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WO2025090631A1 - Vecteurs aav pour l'administration d'apoe2 - Google Patents

Vecteurs aav pour l'administration d'apoe2 Download PDF

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Publication number
WO2025090631A1
WO2025090631A1 PCT/US2024/052596 US2024052596W WO2025090631A1 WO 2025090631 A1 WO2025090631 A1 WO 2025090631A1 US 2024052596 W US2024052596 W US 2024052596W WO 2025090631 A1 WO2025090631 A1 WO 2025090631A1
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sequence
apoe2
aav
transgene
vector
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Inventor
Paul T. RANUM
Beverly L. Davidson
Megan S. KEISER
Yonghong CHEN
Ellie CARRELL
Luis TECEDOR
Rosemary JACKSON
Bradley HYMAN
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General Hospital Corp
Childrens Hospital of Philadelphia CHOP
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General Hospital Corp
Childrens Hospital of Philadelphia CHOP
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present disclosure relates generally to the fields of medicine, virology, and neurology. More particularly, it concerns targeting peptides that target delivery of viral vectors to distinct structures in the brain, particularly for the delivery of the s2 allele of apolipoprotein E (ApoE2).
  • ApoE2 apolipoprotein E
  • AD Alzheimer's disease
  • symptoms can include problems with language, disorientation, mood swings, loss of motivation, self-neglect, and behavioral issues. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the typical life expectancy following diagnosis is less than a decade.
  • the cause of Alzheimer's disease is poorly understood, with many environmental and genetic risk factors associated with its development. However, the strongest genetic risk factor is from an allele of apolipoprotein E. Genetic therapies to modify apolipoprotein E expression, however, are limited because transduction of ependyma and deep brain structures in NHP brain is poorly achievable with current AAV serotypes.
  • Adeno-associated viruses represent strong therapeutic candidates for the treatment of neurodegenerative disease.
  • AAVs are non-enveloped, single-stranded DNA viruses that can infect both dividing and non-dividing cells. Following infection, the virus does not exhibit robust integration within the host genome but persists as an episome in the cell nucleus.
  • Expression of AAV cargoes is controlled spatially at the level of the packaging capsid and by the transgene promoter. Because use of AAV for the treatment of disease may necessitate intervention in diseased tissue, a problem can arise in that target tissue that contains a different gene expression profile than its healthy counterpart. Finding the correct promoter sequence to drive therapeutic transgene expression is an important goal.
  • AAV vector variants including rational design and directed evolution.
  • the rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, such as tyrosine mutations on the capsid surface for increasing transduction efficiency.
  • the directed evolution approach does not require any knowledge of capsid structure and is done through random mutagenesis, capsid shuffling, or random peptide insertions.
  • These strategies generally use in vitro systems or mice, which are ideal for cellbased or mouse studies, but do not imply translation to the clinic. In fact, no AAV variants target distinct brain structures specifically or efficiently. As such, AAV variants that are capable of targeting distinct brain structures are needed.
  • the present disclosure provides modified AAV vectors which express the s2 allele of apolipoprotein E (ApoE2).
  • AAV1 modified adeno associated virus 1
  • the capsid protein comprises, i.e. encapsulates, a nucleic acid molecule comprising a sequence encoding the modified AAV genome comprising and a transgene expressing ApoE2, and optionally comprising a replication (rep) gene, and a capsid (cap) gene.
  • the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.
  • the targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long.
  • the linker sequences may be SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.
  • the sequence encoding the ApoE2 transgene may be operably linked to a polyadenylation signal.
  • the sequence encoding the ApoE2 transgene may be operably linked to a promoter derived from the human von Willebrand factor domain containing 3A (VWA3A promoter), also referred to as human promoter Ependymal promoter (hprEp) promoter .
  • VWA3A promoter human von Willebrand factor domain containing 3A
  • hprEp human promoter Ependymal promoter
  • the hprEp promoter is described in Carrell et al. (2023) “VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF”, Mol Ther Nucleic Acids 33:296- 304, the entirety of the contents of which are incorporated by reference herein.
  • the adeno associated virus may comprise sequences at least 95% identical to the sequences in Table 1 which together describe an AAV-Ep.hprEp.hAPOE2 AAV vector:
  • Table 1 AAV-Ep.hprEp.hAPOE2 AAV vector
  • aspects of the disclosure include a pharmaceutical composition comprising the AAV vectors of the disclosure.
  • vectors of the disclosure are useful for the treatment of Alzheimer’s diseases in a subject.
  • the present disclosure provides methods of treating a subject suffering from Alzheimer’s disease, the method comprising administering to the subject a modified AAV1 vector comprising an EP+ capsid protein comprising the targeting peptide ERDRTRG (SEQ ID NO: 1), and a nucleic acid molecule comprising a sequence encoding the modified AAV genome comprising a transgene expressing ApoE2, and optionally comprising a replication (rep) gene, and capsid (cap) gene.
  • ERDRTRG targeting peptide ERDRTRG
  • FIG. 1 is a western blot of ApoE expression in knockout mice following delivery.
  • FIG. 2 is a chart of ApoE expression in knockout mice following virus delivery.
  • FIG. 3 is a western blot of hprEp driven expression of ApoE2.
  • FIG. 4 is a chart showing hprEp driven expression of ApoE2.
  • FIG. 5A is a plot showing dose dependent increases in viral copies.
  • FIG. 5B is a plot of dose dependent increases in ApoE expression.
  • FIG. 6 is an image of tissue stains of plaque deposition following AAV-EP+ delivery.
  • FIGS. 7A-D are charts showing plaque deposition following AAV-EP+ delivery.
  • FIG. 8 is an image of tissue stains of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 9 is a graph of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. IDA is a chart of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 1 OB is a graph of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 11A is graph of Ibal expression following AAV-EP+ delivery.
  • FIG. 1 IB is graph of Gfap expression following AAV-EP+ delivery.
  • FIG. 12 is an RNAscope image of P2ry 12 and Clec7a transcript expression following AAV-EP+ delivery.
  • FIG. 13A is a chart of P2ryl2 expression following AAV-EP+ delivery.
  • FIG. 13B is a chart of Clec7a expression following AAV-EP+ delivery.
  • FIG. 14 is an image from immunohistochemistryfor PSD95 and A in ApoE4 animals following dose dependent AAV -EP+ delivery.
  • FIGS. 15A-C are graphs/charts of synapse density following dose dependent AAV- EP+ delivery.
  • FIG. 16 is RNAscope image of ApoE transcripts (red) expressed in the ependymal cell layer.
  • FOXJ 1 green
  • DAPI blue
  • Scale bar 100 pm.
  • the present disclosure provides modified AAV vectors which express the a2 allele of apolipoprotein E (ApoE2).
  • the present disclosure provides modified AAV vectors which express the e2 allele of apolipoprotein E (ApoE2). in subject.
  • Aspects of the disclosure provide a modified adeno associated virus 1 (AAV1) vector comprising a capsid protein that comprises a targeting peptide and a nucleic acid molecule comprising a sequence encoding the modified AAV genome comprising a replication (rep) gene, a capsid (cap) gene, and an ApoE2 transgene.
  • AAV1 vector comprising a capsid protein that comprises a targeting peptide and a nucleic acid molecule comprising a sequence encoding the modified AAV genome comprising a replication (rep) gene, a capsid (cap) gene, and an ApoE2 transgene.
  • ApoE is a protein involved in the metabolism of fats in the body of mammals. A subtype is implicated in Alzheimer’s disease and cardiovascular disease. ApoE belongs to a family of fat-binding proteins called apolipoproteins. In the circulation, it is present as part of several classes of lipoprotein particles, including chylomicron remnants, VLDL, IDL, and some HDL. ApoE interacts significantly with the low-density lipoprotein receptor (LDLR), which is essential for the normal processing (catabolism) of triglyceride-rich lipoproteins. In peripheral tissues, ApoE is primarily produced by the liver and macrophages, and mediates cholesterol metabolism.
  • LDLR low-density lipoprotein receptor
  • ApoE is mainly produced by astrocytes and transports cholesterol to neurons via ApoE receptors, which are members of the low-density lipoprotein receptor gene family.
  • ApoE is the principal cholesterol carrier in the brain.
  • ApoE is required for cholesterol transportation from astrocytes to neurons.
  • ApoE qualifies as a checkpoint inhibitor of the classical complement pathway by complex formation with activated Clq.
  • ApoE is a protein involved in the metabolism of fats in the body of mammals. A subtype is implicated in Alzheimer's disease and cardiovascular disease.
  • ApoE is 299 amino acids long and contains multiple amphipathic a-helices.
  • a hinge region connects the N- and C-terminal regions of the protein.
  • the N-terminal region (residues 1-167) forms an anti-parallel four-helix bundle such that the non-polar sides face inside the protein.
  • the C-terminal domain (residues 206-299) contains three a-helices which form a large exposed hydrophobic surface and interact with those in the N-terminal helix bundle domain through hydrogen bonds and salt-bridges.
  • the C-terminal region also contains a low-density lipoprotein receptor (LDLR)-binding site.
  • LDLR low-density lipoprotein receptor
  • ApoE is polymorphic, with three major alleles (epsilon 2, epsilon 3, 25 and epsilon 4): ApoE-s2 (cysl l2, cysl58), APOE-s3 (cysl l2, argl58), and ApoE-e4 (argl l2, argl58). Although these allelic forms differ from each other by only one or two amino acids at positions 112 and 158, these differences alter ApoE structure and function.
  • the E4 variant was the largest known genetic risk factor for late-onset sporadic Alzheimer’s disease (AD) in a variety of ethnic groups.
  • AD late-onset sporadic Alzheimer’s disease
  • the E4 variant does not correlate with risk in every population.
  • Nigerian people have the highest observed frequency of the ApoE4 allele in world populations, but Alzheimer’s disease is rare among them. This may be due to their low cholesterol levels.
  • Caucasian and Japanese carriers of two E4 alleles have between 10 and 30 times the risk of developing Alzheimer’s disease by 75 years of age, as compared to those not carrying any E4 alleles. This may be caused by an interaction with amyloid.
  • Alzheimer's disease is characterized by build-ups of aggregates of the peptide beta-amyloid.
  • Apolipoprotein E enhances proteolytic break-down of this peptide, both within and between cells.
  • the isoform ApoE-s4 is not as effective as the others at promoting these reactions, resulting in increased vulnerability to Alzheimer’s disease in individuals with that gene variation.
  • AD patients Although 40-65% of AD patients have at least one copy of the s4 allele, ApoE4 is not a determinant of the disease. At least one-third of patients with Alzheimer’s disease are ApoE4 negative and some ApoE4 homozygotes never develop the disease. Yet those with two s4 alleles have up to 20 times the risk of developing AD.
  • the ApoE2 allele may serve a protective role in Alzheimer’s disease.
  • the genotype most at risk for Alzheimer's disease and at an earlier age is ApoE4,4.
  • genotype ApoE3,3 as a benchmark (with the persons who have this genotype regarded as having a risk level of 1.0) and for white populations only, individuals with genotype ApoE4,4 have an odds ratio of 14.9 of developing Alzheimer's disease.
  • Individuals with the APpo3,4 genotype face an odds ratio of 3.2, and people with a copy of the 2 allele and the 4 allele (ApoE2,4), have an odds ratio of 2.6.
  • Persons with one copy each of the 2 allele and the 3 allele (ApoE2,3) have an odds ratio of 0.6.
  • Persons with two copies of the 2 allele (ApoE2,2) also have an odds ratio of 0.6.
  • aspects of the disclosure provide a nucleic acid encoding ApoE, an ApoE that has substantial identity to wildtype ApoE, or a variant, mutant and/or or fragment of ApoE, thereby treating Alzheimer's disease.
  • the therapeutic transgene may be ApoE2 and the subject suffers from or is at an increased risk of developing Alzheimer’s Disease as compared to the populational average.
  • Administration may be direct intracerebroventricular or intraparenchymal injection.
  • the modified AAV may be administered more than once, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.
  • the modified AAV may be administered monthly, 10 every other month, every three months, every four months, every six months or annually.
  • the method may further comprise providing a non-AAV therapy to said subject.
  • a method of reducing or impairing microglial inflammation comprise delivering ApoE2 to microglia in a subject in need thereof.
  • the delivering of ApoE2 to the microglial comprises administering to said subject a modified AAV as defined herein or a pharmaceutical formulation comprising the same, wherein the therapeutic transgene is ApoE2.
  • the microglial inflammation may be caused by or associated with a neurodegenerative disease, such as Huntington’s disease, Parkinson’s disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, progressive supranuclear palsy, amyotrophic lateral sclerosis, multiple sclerosis, Batten disease, and Creutzfeldt- Jakob disease.
  • Microglial inflammation may be caused by or associated with Alzheimer’ s disease.
  • the administration may be by direct intracerebroventricular or intraparenchymal injection of ApoE2 or a modified AAV, and may involve more than one administration, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, and/or monthly, every other month, every three months, every four months, every six months or annually.
  • the method may further comprise providing a non-AAV ApoE2 therapy to said subject.
  • AAV adeno-associated virus
  • AAV genomes can exist in an extrachromosomal state without integrating into host cellular genomes; possess a broad host range; transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes.
  • AAV viral particles are heat stable; resistant to solvents, detergents, changes in pH, and temperature, and can be column purified and/or concentrated on CsCl gradients or by other means.
  • the AAV genome comprises a single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed.
  • ssDNA single-stranded deoxyribonucleic acid
  • the approximately 4.7 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity.
  • An AAV “genome” refers to a recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle.
  • An AAV particle often comprises an AAV genome packaged with AAV capsid proteins.
  • the AAV vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid.
  • an AAV vector “genome” refers to nucleic acid that is packaged or encapsulated by AA V capsid proteins.
  • the AAV virion is a non-enveloped, icosahedral particle approximately 25 nm in diameter that comprises an AAV capsid.
  • the AAV particle comprises an icosahedral symmetry comprised of three related capsid proteins, VP1, VP2 and VP3, which interact together to form the capsid.
  • the genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF.
  • ORFs open reading frames
  • the right ORF often encodes the capsid proteins VP 1, VP2, and VP3. These proteins are often found in a ratio of 1:1 :10 respectively, but may be in varied ratios, and are all derived from the righthand ORF.
  • the VP1, VP2 and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1 which is translated from an alternatively spliced message results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any single- stranded progeny DNA or infectious particles.
  • the genome of an AAV particle encodes one, two or all three VP1, VP2 and VP3 polypeptides.
  • the left ORF often encodes the non- structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes.
  • Two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19.
  • Rep68 and Rep78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities.
  • Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites.
  • the genome of an AAV e.g., an rAA V
  • the genome of an AAV does not encode the Rep proteins.
  • one or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a polypeptide.
  • the ends of the AAV genome comprise short-inverted terminal repeats (ITR) which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication.
  • the genome of an AAV comprises one or more (e.g., a pair of) ITR sequences that flank a single stranded viral DNA genome.
  • the ITR sequences often have a length of about 145 bases each.
  • two elements have been described which are believed to be central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs).
  • the repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation.
  • recombinant as a modifier of vector, such as recombinant viral, e.g., lentivirus or parvovirus (e.g., AA V) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature.
  • a recombinant vector such as an AAV, retroviral, or lentiviral vector would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted within the viral genome.
  • nucleic acid sequence e.g., gene
  • a nucleic acid e.g., gene
  • RNA cloned into a vector with or without 5', 3’ and/or intron regions that the gene is normally associated within the viral genome.
  • recombinant is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission.
  • a recombinant viral “vector” is derived from the wild type genome of a virus by using molecular methods to remove part of the wild type genome from the virus, and replacement with a non-native nucleic acid, such as a nucleic acid sequence.
  • a non-native nucleic acid such as a nucleic acid sequence.
  • ITR inverted terminal repeat
  • a “recombinant” viral vector e.g., rAAV
  • a viral genome since part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”
  • an AAV (e.g., a rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair ofITRs that flank (i.e., are at each 5' and 3' end) of a nucleic acid sequence that at least encodes a polypeptide having function or activity.
  • An AAV vector (e.g., rAAV vector) can be packaged and is referred to herein as an “AAV particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo.
  • an AAV particle is a rAAV particle.
  • a rAAV particle often comprises a rAAV vector, or a portion thereof.
  • a rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles).
  • rAAV particles typically comprise proteins that encapsulate or package the rAAV vector genome (e.g., capsid proteins). It is noted that reference to a rAA V vector can also be used to reference a rAAV particle.
  • AAV particle e.g., rAAV particle
  • a rAAV particle, and/or genome comprised therein can be derived from any suitable serotype or strain of AAV.
  • a rAAV particle, and/or genome comprised therein can be derived from two or more serotypes or strains of AAV.
  • a rAAV can comprise proteins and/or nucleic acids, or portions thereof, of any serotype or strain of AA V, wherein the AA V particle is suitable for infection and/or transduction of a mammalian cell.
  • Nonlimiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA V7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rhlO and AAV-2i8.
  • a plurality of rAAV particles comprises particles of, or derived from, the same strain or serotype (or subgroup or variant). In certain embodiments a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., of different serotypes and/or strains).
  • serotype is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
  • AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.
  • a rAAV vector based upon a first serotype genome corresponds to the serotype of one or more of the capsid proteins that package the vector.
  • the serotype of one or more AAV nucleic acids (e.g., ITRs) that comprises the AAV vector genome corresponds to the serotype of a capsid that comprises the rAAV particle.
  • a rAAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from the serotype of one or more of the AAV capsid proteins that package the vector.
  • a rAAV vector genome can comprise AAV 1 derived nucleic acids (e.g., ITRs), whereas at least one or more of the three capsid proteins are derived from a different serotype, e.g., an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 or AAV-2i8 serotype or variant thereof.
  • a rAAV particle or a vector genome thereof related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 or AAV-2i8 particle.
  • a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%,
  • a rAAV particle or a vector genome thereof related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AA V2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 or AAV-2i8 serotype.
  • a method herein comprises use, administration or delivery of an rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVIO, rAAVl 1, rAAV12, rRhlO, rRh74 or rAAV-2i8 particle.
  • a method herein comprises use, administration or delivery of a rAAV 1 particle.
  • a rAAV 1 particle comprises an AAV 1 capsid.
  • a rAAVl particle comprises one or more capsid proteins (e.g., VP1, VP2 and/or VP3) that are at least 60%, 65%, 70%, 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV1 particle.
  • capsid proteins e.g., VP1, VP2 and/or VP3
  • a rAAV2 particle comprises VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV 1 particle.
  • a rAAV2 particle is a variant of a native or wild-type AAV1 particle.
  • one or more capsid proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to capsid protein(s) of a native or wild-type AAV 1 particle.
  • a rAAV particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV 12, AAV-rh74, or AAV-rhlO or AAV-2i8, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and/or packaging, if desired).
  • ITRs
  • a rAAVl particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV1 particle, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and/or packaging, if desired).
  • ITRs e.g., a pair of ITRs
  • a rAAV particle can comprise an ITR having any suitable number of “GAGC” repeats.
  • an ITR of an AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more “GAGC” repeats.
  • a rAAV2 particle comprises an ITR comprising three “GAGC” repeats.
  • a rAAV2 particle comprises an ITR which has less than four “GAGC” repeats.
  • a rAAV2 particle comprises an ITR which has more than four “GAGC” repeats.
  • an ITR of a rAAV2 particle comprises a Rep binding site wherein the fourth nucleotide in the first two “GAGC” repeats is a C rather than a T.
  • Exemplary suitable length of DNA can be incorporated in rAAV vectors for packaging/encapsidation into a rAAV particle can about 5 kilobases (kb) or less.
  • length of DNA is less than about 5kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.
  • rAAV vectors that include a nucleic acid sequence that directs the expression of an RNAi or polypeptide can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989).
  • Recombinant AAV vectors are typically packaged into transduction competent AAV particles and propagated using an AAV viral packaging system.
  • a transduction competent AAV particle is capable of binding to and entering a mammalian cell and subsequently delivering a nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell.
  • a nucleic acid cargo e.g., a heterologous gene
  • an intact rAAV particle that is transduction-competent is configured to transduce a mammalian cell.
  • a rAAV particle configured to transduce a mammalian cell is often not replication competent and requires additional protein machinery to self-replicate.
  • a rAAV particle that is configured to transduce a mammalian cell is engineered to bind and enter a mammalian cell and deliver a nucleic acid to the cell, wherein the nucleic acid for delivery is often positioned between a pair of AA V ITRs in the rAAV genome.
  • Suitable host cells for producing transduction competent AAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells that can be, or have been, used as recipients of a heterologous rAAV vectors.
  • Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used.
  • a modified human embryonic kidney cell line e.g., HEK293
  • the modified HEK293 cell line is readily transfected and provides a particularly convenient platform in which to produce rAAV particles. Methods of generating high titer AAV particles capable of transducing mammalian cells are known in the art.
  • AAV helper functions are introduced into the host cell by transfecting the host cell with an AA V helper construct either prior to, or concurrently with, the transfection of an AAV expression vector.
  • AAV helper constructs are thus sometimes used to provide at least transient expression of AA V rep and/or cap genes to complement missing AAV functions necessary for productive AAV transduction.
  • AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AA V helper constructs have been described, such as the commonly used plasmids pAAV/Ad and PIM29+45 which encode both Rep and Cap expression products.
  • a number of other vectors are known which encode Rep and/or Cap expression products.
  • Viral vectors may be administered directly to patients (in vivo) or 5 they can be used to treat cells in vitro or ex vivo, and then administered to patients.
  • the term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid.
  • Vectors such as viral vectors, can be used to introduce/transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells.
  • any suitable cell or mammal can be administered or treated by a method or use described herein.
  • a mammal in need of a method described herein is suspected of having or expressing an abnormal or aberrant protein that is associated with a disease state.
  • the mammalian recipient may have a condition that is amenable to gene replacement therapy.
  • gene replacement therapy refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ.
  • Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
  • a mammal is a human.
  • a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like).
  • a non-rodent mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a mammal can be an animal disease model, for example, animal models having or expressing an abnormal or aberrant protein that is associated with a disease state or animal models with insufficient expression of a protein, which causes a disease state.
  • Mammals (subjects) treated by a method or composition described herein include adults (18 years or older) and children (less than 18 years of age).
  • Adults include the elderly. Representative adults are 50 years or older. Children range in age from 1-2 years old, or from 2-10 4, 4-6, 6-18, 8-10, 10-12, 12-15 and 15-18 years old. Children also include infants. Infants typically range from 1 -12 months of age.
  • a method includes administering a plurality of viral particles to a mammal as set forth herein, where severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, decreased, reduced, prevented, inhibited or delayed.
  • a method includes administering a plurality of viral particles to a mammal to treat an adverse symptom of a disease state, such as a neuro-degenerative disease.
  • a method includes administering a plurality of viral particles to a mammal to stabilize, delay or prevent worsening, or progression, or reverse and adverse symptom of a disease state, such as a neuro-degenerative disease.
  • a method includes administering a plurality of viral particles to the central nervous system, or portion thereof as set forth herein, of a mammal and severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, are decreased, reduced, prevented, inhibited or delayed by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days.
  • a symptom or adverse effect comprises an early stage, middle or late-stage symptom; a behavior, personality or language symptom; swallowing, movement, seizure, tremor or fidgeting symptom; ataxia; and/or a cognitive symptom such as memory, ability to organize.
  • a method includes administering or delivering AAV-ApoE2 particles to a mammal and administering one or more immunosuppressive agents to the mammal. In certain embodiments a method includes administering or delivering AAV- ApoE2 particles to a mammal and administering 2, 3, 4 or more immunosuppressive agents to the mammal. In certain embodiments a method includes administering or delivering AAV- ApoE2 particles to a mammal and administering two immunosuppressive agents to the mammal. In one representative embodiment, a method of treating a mammal includes administering or delivering AAV-ApoE2 particles to a mammal and administering first and second immunosuppressive agents to the mammal.
  • each immunosuppressive agent is distinct and/or different (e.g., each agent differs in structure and/or mechanism of action).
  • An “agent” refers to an active pharmaceutical ingredient.
  • an immunosuppressive agent is an anti-inflammatory agent.
  • an immunosuppressive agent is mycophenolate, or a derivative thereof.
  • An example of such a mycophenolate derivative is mycophenolate mofetil (MMF).
  • an immunosuppressive agent is cyclosporine or a derivative thereof.
  • a first immunosuppressive agent comprises cyclosporine and a second immunosuppressive agent comprises mycophenolate, or a derivative thereof (e.g., MMF).
  • a first immunosuppressive agent comprises cyclosporine and a second immunosuppressive agent comprises MMF.
  • an immunosuppressive agent is administered before, during and/or after administration of AAV-ApoE2 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered concurrently with administration of AA V -TPP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered after administration of AAV-ApoE2 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1 to about 60 minutes after, about 1 to about 24 hours after, about 1 to about 100 days after, about 1 to about 12 months after, or about 1 to about 5 years after administration of AAV-ApoE2 particles to a mammal.
  • an immunosuppressive agent is administered before administration of AA V- TPP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1 to about 60 minutes before, about 1 to about 24 hours before, about 1 to about 100 days before, or about 1 to about 3 months before administration of AAV-ApoE2 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 days before administration of AAV-ApoE2 particles to a mammal.
  • an immunosuppressive agent is administered at predetermined intervals before, during and/or after administration of AAV-ApoE2 particles to a mammal (e.g., once a day, twice a day, three times a day, every other day, weekly, biweekly, bimonthly, combination thereof or the like).
  • a first immunosuppressive agent is administered to a mammal at least about 1 to about 7 days before, or about 1 , about 2, about 3, about 4 or about 5 weeks before administration of AAV-ApoE2 particles to a mammal and a second immunosuppressive agent is administered about 1 to about 7 days before, about 1 , about 2, about 3, about 4 or about 5 weeks before, during and/or within about 10, about 20, about 30, about 40, 19 about 50, about 100, about 200, about 300, about 350, about 400 or about 500 days after administration of AAV-ApoE2 particles to the mammal.
  • cyclosporine is administered to a mammal at least about I to about 7 days before, or about 1, about 2, about 3, about 4 or about 5 weeks before administration of AAV-ApoE2 particles to a mammal, and mycophenolate or a derivative thereof (e.g., MMF) is administered about 1 to about 7 days before, about 1, about 2, about 3, about 4 or about 5 weeks before, during and/or within about 10, about 20 20, about 30, about 40, about 50, about 100, about 200, about 300, about 350, about 400 or about 500 days after administration of AAV-ApoE2 particles to the mammal.
  • mycophenolate or a derivative thereof e.g., MMF
  • cyclosporine is administered about 1 to about 7 days before, or about 1, about 2, about 3, about 4 or about 5 weeks before administration of AAV- ApoE2 particles and at regular intervals after treatment, and mycophenolate or a derivative thereof (e.g., MMF) is administered once at about 1 to about 7 days before, about 1, about 2, about 3, about 4 or about 5 weeks before, during and/or within about 10 to about 40 days after administration of AAV-ApoE2 particles to the mammal.
  • mycophenolate or a derivative thereof e.g., MMF
  • An immunosuppressive agent can be administered at any suitable dose.
  • cyclosporine is administered at a dosage of about 1 to about 50 mg/kg, about 1 to about 20 mg/kg, or about 5 to about 10 mg/kg at a frequency of once, twice or three times a day, 30 to once every other day.
  • cyclosporine is administered at about 10 mg/kg twice a day.
  • cyclosporine is administered at about 10 mg/kg twice a day for a period of at least about 1, about 2, about 3, about 4 or about 5 months.
  • a dosage of cyclosporine is tapered down to a dose of less than about 5 mg/kg, or less than about 2 mg/kg about 1 to about 2 months after the administration of AAV-ApoE2 particles to a mammal.
  • mycophenolate or a derivative thereof is administered at a dosage of about 1 to about 100 mg/kg, about 1 to about 50 mg/kg, about 1 to about 25 mg/kg, or about 5 to about 20 mg/kg at a frequency of once, twice or three times a day, to once every other day.
  • mycophenolate or a derivative thereof is administered at about 10 to about 20 mg/kg once a day.
  • a dosage of mycophenolate or a derivative thereof is reduced down to a dose of less than about 5 mg/kg, or less than about 2 mg/kg about 1 to about 2 months after the administration of AAV-ApoE2 particles to a mammal.
  • An immunosuppressive agent can be formulated in any suitable formulation suitable for a particular route of administration. Various pharmaceutically acceptable formulations of immunosuppressive agents are commercially available and readily obtainable by a medical practitioner.
  • An immunosuppressive agent can be administered by any suitable route.
  • an immunosuppressive agent is administered orally.
  • mycophenolate or a derivative thereof, such as Mycophenolate Mofetil (MMF) is administered orally.
  • cyclosporine is administered orally.
  • An immunosuppressive agent can also be administered parenterally (e.g., intramuscularly, intravenously, subcutaneously), or administered by injection to the brain, spinal cord, or a portion thereof (e.g., injected into the CSF).
  • a method includes administering one or more (e.g., a plurality of) AAV-ApoE2 particles to the central nervous system of a mammal (e.g., a mammal having a LSD).
  • the central nervous system includes brain, spinal cord and cerebral spinal fluid (CSF).
  • a method includes administering one or more AAV-ApoE2 particles to the brain or spinal cord or CSF of a mammal.
  • AAV-ApoE2 particles are administered to a portion of brain or spinal cord.
  • a composition including AAV-ApoE2 particles and an immunosuppressive agent are administered to a mammal's cistema magna and/or to the mammal’s brain ventricle, subarachnoid space, and/or intrathecal space, and/or ependyma.
  • AAV-ApoE2 particles can be delivered directly to the cisterna magna, intraventricular space, a brain ventricle, subarachnoid space, intrathecal space or ependyma.
  • a method includes administering one or more AAV-ApoE2 particles to the ependyma of a mammal.
  • AAV-ApoE2 particles are administered to one or more cells that contact the CSF in a mammal, for example by contacting cells with AAV-ApoE2 particles.
  • cells that contact the CSF include ependymal cells, pial cells, endothelial cells and/or meningeal cells.
  • AAV-ApoE2 particles are administered to ependymal cells.
  • AAV-ApoE2 particles are delivered to ependymal cells, for example by contacting ependymal cells with AAV-ApoE2 particles.
  • AAV-ApoE2 particles are delivered locally.
  • “Local delivery” refers to delivery of an active agent directly to a target site within a mammal (e.g., directly to a tissue or fluid).
  • an agent can be locally delivered by direct injection into an organ, tissue or specified anatomical location.
  • one or more AAV- ApoE2 particles are delivered or administered by direct injection to the brain, spinal cord, or a tissue or fluid thereof (e.g., CSF, such as ependymal cells, pial cells, endothelial cells and/or meningeal cells).
  • AA V-TPP1 particles can be directly delivered, by way of direct injection, to the CSF, cistema magna, intraventricular space, a brain ventricle, subarachnoid space and/or intrathecal space and/or ependyma.
  • AAV- ApoE2 particles are contacted with a tissue, fluid or cell of the brain or spinal cord by direct injection into a tissue or fluid of the brain or spinal cord.
  • AAV- ApoE2 particles are not delivered systemically by, for example, intravenous, subcutaneous, or intramuscular injection, or by intravenous infusion.
  • AAV-ApoE2 particles are delivered to a tissue or fluid of the brain or spinal cord by stereotactic injection.
  • one or more AAV-ApoE2 particles are delivered or administered by direct injection of AAV-ApoE2 particles to the brain, spinal cord, or a tissue or fluid thereof (e.g., CSF such as ependyma).
  • AA V-TPP particles transduce ependymal cells, pial cells, endothelial cells and/or meningeal cells.
  • an effective amount of AAV-ApoE2 particles can be empirically determined.
  • Administration can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • Effective doses of administration can be determined by those of skill in the art and may vary according to the AA V serotype, viral titer and the weight, condition and species of mammal being treated. Single and multiple administrations can be carried out with the dose level, target and timing being selected by the treating physician.
  • a plurality of AAV-TPP 1 particles are administered.
  • a plurality of AAV particle refers to about IxlO 5 to about IxlO 8 particles.
  • AAV-ApoE2 particles are administered at a dose of about IxlO 5 to about IxlO 16 vg/ml in about 1 to about 5 ml; at a dose of about 1 to about 3 ml of IxlO 7 to about IxlO 14 vg/ml; or at a dose of about 1 to about 2 ml of IxlO 8 to about IxlO 13 vg/ml.
  • AAV-ApoE2 particles are administered at a dose of about IxlO 8 to about IxlO 15 vg/kg body weight of the mammal being treated.
  • AAV- ApoE2 particles can be administered at a dose of about IxlO 8 vg/kg, about 5xl0 8 vg/kg, about IxlO 9 vg/kg, about 5xl0 9 vg/kg, about IxlO 10 vg/kg, about 5xlO 10 vg/kg, about IxlO 11 vg/kg, about 5xlO n vg/kg, about lx 10 12 vg/kg, about 5xl0 12 vg/kg, about IxlO 13 vg/kg, about 5xlO 13 vg/kg, about IxlO 14 vg/kg, about 5xl0 14 vg/kg, or about IxlO 15 vg/kg body weight of the mammal being treated.
  • Administration of AAV-ApoE2 particles may be in one or more doses. Multiple doses may be administered as is required to maintain adequate enzyme activity, for example.
  • pharmaceutically acceptable and “physiologically acceptable” mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • a pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects.
  • Such composition, “pharmaceutically acceptable” and “physiologically acceptable” formulations and compositions can be sterile. Such pharmaceutical formulations and compositions may be used, for example, in administering a viral particle to a subject.
  • Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • solvents aqueous or non-aqueous
  • solutions aqueous or non-aqueous
  • emulsions e.g., oil-in-water or water-in-oil
  • suspensions syrups, elixirs, dispersion and suspension media
  • coatings isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions typically contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions suitable for injection or infusion of AAV-ApoE2 particles can include sterile aqueous solutions or dispersions which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate form should be a sterile fluid and stable under the conditions of manufacture, use and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • Isotonic agents for example, sugars, buffers or salts (e.g., sodium chloride) can be included.
  • Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solutions or suspensions of AAV-ApoE2 particles can optionally include the following components: a sterile diluent such as water for injection, saline solution, such as phosphate buffered saline (PBS), artificial CSF, fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), glycerin, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, such as phosphate buffered saline (PBS),
  • compositions and delivery systems appropriate for the compositions, methods and uses of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklasa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., 2004 ).
  • AAV-ApoE2 particles and compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage unit forms are dependent upon the amount of AA V-TPP1 particles necessary to produce the desired effect(s).
  • the amount necessary can be formulated in a single dose or can be formulated in multiple dosage units.
  • the dose may be adjusted to a suitable AAV-ApoE2 particles concentration, optionally combined with an anti-inflammatory agent, and packaged for use.
  • compositions will include sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate symptoms of a disease state in question or an amount sufficient to confer the desired benefit.
  • Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol.
  • salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • a “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect).
  • Unit dosage forms may be within, for example, ampules and 20 vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • AA V-TPP1 particles will contain an effective amount of the rAAV particles in a vehicle, the effective amount being readily determined by one skilled in the art.
  • the AA V-TPP1 particles may typically range from about 1 % to about 95% (w/w) of the composition, or even higher if suitable.
  • the quantity to be administered depends upon factors such as the age, weight and physical condition of the mammal or the human subject considered for treatment. Effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • a “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and/or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • the promoter comprises a sequence having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the nucleic acid set forth in wild-type human ependymal promoter.
  • An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5' ->3' or 3’ - >5') and may be capable of functioning even when positioned either upstream or downstream of the promoter.
  • Promoters and/or enhancers may be derived in their entirety from a native gene or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments.
  • a promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate/control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.
  • transgene is used herein to conveniently refer to a nucleic acid sequence/polynucleotide that is intended or has been introduced into a cell or organism.
  • Transgenes include any nucleic acid, such as a gene that encodes an inhibitory RNA polypeptide or protein (e.g., TTP1) and are generally heterologous with respect to naturally occurring genomic sequences.
  • transduce refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (e.g., a viral particle).
  • a vector e.g., a viral particle
  • Introduction of a trans gene into a cell by a viral particle can therefore be referred to as “transduction” of the cell.
  • the trans gene may or may not be integrated into genomic nucleic acid of a transduced cell. If an introduced trans gene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced trans gene may exist in the recipient cell or host organism extra chromosomally, or only transiently.
  • a “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction.
  • a “transduced” cell is a cell into which, or a progeny thereof in which a transgene has been introduced.
  • a transduced cell can be propagated, transgene transcribed and the encoded protein expressed.
  • a transduced cell can be in a mammal.
  • modify or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence.
  • a particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, e.g., a missense or nonsense mutation.
  • a “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type.
  • the sequence may be genetically modified without altering the encoded protein sequence.
  • the sequence may be genetically modified to encode a variant protein, e.g., a variant ApoE2 protein.
  • a nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein.
  • codons of such a nucleic acid variant will be changed without altering the amino acids of a ApoE2 protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of a protein encoded thereby.
  • polypeptides encoded by a “nucleic acid” or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild- type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide (e.g., ApoE2) retains some degree of function or activity.
  • polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal.
  • Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., about I to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 10 or more nucleotides or residues).
  • nucleotide or amino acid substitutions e.g., about I to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 10 or more nucleotides or residues).
  • an amino acid modification is a conservative amino acid substitution or a deletion.
  • a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (e.g., wild-type sequence).
  • Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors, to the central nervous system, such as to distinct brain regions.
  • variants are a sequence that is substantially similar to the sequence of the native molecule.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.
  • nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, e.g., 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81 %-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.
  • the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type).
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 5 77%, 78%, or 79%, or at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91 %, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • substantially identical in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91 %, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
  • An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide.
  • a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those predisposed (e.g., as determined by a genetic assay).
  • kits with packaging material and one or more components therein typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • a kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, and/or viral particles.
  • kits refers to a physical structure housing one or more components of the kit.
  • Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
  • Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.
  • Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.
  • Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD- ROM/RAM, DVD, MP3, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH memory, hybrids and memory type cards. VII. Examples
  • This mouse model has 2 mutations that cause human familial Alzheimer's disease (in APP and PSI) as well as a knock in of human ApoE4 into the mouse ApoE locus. It develops amyloid plaques starting at 4 months of age, which are sometimes surrounded by activated microglia.
  • FIG. 1 is a western blot of ApoE expression in knockout mice AAV-EP+ delivery.
  • FIG. 2 is a chart of ApoE expression in knockout mice following AAV-EP+delivery.
  • FIG. 3 is a western blot of hprEp driven expression of ApoE2.
  • FIG. 4 is a chart showing hprEp driven expression of ApoE2.
  • the hprEp promoter drives higher expression than unbiquitous CMV immediate enhancer/p-actin (CAG) promoter in mice.
  • ApoE-/- (null) mice were injected with 6e9 vg of AAV-EP+ to the lateral ventricle under either a ubiquitous CAG promoter or the hprEp promoter to their right lateral ventricle at equal doses.
  • Adult ApoE-/- mice were injected with 6e9 vg to lateral ventricle.
  • Protein was extracted from ependymal tissues microdissected from all animals and subjected to automated Western blot technology (WES). From the intensity of the bands, ApoE2 driven by the hprEp promoter expressed higher amounts of ApoE2 protein than the CAG promoter.
  • WES Western blot technology
  • FIG. 5A is a plot showing dose dependent increases in viral copies.
  • FIG. 5B is a plot of dose dependent increases in ApoE expression.
  • FIG. 6 is an image of tissue stains of plaque deposition following AAV-EP+ delivery.
  • FIGS. 7A-E are charts showing plaque deposition following AAV-EP+ delivery.
  • AAV EP+ delivery reduced plaque deposition, number, and size in a dose-dependent manner, as shown by immunohistochemistry for ThioS in the cortex of dosed APP/PS l/ApoE4 animals.
  • N indicated as each mouse is an individual dot with open circles as females and closed as males. Post Hoc tests are shown as Dunnett’s multiple comparisons test comparing with vehicle, p * p ⁇ 0.05, ** p
  • FIG. 8 is an image of tissue stains of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 9 is a graph of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 10A is a chart of microgliosis near plagues following AAV-EP+ delivery.
  • FIG. 10B is a graph of microgliosis near plagues following AAV-EP+ delivery.
  • ApoE2 reduced microgliosis near plaques as shown by immunohistochemistry for IBA1 and Ap in the cortex of dosed APP/PSl/ApoE4 animals. Images were scored for level of Ibal intensity by two independent blinded investigators.
  • FIG. 11A is graph of Ibal expression following AAV-EP+ delivery.
  • FIG. 11B is graph of Gfap expression following AAV-EP+ delivery. No significant change in Ibal expression was shown due to ApoE2 expression (relative to -actin). Gfap expression relative to P-actin was restored to vehicle levels with ApoE2 2E10vg, and elevated with ApoE2 7e9 vg and 7el0 vg.
  • FIG. 12 is an RNAscope image of P2ryl2 and Clec7a transcript expression following AAV-EP+ delivery.
  • FIG. 13 A is a chart of P2ryl2 expression following AAV-EP+ delivery.
  • FIG. 13B is a chart of Clec7a expression following AAV-EP+ delivery.
  • FIG. 14 is an image from immunohistochemistry for PSD95 and Ap in ApoE4 animals following dose dependent AAV-EP+ delivery.
  • FIGS. 15A-C are graphs/charts of synapse density following dose dependent AAV- EP+ delivery.
  • APAAV-EP+ delivery reduced synaptic loss as shown by immunohistochemistry for PSD95 and Ap in the cortex of dosed APP/PSl/ApoE4 animals.
  • N indicated as each mouse is an individual dot with open circles as females and closed as males. Post Hoc tests are shown as Dunnett's multiple comparisons test comparing with vehicle, p * p ⁇ 0.05.
  • NHP Study experimental design Intracerebroventricular injection of AAV-Ep+ capsid was administered with hprEp driving ApoE2 into wild type African green monkeys at lel3 or 3el3 vg/ animal. Tissue was collected 150 days post injection. ApoE expression is confirmed in the ependymal cell layer by RNAscope. The ependymal cells are identified by expression of the marker FOXJ1. As shown in FIG. 16, the ApoE probe detects both human and NHP ApoE, but ApoE is not endogenously expressed in the ependyma, therefore ApoE expression in the ependyma is due to AAV-Ep+ viral transduction.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Abstract

La présente divulgation concerne des vecteurs AAV modifiés qui expriment l'allèle ε2 de l'apolipoprotéine E (ApoE2) chez un sujet. Des aspects de la divulgation concernent un vecteur de virus adéno-associé modifié 1 (AAV1) comprenant une protéine de capside qui comprend un peptide de ciblage et une molécule d'acide nucléique comprenant une séquence codant pour le génome d'AAV modifié comportant un gène de réplication (rep), un gène de capside (cap) et un transgène ApoE2.
PCT/US2024/052596 2023-10-23 2024-10-23 Vecteurs aav pour l'administration d'apoe2 Pending WO2025090631A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US10407724B2 (en) * 2012-02-09 2019-09-10 The Hospital For Sick Children Methods and compositions for screening and treating developmental disorders
WO2021102234A1 (fr) * 2019-11-22 2021-05-27 The Children's Hospital Of Philadelphia Variants de vecteurs viraux adéno-associés
WO2023109911A1 (fr) * 2021-12-15 2023-06-22 National Institute Of Biological Sciences, Beijing Microglie ayant un car et son utilisation
WO2023198745A1 (fr) * 2022-04-12 2023-10-19 Uniqure Biopharma B.V. Régulation d'apoe par un acide nucléique

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Publication number Priority date Publication date Assignee Title
US10407724B2 (en) * 2012-02-09 2019-09-10 The Hospital For Sick Children Methods and compositions for screening and treating developmental disorders
WO2021102234A1 (fr) * 2019-11-22 2021-05-27 The Children's Hospital Of Philadelphia Variants de vecteurs viraux adéno-associés
WO2023109911A1 (fr) * 2021-12-15 2023-06-22 National Institute Of Biological Sciences, Beijing Microglie ayant un car et son utilisation
WO2023198745A1 (fr) * 2022-04-12 2023-10-19 Uniqure Biopharma B.V. Régulation d'apoe par un acide nucléique

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CARRELL ELLIE M., CHEN YONG HONG, RANUM PAUL T., COFFIN STEPHANIE L., SINGH LARRY N., TECEDOR LUIS, KEISER MEGAN S., HUDRY ELOISE,: "VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF", MOLECULAR THERAPY-NUCLEIC ACIDS, CELL PRESS, US, vol. 33, 1 September 2023 (2023-09-01), US , pages 296 - 304, XP093312619, ISSN: 2162-2531, DOI: 10.1016/j.omtn.2023.07.016 *
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