
  • formulations may include polymers and complexes with a metal cation (See e.g., U.S. Pat. Nos.6265389 and 6555525, each of which is herein incorporated by reference in its entirety).
  • Formulations of the disclosure may also include one or more pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • Solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
  • solvents examples include ethanol, water (for example, mono-, di-, and tri-hydrates), N- methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N’-dimethylformamide (DMF), N,N’-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl- 3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like.
  • NMP N- methylpyrrolidinone
  • DMSO dimethyl sulfoxide
  • DMF N,N’-dimethylformamide
  • DMAC N,N’-dimethylacetamide
  • DMEU 1,3-dimethyl-2-imidazolidinone
  • the AAV particles described herein may be formulated in phosphate buffered saline (PBS), in combination with an ethylene oxide/propylene oxide copolymer (also known as Pluronic or poloxamer).
  • PBS phosphate buffered saline
  • an ethylene oxide/propylene oxide copolymer also known as Pluronic or poloxamer.
  • the AAV particles described herein may be formulated in PBS with 0.001% Pluronic acid (F-68) (poloxamer 188) at a pH of about 7.0.
  • the AAV particles described herein may be formulated in PBS with 0.001% Pluronic acid (F-68) (poloxamer 188) at a pH of about 7.3.
  • the AAV particles of the described herein may be formulated in PBS with 0.001% Pluronic acid (F-68) (poloxamer 188) at a pH of about 7.4.
  • the AAV particles described herein may be formulated in a solution comprising sodium chloride, sodium phosphate and an ethylene oxide/propylene oxide copolymer.
  • the AAV particles described herein may be formulated in a solution comprising sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic and poloxamer 188/Pluronic acid (F-68).
  • the AAV particles described herein may be formulated in a solution comprising sodium chloride, potassium phosphate monobasic, sodium phosphate dibasic, and poloxamer 188/Pluronic acid (F-68).
  • AAV particles are formulated in a solution comprising about 200mM sodium chloride (NaCl), about 1mM potassium phosphate monobasic (KH 2 PO 4 ), about 3mM sodium phosphate dibasic (Na 2 HPO 4 ), and about 0.001% Pluronic F- 68/poloxamer 188 at a pH of about 7.4.
  • the concentration of sodium chloride in the final solution may be 150mM-250mM.
  • the concentration of sodium chloride in the final solution may be 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, or any concentration in between.
  • the concentration of potassium phosphate monobasic in the final solution may be 0.01mM-3mM.
  • the concentration of potassium phosphate monobasic in the final solution may be 0.01mM, 0.5mM, 1mM, 2mM, 3mM, or any concentration in between.
  • the concentration of sodium phosphate dibasic in the final solution may be 1mM-10mM.
  • the concentration of sodium phosphate dibasic in the final solution may be 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM or any concentration in between.
  • the concentration of Pluronic F-68 (poloxamer 188) may be 0.0001%-1%.
  • the concentration of Pluronic F-68 (poloxamer 188) may be 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, or 1%.
  • the final solution may have a pH of 6.8-7.7.
  • Non-limiting examples for the pH of the final solution include a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7.
  • the formulation may be 10mM Na2HPO4, 2 mM KH2PO4, 2.7mM KCl, 192 mM NaCl and 0.001% Pluronic F-68 at pH 7.4.
  • the formulation may be 200mM NaCl, 1mM KH 2 PO 4 , 3mM Na 2 HPO 4 , 0.001% Pluronic F-68 at pH 7.4. V.
  • the AAV particles may be administered to a subject in a therapeutically effective amount to reduce the symptoms of disease, such as a neurological disease (affecting either the CNS or PNS), of a subject (e.g., determined using a known evaluation method).
  • the subject is a mammal.
  • a mammal may include, a mouse, a non-human primate, and/or a human.
  • Administration [0452]
  • the AAV particles described herein may be administered by any delivery route which results in a therapeutically effective outcome.
  • intravenous intravenous
  • CNS intraparenchymal
  • brain intraparenchymal
  • spinal cord intraparenchymal
  • DSG intracranial, intrastriatal, intrathalamic, enteral (into the intestine), gastroenteral, epidural (into the dura mater), oral (by way of the mouth), transdermal, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), sub-pial (between pia and CNS parenchyma), intracarotid arterial (into the intracarotid artery), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous bolus, intravenous drip, intra-arterial (into an artery), systemic, intramuscular (into a muscle), intracardiac (into the heart), intraosse
  • the AAV particles described herein are administered by intraparenchymal injection.
  • the AAV particles are delivered directly to the target tissue.
  • AAV particles described herein may be administered by intraparenchymal delivery to central nervous system tissue, such as but not limited to, the putamen, the thalamus, and/or the cortex.
  • AAV particles described herein may be administered by intraparenchymal delivery to the peripheral nervous system tissue, such as, but not limited to, the dorsal root ganglia.
  • AAV particles may be administered systemically.
  • AAV particles are administered intravenously.
  • Intravenous administration encompasses the use of any vein of the subject for the delivery of AAV particles.
  • target veins include, median cubital vein, orbital veins (superior or inferior ophthalmic, saphenous veins (greater or lesser), internal jugular, and/or femoral vein.
  • the target vein may be the tail vein or the orbital veins.
  • the AAV particles and compositions comprising AAV particles may be administered in a way that leverages the vascular connectivity of the central nervous system, such as, but not limited to, by intravenous administration.
  • the AAV particles described herein may be administered via intravenous delivery to cells of the central nervous system (CNS), such as, but not limited to, neurons, astrocytes, microglia and/or oligodendrocytes.
  • CNS central nervous system
  • the AAV particles described herein may be administered via intravenous delivery to cells of the peripheral nervous system (PNS), such as, but not limited to, neurons and/or Schwann cells.
  • PNS peripheral nervous system
  • the AAV particles described herein may be administered via intravenous delivery to DRG neurons.
  • the AAV particles may be delivered by injection into a CSF pathway (e.g., intrathecal or intraventricular).
  • the AAV particles described herein may be administered to a subject by intracranial delivery (See, e.g., U. S. Pat. No.8,119,611; the content of which are incorporated herein by reference in their entirety).
  • the AAV particle may be administered to the CNS or PNS in a therapeutically effective amount to improve function and/or survival for a subject with a neurological disease.
  • the vector may be administered intravenously.
  • the AAV particles described herein may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.
  • the AAV particles may be formulated with any appropriate and pharmaceutically acceptable excipient.
  • the AAV particles described herein may be delivered to a subject via a single route of administration. In other embodiments, the AAV particles may be delivered to a subject via more than one route of administration.
  • the AAV particles described herein may be delivered to a subject via a multi-site route of administration. AAV particles may be administered at 2, 3, 4, 5 or more than 5 sites.
  • two or more compositions comprising AAV particles may be administered to the same subject.
  • the two or more AAV particle compositions may be administered via the same route.
  • the two or more AAV particle compositions may be administered via different routes.
  • Two or more compositions comprising AAV particles may be administered simultaneously, or at different times.
  • one composition comprising AAV particles may be administered by an intraparenchymal route, while another composition comprising AAV particles may be administered by an intravenous route.
  • AAV particles may be administered by injection.
  • AAV particles are administered by infusion.
  • AAVs may be administered as a bolus.
  • Injection sites for AAV particle administration may include, but are not limited to, the putamen, thalamus, cortex, hippocampus, dorsal root ganglia, and deep cerebellar nuclei.
  • administration of the AAV particles described herein occurs only once, and serves as a one-time treatment. In other embodiments, administration of the AAV particles occurs more than once.
  • Dosing [0470] The present disclosure provides methods of administering AAV particles described herein to a subject in need thereof.
  • the pharmaceutical, diagnostic, or prophylactic AAV particles and compositions described herein may be administered to a subject using any amount and any route of administration effective for preventing, treating, managing, or diagnosing diseases, disorders and/or conditions.
  • compositions in accordance with the disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate diagnostic dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific AAV particle employed; the duration of the treatment; drugs used in combination or coincidental with the specific AAV particle employed; and like factors well known in the medical arts.
  • AAV particle pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight to obtain the desired therapeutic, diagnostic, or prophylactic effect.
  • AAV particle pharmaceutical compositions in accordance with the present disclosure may be administered at about 0.1-600 ⁇ l/site, or about 0.1 to about 0.5 ⁇ l/site, about 0.5 to about 1 ⁇ l/site, about 1 to about 10 ⁇ l/site, about 10 to about 600 ⁇ l/site, about 50 to about 500 ⁇ l/site, about 100 to about 400 ⁇ l/site, about 120 to about 300 ⁇ l/site, about 140 to about 200 ⁇ l/site, about 160 ⁇ l/site.
  • AAV particles may be administered at 0.5 ⁇ l/site, 50 ⁇ l/site, 150 ⁇ l/site, 160 ⁇ l/site or 250 ⁇ l/site.
  • delivery of AAV particle compositions to cells may comprise a total concentration per subject between about 1x10 6 VG (viral genome) and about 1x10 16 VG.
  • delivery may comprise a composition concentration of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 1.5x10 10 , 2x10 10 , 2.5x10 10 , 3
  • delivery of AAV particle compositions to cells may comprise a total concentration per subject between about 1x10 6 VG/kg and about 1x10 16 VG/kg.
  • delivery may comprise a composition concentration of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 8 , 7x10 8 , 8
  • the delivery comprises a composition concentration of 1x10 13 VG/kg. In some embodiments, the delivery comprises a composition concentration of 2.1x10 12 VG/kg. In some embodiments, the delivery comprises a composition concentration of 1x10 13 VG/kg. In some embodiments, the delivery comprises a composition concentration of 6.7x10 12 VG/kg.
  • the delivery comprises a composition concentration some embodiments, the delivery comprises a composition concentration some embodiments, the delivery comprises a composition concentration some embodiments, the delivery comprises a composition concentration some embodiments, the delivery comprises a composition concentration some embodiments, the delivery comprises a composition concentration [0477]
  • delivery of AAV particle compositions to cells may comprise a total concentration per site between about 1x10 6 VG/site and about 1x10 16 VG/site.
  • delivery may comprise a composition concentration of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9
  • delivery of AAV particles to cells of the central nervous system may comprise a total dose between about 1x10 6 VG and about 1x10 16 VG.
  • delivery may comprise a total dose of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 8 , 7x10 8 , 8x10 8
  • the total dose is 1x10 13 VG.
  • the total dose is 2.1x10 12 VG.
  • the total dose is 6.3x10 12 VG.
  • delivery of the compositions comprising the AAV particles in accordance with the present disclosure to cells may comprise a total concentration between about 1x10 6 VG/mL and about 1x10 16 VG/mL.
  • delivery may comprise a composition concentration of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9
  • delivery of AAV particles to cells of the central nervous system may comprise a composition concentration between about 1x10 6 VG/mL and about 1x10 16 VG/mL.
  • delivery may comprise a composition concentration of about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 8 , 7x10 8
  • the delivery comprises a composition concentration of 1x10 13 VG/mL. In some embodiments, the delivery comprises a composition concentration of 2.1x10 12 VG/mL. In some embodiments, the delivery comprises a composition concentration of 1x10 13 VG/mL. In some embodiments, the delivery comprises a composition concentration of 2x10 13 VG/mL. In some embodiments, the delivery comprises a composition concentration of 3x10 11 VG/mL. In some embodiments, the delivery comprises a composition concentration of 3x10 12 VG/mL. In some embodiments, the delivery comprises a composition concentration of 6.3x10 12 VG/mL. In some embodiments, the delivery comprises a composition concentration of 3x10 13 VG/mL.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • multiple administrations e.g., split dosing regimens such as those described herein may be used.
  • a “split dose” is the division of “single unit dose” or total daily dose into two or more doses, e.g., two or more administrations of the “single unit dose”.
  • a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • the desired dosage of the AAV particles described herein may be administered as a “pulse dose” or as a “continuous flow”.
  • a “pulse dose” is a series of single unit doses of any therapeutic administered with a set frequency over a period of time.
  • a “continuous flow” is a dose of therapeutic administered continuously for a period of time in a single route/single point of contact, i.e., continuous administration event.
  • a total daily dose, an amount given or prescribed in 24 hour period may be administered by any of these methods, or as a combination of these methods, or by any other methods suitable for a pharmaceutical administration.
  • delivery of the AAV particles described herein to a subject provides regulating activity of a target gene in a subject.
  • the regulating activity may be an increase in the production of the target protein in a subject or the decrease of the production of target protein in a subject.
  • the regulating activity can be for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.
  • the AAV particles described herein may be administered to a subject using a single dose, one-time treatment.
  • the dose of the one-time treatment may be administered by any methods known in the art and/or described herein.
  • a “one-time treatment” refers to a composition which is only administered one time. If needed, a booster dose may be administered to the subject to ensure the appropriate efficacy is reached.
  • a booster may be administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years after the one-time treatment.
  • Delivery [0486]
  • the AAV particles or pharmaceutical compositions described herein are delivered by intravenous injection.
  • the AAV particles or pharmaceutical compositions described herein may be administered or delivered using the methods for treatment of disease described in US Patent No.8,999,948, or International Publication No. WO2014178863, the contents of each of which are herein incorporated by reference in their entirety.
  • the AAV particles or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering gene therapy in Alzheimer’s Disease or other neurodegenerative conditions as described in US Application No.20150126590, the contents of which are herein incorporated by reference in their entirety.
  • the AAV particles or pharmaceutical compositions described herein may be administered or delivered using the methods for delivery of a CNS gene therapy as described in US Patent Nos.6,436,708, and 8,946,152, and International Publication No. WO2015168666, the contents of each of which are herein incorporated by reference in their entirety.
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering proteins using AAV vectors described in European Patent Application No. EP2678433, the contents of which are herein incorporated by reference in their entirety.
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering DNA to the bloodstream described in US Patent No.
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering a payload to the central nervous system described in US Patent No. US 7,588,757, the contents of which are herein incorporated by reference in their entirety.
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering a payload described in US Patent No. US 8,283,151, the contents of which are herein incorporated by reference in their entirety.
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering a payload using a glutamic acid decarboxylase (GAD) delivery vector described in International Patent Publication No. WO2001089583, the contents of which are herein incorporated by reference in their entirety.
  • GAD glutamic acid decarboxylase
  • the AAV particle or pharmaceutical compositions described herein may be administered or delivered using the methods for delivering a payload to neural cells described in International Patent Publication No. WO2012057363, the contents of which are herein incorporated by reference in their entirety.
  • the present disclosure provides a method of delivering to a cell or tissue any of the above-described AAV particles, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions.
  • the method of delivering the AAV particle to a cell or tissue can be accomplished in vitro, ex vivo, or in vivo.
  • the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.
  • the subject is a human.
  • the human subject is a patient with a disease, for example, a neurological disease, or a disease associated with the central or peripheral nervous system or components thereof.
  • the AAV particles may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents.
  • compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the present disclosure encompasses the delivery of pharmaceutical, prophylactic, research, or diagnostic compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • kits for conveniently and/or effectively carrying out the methods described herein.
  • kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • Any of the targeting peptides and/or associated AAV particles described herein may be comprised in a kit.
  • kits may further include reagents and/or instructions for creating and/or synthesizing compounds and/or compositions described herein.
  • kits may also include one or more buffers.
  • kits described herein may include components for making protein or nucleic acid arrays or libraries and thus, may include, for example, solid supports.
  • kit components may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one kit component, (labeling reagent and label may be packaged together), kits may also generally contain second, third or other additional containers into which additional components may be separately placed.
  • kits may also comprise second container means for containing sterile, pharmaceutically acceptable buffers and/or other diluents.
  • various combinations of components may be comprised in one or more vial.
  • Kits described herein may also typically include means for containing compounds and/or compositions described herein , e.g., proteins, nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which desired vials are retained.
  • kit components are provided in one and/or more liquid solutions.
  • liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred.
  • kit components may be provided as dried powder(s).
  • reagents and/or components are provided as dry powders, such powders may be reconstituted by the addition of suitable volumes of solvent.
  • solvents may also be provided in another container means.
  • labeling dyes are provided as dried powders.
  • 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits described herein.
  • kits may include instructions for employing kit components as well the use of any other reagent not included in the kit. Instructions may include variations that may be implemented.
  • Devices [0505] In some embodiments, the AAV particles described herein are delivered by catheter. [0506] In some embodiments, the AAV particles described herein are delivered by syringe and/or syringe pump. [0507] In some embodiments, the AAV particles described herein are delivered by cannula. [0508] In some embodiments, the AAV particles described herein are delivered by a device, wherein the device is further placed in a mechanism to direct or target the trajectory.
  • the AAV particles may be delivered to a subject using a device to deliver the AAV particles and a head fixation assembly.
  • the head fixation assembly may be, but is not limited to, any of the head fixation assemblies sold by MRI interventions.
  • the head fixation assembly may be any of the assemblies described in US Patent Nos.8099150, 8548569, and 9031636 and International Patent Publication Nos. WO201108495 and WO2014014585, the contents of each of which are incorporated by reference in their entireties.
  • a head fixation assembly may be used in combination with an MRI compatible drill such as, but not limited to, the MRI compatible drills described in International Patent Publication No.
  • the AAV particles may be delivered using a method, system and/or computer program for positioning of an apparatus to a target point on a subject to deliver the AAV particles.
  • the method, system and/or computer program may be the methods, systems and/or computer programs described in US Patent No. 8340743, the contents of which are herein incorporated by reference in their entirety.
  • the method may include: determining a target point in the body and a reference point, wherein the target point and the reference point define a planned trajectory line (PTL) extending through each; determining a visualization plane, wherein the PTL intersects the visualization plane at a sighting point; mounting the guide device relative to the body to move with respect to the PTL, wherein the guide device does not intersect the visualization plane; determining a point of intersection (GPP) between the guide axis and the visualization plane; and aligning the GPP with the sighting point in the visualization plane.
  • the AAV particles may be delivered to a subject using a convention-enhanced delivery device. Non-limiting examples of targeted delivery of drugs using convection are described in US Patent Publication Nos.
  • a subject may be imaged prior to, during and/or after delivery of the AAV particles.
  • the imaging method may be a method known in the art and/or described herein, such as but not limited to, magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • imaging may be used to assess therapeutic effect.
  • imaging may be used for assisted delivery of AAV particles.
  • the AAV particles may be delivered using an MRI-guided device.
  • Non-limiting examples of MRI-guided devices are described in US Patent Nos. 9055884, 9042958, 8886288, 8768433, 8396532, 8369930, 8374677, and 8175677 and US Patent Application No. US20140024927 the contents of each of which are herein incorporated by reference in their entireties.
  • the MRI-guided device may be able to provide data in real time such as those described in US Patent Nos. 8886288 and 8768433, the contents of each of which is herein incorporated by reference in its entirety.
  • the MRI-guided device or system may be used with a targeting cannula such as the systems described in US Patent Nos.8175677 and 8374677, the contents of each of which are herein incorporated by reference in their entireties.
  • the MRI-guided device includes a trajectory guide frame for guiding an interventional device as described, for example, in US Patent No. 9055884 and US Patent Application No. US20140024927, the contents of each of which are herein incorporated by reference in their entireties.
  • the AAV particles may be delivered using an MRI- compatible tip assembly. Non-limiting examples of MRI-compatible tip assemblies are described in US Patent Publication No.
  • the AAV particles may be delivered using a cannula which is MRI-compatible.
  • MRI-compatible cannulas include those taught in International Patent Publication No. WO2011130107, the contents of which are herein incorporated by reference in its entirety.
  • the AAV particles may be delivered using a catheter which is MRI-compatible.
  • Non-limiting examples of MRI-compatible catheters include those taught in International Patent Publication No. WO2012116265, US Patent No.8825133 and US Patent Publication No. US20140024909, the contents of each of which are herein incorporated by reference in their entireties.
  • the AAV particles may be delivered using a device with an elongated tubular body and a diaphragm as described in US Patent Publication Nos. US20140276582 and US20140276614, the contents of each of which are herein incorporated by reference in their entireties.
  • the AAV particles may be delivered using an MRI compatible localization and/or guidance system such as, but not limited to, those described in US Patent Publication Nos. US20150223905 and US20150230871, the contents of each of which are herein incorporated by reference in their entireties.
  • the MRI compatible localization and/or guidance systems may comprise a mount adapted for fixation to a patient, a targeting cannula with a lumen configured to attach to the mount so as to be able to controllably translate in at least three dimensions, and an elongate probe configured to snugly advance via slide and retract in the targeting cannula lumen, the elongate probe comprising at least one of a stimulation or recording electrode.
  • the AAV particles may be delivered to a subject using a trajectory frame as described in US Patent Publication Nos. US20150031982 and US20140066750 and International Patent Publication Nos.
  • Adeno-associated virus As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependovirus genus comprising any particle, sequence, gene, protein, or component derived therefrom.
  • AAV Particle As used herein, an “AAV particle” is a virus which comprises a capsid and a viral genome with at least one payload region and at least one ITR.
  • AAV particles as described herein are AAV particles comprising a parent capsid sequence with at least one targeting peptide insert.
  • AAV particles of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences.
  • AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary).
  • the AAV particle may be replication defective and/or targeted.
  • the AAV particle may have a targeting peptide inserted into the capsid to enhance tropism for a desired target tissue. It is to be understood that reference to the AAV particles of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.
  • Administering refers to providing a pharmaceutical agent or composition to a subject.
  • Amelioration refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of neurodegeneration disorder, amelioration includes the reduction of neuron loss.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.
  • mammals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.
  • the animal is a transgenic animal, genetically-engineered animal, or a clone.
  • Antisense strand As used herein, the term “the antisense strand” or “the first strand” or “the guide strand” of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19- 22 nucleotides of the mRNA of a gene targeted for silencing.
  • the antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.
  • target-specific silencing e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Capsid refers to the protein shell of a virus particle.
  • Complementary and substantially complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenine.
  • the polynucleotide strands exhibit 90% complementarity.
  • the term “substantially complementary” means that the siRNA has a sequence (e.g., in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA.
  • control elements refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and/or translated in an appropriate host cell.
  • Delivery refers to the act or manner of delivering an AAV particle, a compound, substance, entity, moiety, cargo or payload.
  • Element refers to a distinct portion of an entity.
  • an element may be a polynucleotide sequence with a specific purpose, incorporated into a longer polynucleotide sequence.
  • Encapsulate As used herein, the term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein often encapsulates a viral genome.
  • Engineered As used herein, embodiments of the disclosure are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
  • Effective Amount As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.
  • expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5 ⁇ cap formation, and/or 3 ⁇ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Feature refers to a characteristic, a property, or a distinctive element.
  • a “formulation” includes at least one AAV particle (active ingredient) and an excipient, and/or an inactive ingredient.
  • Fragment A “fragment,” as used herein, refers to a portion.
  • an antibody fragment may comprise a CDR, or a heavy chain variable region, or a scFv, etc.
  • Functional As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Gene expression refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide.
  • measurement of “gene expression” this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.
  • Homology As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids.
  • homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4–5 uniquely specified amino acids.
  • two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference.
  • Inhibit expression of a gene means to cause a reduction in the amount of an expression product of the gene.
  • the expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • Insert may refer to the addition of a targeting peptide sequence to a parent AAV capsid sequence. An “insertion” may result in the replacement of one or more amino acids of the parent AAV capsid sequence. Alternatively, an insertion may result in no changes to the parent AAV capsid sequence beyond the addition of the targeting peptide sequence.
  • Inverted terminal repeat As used herein, the term “inverted terminal repeat” or “ITR” refers to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids.
  • Library As used herein, the term “library” refers to a diverse collection of linear polypeptides, polynucleotides, viral particles, or viral vectors. As examples, a library may be a DNA library or an AAV capsid library.
  • Neurological disease As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).
  • Naturally Occurring As used herein, “naturally occurring” or “wild-type” means existing in nature without artificial aid, or involvement of the hand of man.
  • Open reading frame As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.
  • Parent sequence As used herein, a “parent sequence” is a nucleic acid or amino acid sequence from which a variant is derived. In some embodiments, a parent sequence is a sequence into which a heterologous sequence is inserted. In other words, a parent sequence may be considered an acceptor or recipient sequence.
  • a parent sequence is an AAV capsid sequence into which a targeting sequence is inserted.
  • Particle As used herein, a “particle” is a virus comprised of at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.
  • Patient As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
  • Payload region is any nucleic acid sequence (e.g., within the viral genome) which encodes one or more “payloads” as described herein.
  • a payload region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is an RNAi agent or a polypeptide.
  • Payloads as described herein may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc.
  • Peptide As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • Pharmaceutically acceptable The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term “preventing” or “prevention” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
  • Prophylactic As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.
  • Prophylaxis As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.
  • Region As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three- dimensional area, an epitope and/or a cluster of epitopes. In some embodiments, regions comprise terminal regions.
  • terminal region refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini. [0561] In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three- dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5’ and/or 3’ termini.
  • RNA or RNA molecule refers to a polymer of ribonucleotides
  • DNA or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized.
  • DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • mRNA or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
  • RNA interfering or RNAi refers to a sequence specific regulatory mechanism mediated by RNA molecules which results in the inhibition or interfering or “silencing” of the expression of a corresponding protein-coding gene.
  • RNAi has been observed in many types of organisms, including plants, animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. RNAi is controlled by the RNA- induced silencing complex (RISC) and is initiated by short/small dsRNA molecules in cell cytoplasm, where they interact with the catalytic RISC component argonaute. The dsRNA molecules can be introduced into cells exogenously.
  • RISC RNA- induced silencing complex
  • RNAi agent refers to an RNA molecule, or its derivative, that can induce inhibition, interfering, or “silencing” of the expression of a target gene and/or its protein product.
  • An RNAi agent may knock-out (virtually eliminate or eliminate) expression, or knock-down (lessen or decrease) expression.
  • the RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA.
  • sample refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.
  • a sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.
  • Self-complementary viral particle As used herein, a “self-complementary viral particle” is a particle comprised of at least two components, a protein capsid and a self- complementary viral genome enclosed within the capsid.
  • Sense Strand As used herein, the term “the sense strand” or “the second strand” or “the passenger strand” of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand. The antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure.
  • a “siRNA duplex” includes a siRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a siRNA strand having sufficient complementarity to form a duplex with the other siRNA strand.
  • Similarity refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Short interfering RNA or siRNA As used herein, the terms “short interfering RNA,” “small interfering RNA” or “siRNA” refer to an RNA molecule (or RNA analog) comprising between about 5-60 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi.
  • a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogs, such as between about 16-25 nucleotides (or nucleotide analogs), between about 18-23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nucleotides (or nucleotide analogs), and between about 19-24 nucleotides (or nucleotide analogs).
  • nucleotides or nucleotide analogs such as between about 16-25 nucleotides (or nucleotide analogs), between about 18-23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nu
  • short siRNA refers to a siRNA comprising 5- 23 nucleotides, preferably 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides.
  • long siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides.
  • Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi.
  • siRNAs may, in some instances, include more than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi or translational repression absent further processing, e.g., enzymatic processing, to a short siRNA.
  • siRNAs can be single stranded RNA molecules (ss- siRNAs) or double stranded RNA molecules (ds-siRNAs) comprising a sense strand and an antisense strand which hybridized to form a duplex structure called an siRNA duplex.
  • Subject refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • plants e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • Substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • Targeting peptide refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein. As a non-limiting example, the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism.
  • Target cells refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.
  • Therapeutic Agent refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • Therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • a therapeutically effective amount is provided in a single dose.
  • therapeutically effective outcome means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • Treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition.
  • “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • Vector refers to any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule.
  • vectors may be plasmids.
  • vectors may be viruses.
  • An AAV particle is an example of a vector.
  • Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequences.
  • the heterologous molecule may be a polynucleotide and/or a polypeptide.
  • Viral Genome As used herein, the terms “viral genome” or “vector genome” refer to the nucleic acid sequence(s) encapsulated in an AAV particle.
  • a viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.
  • the present disclosure is further illustrated by the following non-limiting examples. EXAMPLES Example 1.
  • CREATE Use of CREATE in human primate to identify CNS targeting peptides and associated capsids
  • the CREATE method for generating libraries of targeting peptides with enhanced tropism for CNS tissues in mice has been previously described in Deverman et al (Nature Biotechnology 34(2):204-209 (2016), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017), and International Patent Publication Nos. WO2015038958 and WO2017100671), the contents of each of which are herein incorporated by reference in their entirety.
  • This method was used to identify targeting peptides, such as PHP.B and PHP.N (or PHP.eB), showing enhanced tropism to CNS tissues in the mouse, when compared to AAV9.
  • the AAV capsid libraries were produced by triple transfection and purified by iodixanol gradient ultracentrifugation.
  • the CREATE in NHP workflow involved the following steps 1) an AAV DNA library was generated, 2) an AAV capsid library was prepared by triple transfection, 3) the AAV library was delivered intravenously to NHP on Day 0, 4) AAV-Cre was delivered to the same NHP by intracranial or intraparenchymal infusion on Day 7, 5) the NHP underwent necropsy and tissue collection on Day 20, 6) capsid DNA was recovered by cre-dependent DNA recovery and subjected to next generation sequencing (NGS). For second round CREATE, these steps were repeated with DNA libraries generated from materials recovered from the first round.
  • NGS next generation sequencing
  • AAV-Syn-Cre vectors were administered intraparenchymally to the striatum and thalamus of three adult (2-11yr, 4-8kg) non-human primates (macaca mulatta or rhesus macaque), while the AAV capsid library was administered intravenously as shown in the study design of Table 4, below.
  • the AAV particles were delivered in an excipient comprising 200mM NaCl, 1mM KH 2 PO 4 , 3mM Na 2 HPO 4 , 0.001% Pluronic F-68 at pH 7.4.
  • Table 4 Study design for AAV9-7mer library in NHP [0586] Five animals were selected based on results of neutralizing antibody assays for anti-AAV antibodies to AAV1 and AAV9. Minimal or no detectable capsid specific antibodies was required for selection. Two of these five animals served as alternate study animals. Study subjects underwent a pre-project physical examination. [0587] On Day 0 of the study, each animal received intravenous infusion of the AAV9- 7mer library. On Day 7 ⁇ 1, animals had bilateral intracranial infusion of AAV-Syn-Cre vectors to the putamen and thalamus, using MRI-guided convection enhanced delivery.
  • AAV9-7mer libraries or AAV-Syn-Cre containing compositions were thawed at 2-8C.
  • the excipient detailed above was used as a vehicle control.
  • Dosing solutions were gently centrifuged to collect material from the vial cap then transferred to the syringe/needle for delivery. Dosing solutions were kept at room temperature until administration, but not longer than for 2 hours.
  • Gadoteridol ProHance, Bracco Diagnostics Inc
  • a skull mounted cannula guide ball array was temporarily secured to the skull with titanium screws over each burr hole.
  • animals were transferred to the MRI suite.
  • MRI was used to align the cannula guides with putamen and thalamus targets ipsilateral (same side of the brain/body) to each cannula guide.
  • Repeat MRI was used to visually monitor infusions.
  • Dosing solutions were delivered via an adjustable tip 16G cannula (MRI Interventions Inc) primed with the dosing solution then guided into each target site through the skull mounted cannula arrays.
  • Cannula were connected via microbore extension lines (Smiths Medical) to a syringe mounted on a syringe pump (Harvard apparatus). Ascending infusion rates (up to 10 ⁇ L/min) were used to deliver dose volumes (150 ⁇ L/putamen; 250 ⁇ L/thalamus) to the putamen and thalamus. Serial MRI scans were used to monitor infusate distribution. [0592] Immediately after MRI-CED dosing, animals were transferred back to the operating room where the cannula guide system was removed and the wound site closed, using a vicryl suture. Animals were provided pre- and post-operative medications, allowed to recover from anesthesia and returned to their home cages.
  • Samples were collected from brain, spinal cord, 12 dorsal root ganglia and associated roots (4 each from cervical, thoracic and lumbar spinal cord), liver, heart (left ventricle and right atrium), gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal glands, stomach, sciatic nerve, saphenous nerve, thyroid gland, eyes (with optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, skin of the leg, superior cervical ganglia, urinary bladder, ovaries, uterus, prostate gland, testes, and/or any sites identified as having a lesion, and fresh frozen for biochemical analysis.
  • Sample volumes listed in the Serum column refer to the volume of whole blood which was then processed to serum and split to 2 aliquots and stored at -60C or below.
  • CSF samples were collected after animals were immobilized with ketamine (10mg/kg; IM) and dexmedetomidine (15ug/kg; IM). The neck was shaved and flexed and skin prepared. A 23G needle was manually advanced through the skin and the atlanto-occipital membrane into the cerebellomedullary cistern space. Once CSF flow was confirmed in the needle hub, CSF collection began.
  • AAV-G (SEQ ID NO: 659 or 660), comprising targeting peptide SMPREPG (SEQ ID NO: 4) inserted between amino acids at positions 588 and 589 of AAV9 (SEQ ID NO: 2) was selected for further characterization.
  • AAV particles comprising AAV-G capsid (SEQ ID NO: 659 or 660) and a CAG- NLS-eGFP viral genome (SEQ ID NO: 661) were generated by triple transfection.
  • the SMPREPG (SEQ ID NO: 4) targeting peptide was encoded by an codon_1 (NNK) variant comprising SEQ ID NO: 223 for AAV-G particle generation.
  • the CAG-NLSeGFP viral genome read in 5’ to 3’ order, included a 5’ AAV2 inverted terminal repeat (ITR), a CAG promoter, an intron, a nuclear localization signal (NLS), a sequence encoding eGFP, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a polyadenylation sequence (PolyA) and an AAV23’ ITR.
  • ITR AAV2 inverted terminal repeat
  • NLS nuclear localization signal
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • PolyA polyadenylation sequence
  • AAV-G-CAG-NLSeGFP-wpre particles were purified by two rounds of iodixanol gradient ultracentrifugation and analyzed by silver staining. Similar preparations of AAV9 were used for comparison. Analysis by silver staining showed the expected bands for each of VP1, VP2, and VP3 proteins for both AAV9 and AAV-G.
  • AAV vector genome yield (vg/cell) [0601] AAV-G-CAG-NLSeGFP-wpre particles were next tested in HEK293T and Lec2 cells. AAV9 particles were run as comparison. HEK293T and Lec2 cells were seeded in 96- well plates and transduced with AAV-G-CAG-NLSeGFP-wpre or AAV9 particles 24 hours after seeding at multiplicity of infection (MOI) 5e3 or 2e4. Native GFP imaging was conducted 3 days after transduction.
  • MOI multiplicity of infection
  • AAV- G showed greater transduction, as evidenced by brighter and/or more eGFP signal, as compared to that of cells transduced with AAV9 particles. These data showed that AAV-G efficiently transduced HEK293T and Lec2 cells. Native GFP images collected from transduced HEK293T and Lec2 cells using a 10x objective are shown in FIG.1A and 1B, respectively. Scale bars indicate 400 ⁇ m. [0602] AAV-G-CAG-NLSeGFP-wpre particles were then tested in brain microvascular endothelial cells (BMVEC) derived from mouse, cynomolgus monkey and human tissues.
  • BMVEC brain microvascular endothelial cells
  • BMVEC (Catalog # C57-6023, MK-6023, H-6023; Cell Biologics, respectively) were seeded into gelatin or fibronectin-coated 96 well plates and transduced with AAV-G-CAG- NLSeGFP-wpre or AAV9 particles 24 hours after seeding. Native GFP imaging was conducted 3 days after transduction. In each of mouse, monkey and human BMVEC, AAV- G showed greater transduction, as evidenced by brighter and/or more eGFP signal, as compared to that of cells transduced with AAV9 particles. In both the mouse and human BMVEC, eGFP signal in cells transduced with AAV9 particles was negligible, while in monkey BMVEC, limited eGFP signal was seen.
  • FIG.2A, 2B, and 2C Native GFP images collected from mouse, monkey and human BMVEC are shown in FIG.2A, 2B, and 2C, respectively. “BF” and “mock” indicate bright field and untransduced control samples. Scale bars indicate 400 ⁇ m.
  • primary human BMVEC cells were seeded (1e5 cells/well) in a 24- well fibronectin coated plate and transduced with AAV-G-CAG-NLSeGFP-wpre or AAV9 particles 24 hours after seeding at MOI 5e3.
  • Genomic DNA was isolated from the cells 3 days after transduction and vector genome copies (VG/DC) quantified by droplet digital PCR using eGFP probe (FAM) and GAPDH probe (HEX). Data are shown in Table 8 below, with the average summarized in the final row. Table 8.
  • AAV vector genome copies (VG/DC) [0604] As shown in Table 8, AAV-G-CAG-NLSeGFP-wpre particles provided at MOI 5e3 transduced human BMVEC to a greater extent than AAV9 particles as determined by quantification of vector genome quantification by ddPCR. [0605] Finally, AAV-G-CAG-NLSeGFP-wpre particles were tested in human neuroblastoma cells. Again, AAV9 was used as comparison.
  • undifferentiated SH- SY5Y cells were transduced with AAV-G-CAG-NLSeGFP-wpre or AAV9 particles at one of three doses, MOI 5e3, 1.25e4 or 5e4.
  • Native GFP imaging was captured 3 days after transduction.
  • cells transduced with AAV- G particles showed greater eGFP signal than cells transduced with the same dose of AAV9, as shown in FIG.3A.
  • “BF” indicates bright field and the scale bars indicate 400 ⁇ m.
  • AAV-G efficiently transduced human neuroblastoma cells across two different developmental stages.
  • AAV-G consistently showed greater transduction patterns, as compared to AAV9, in each of the cell lines tested: HEK293T, Lec2, BMVEC from mouse, monkey and human, and undifferentiated and differentiated human neuroblastoma cells.
  • Example 3 In vivo testing of targeting peptides and associated capsid variants [0608] Targeting peptides and associated capsids and AAV particles will be tested in vivo.
  • Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps.
  • compositions of the disclosure e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.
  • any particular embodiment of the compositions of the disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

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Abstract

La divulgation concerne des compositions, des procédés et des processus pour la préparation, l'utilisation et/ou la formulation de protéines de capsides de virus adéno-associés, les protéines capsidiques comprenant des inserts peptidiques de ciblage pour un tropisme amélioré vis-à-vis d'un tissu cible.
PCT/US2021/030779 2020-05-06 2021-05-05 Variants de vaa issus de bibliothèques de second tour présentant un tropisme pour des tissus du système nerveux central WO2021226167A1 (fr)

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WO2024086747A1 (fr) 2022-10-19 2024-04-25 Affinia Therapeutics Inc. Aavs recombinants à tropisme et spécificité améliorés

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Publication number Priority date Publication date Assignee Title
US11649459B2 (en) 2021-02-12 2023-05-16 Alnylam Pharmaceuticals, Inc. Superoxide dismutase 1 (SOD1) iRNA compositions and methods of use thereof for treating or preventing superoxide dismutase 1-(SOD1-) associated neurodegenerative diseases
EP4269426A1 (fr) * 2022-04-29 2023-11-01 Ospedale San Raffaele S.r.l. Thérapie génique
WO2023209221A1 (fr) * 2022-04-29 2023-11-02 Ospedale San Raffaele S.R.L. Thérapie génique
WO2024086747A1 (fr) 2022-10-19 2024-04-25 Affinia Therapeutics Inc. Aavs recombinants à tropisme et spécificité améliorés

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