US20230142867A1 - Aav native-neuro platform and use for neuronal disease gene therapy - Google Patents

Aav native-neuro platform and use for neuronal disease gene therapy Download PDF

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US20230142867A1
US20230142867A1 US17/920,483 US202117920483A US2023142867A1 US 20230142867 A1 US20230142867 A1 US 20230142867A1 US 202117920483 A US202117920483 A US 202117920483A US 2023142867 A1 US2023142867 A1 US 2023142867A1
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disease
neuronal
raav vector
raav
goi
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Senthil Ramu
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Aav Gene Therapeutics Inc
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Aav Gene Therapeutics Inc
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Definitions

  • AAV Adeno Associated Virus
  • Efficient and specific delivery to a target organ, such as the brain, is a rate limiting step in many protein-based therapeutics. There are multiple barriers to efficient direct protein delivery to the CNS.
  • BBB blood-brain barrier
  • TJs intercellular tight junctions
  • gene transfer vehicles attempting entry into the CNS are forced to go through the transcellular route.
  • specialized lumen facing efflux transporters which include both the ABC family of transporters and multidrug resistance-related proteins, contribute to the formation of the “transport barrier” that prevents xenobiotic substances from gaining access to the neuronal environment.
  • trancytotic/endosomal sorting Another obstacle that partially prevents endothelial crossing of gene-therapy vehicles is the trancytotic/endosomal sorting, which can lead to degradation.
  • endothelium is the greatest obstacle in restricting access to the CNS, the presence of a complex extracellular space, molecular composition and geometry also significantly impede diffusion of viral-based gene delivery systems. That is, even if a therapy can be designed to target one of these receptors and traverse the BMVEC cytoplasm to be released at the basal cell membrane, only the first of many guardian mechanisms at the BBB has been overcome.
  • the CAG/CBA/CB promoter is generally viewed as a ubiquitous promoter as it works in numerous tissues and cell types. However, the assumption that it is highly active in every cell type may not be entirely warranted in the CNS. Studies have been conducted using AAV vectors with the same capsid but carrying different CNS cell specific promoters or the CBA promoter. The apparent discrepancy in CNS transduction profiles of these different AAV vectors suggests that the CBA promoter may not mediate detectable transgene expression in all cell types in the brain.
  • AAV vectors with numerous tropism carrying the cytomegalovirus (CMV), CBA, Rous sarcoma virus (RSV), or human GUSB promoters to drive transgene expression, in the context of intra-parenchymal delivery to different brain structures.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • human GUSB promoters to drive transgene expression, in the context of intra-parenchymal delivery to different brain structures.
  • AAV vectors for treating neurological diseases have used the CMV immediate early promoter followed by a chimeric intron composed of a CMV splice donor and a human globin splice acceptor, or a version of the CAG/CBA promoter (supra).
  • AAV vectors carrying mammalian promoters such as the mouse phosphoglycerate kinase (PGK) promoter, or the rat neuron-specific enolase (NSE) promoter.
  • PGK mouse phosphoglycerate kinase
  • NSE rat neuron-specific enolase
  • AAV2 vector encoding aromatic amino acid decarboxylase AADC
  • AAV-CMV or AAV-CAG/CBA vectors are their ability to drive strong gene expression in many neurons in the CNS of multiple species.
  • the disparate CNS transduction results between cellular promoters and strong non-mammalian promoters raises the possibility that the current generation of AAV vectors express transgenes at exceptionally high levels in a small percentage of all cells that harbor transcriptionally ready vector genomes in the nucleus.
  • TPP-I human tripeptidyl peptidase
  • the potential for toxicity due to excessive transgene expression is particularly acute in neurological diseases where the expressed proteins are not secreted, and thus the need for high level expression is even less.
  • the development of an AAV gene therapy approach for Rett syndrome represents a significant challenge to the prevalent approach of simply using strong promoters. This disease is caused by mutations in the MeCP2 gene on the X-chromosome. Interestingly, duplication of this gene also leads to neurodevelopmental delays, suggesting that expression of this gene in the CNS need to be tightly controlled, which is a challenge to current AAV gene therapy.
  • the invention described herein provides a gene replacement therapy platform to replace mutated genes by AAV Gene Therapy.
  • exemplary central nervous system (CNS) disease drug protypes are packaged in recombinant AAV vectors for intravenous administration to a patient, in order to express functional genes in native neuronal cells.
  • the technology is designed for intravenous dosing of AAV to express genes precisely in intended cell types by using endogenous/native promoter fused with viral/heterologous enhancer.
  • the gene cassette in the AAV vector of the invention begins with AAV inverted terminal repeat (ITR), followed by proprietary heterologous enhancer, followed by a native promoter of a gene of interest (GOI), 5′UTR, proprietary codon optimized gene of interest (GOI), 3′UTR, and ends in another AAV inverted terminal repeat (ITR).
  • ITR AAV inverted terminal repeat
  • GOI gene of interest
  • 5′UTR native promoter of a gene of interest
  • GOI proprietary codon optimized gene of interest
  • 3′UTR proprietary codon optimized gene of interest
  • a recombinant adeno-associated viral (rAAV) vector comprising: (1) a polynucleotide comprising a gene of interest (GOI), under the transcriptional control of the native/endogenous promoter of said GOI; (2) a heterologous enhancer that enhances transcription of the GOI from the native/endogenous promoter; and, (3) a pair of inverted terminal repeat (ITR) flanking the polynucleotide.
  • GOI gene of interest
  • ITR inverted terminal repeat
  • the GOI is a neuronal gene, or is a gene expressed in neuronal tissues (CNS and/or PNS).
  • the GOI is defective (e.g., loss-of-function) in a neuronal disease or condition.
  • the neuronal disease or condition is a CNS disease or condition.
  • the CNS disease or condition is: Ceroid-Lipofuscinosis, Fucosidosis, Giant Axonal Neuropathy, Globoid cell leukodystrophy, Mucolipidiosis Type IV, Neuronal Ceroid Lipofusinoses, Niemann-Pick Disease, Sandhoff Disease, Sanfilippo syndrome, Tay-Sachs Disease, Hunter Syndrome, Sialidosis, Multiple Sulfatase Deficiency, Childhood Epilepsy, CMT Peripheral Neuropathy, Neuronal Ceroid Lipofusinoses, Aspartylglycosaminuria, Mucopolysaccharidoses, Parkinsons Disease/Symptoms, GM1-Gangliosidosis, Charcot-Marie-Tooth Type 1A, Retts Syndrome, Dannon Disease, Mucopolysaccharidoses (due to GNS, HGSNAT, NAGLU, or ARSB), Sly Syndrome, Biotin basal ganglia disease, Pelizaeus-M
  • the GOI is: CLN3, FUCA1, GAN, GALC, MCOLN1, PPT1, SMPD1, HEXB, SGSH, HEXA, IDS, NEU1, SUMF1, GAT1/SLCA1, FIG. 4 , CLN5, AGA, IDUA, GDNF, GLB1, PMP22/MFN2, MECP2, LAMP2, NAGLU, ARSB, GUSB, SLC19A3, PLP1, TPP1/CLN2, ACY2/ASPA, MANBA, LIPA, CTNS, GNS, HGSNAT, NEUROD1, SLC17A5, CLN6, CLN8, or GM2A.
  • the rAAV has a tropism for neuronal cells, or for a cell in neuronal tissue.
  • the polynucleotide further comprises a 5′-UTR coding region, and a 3′-UTR coding region.
  • the heterologous enhancer is a viral enhancer.
  • the viral enhancer is CMV enhancer.
  • the viral enhancer is SV-40 enhancer.
  • Another aspect of the invention provides a pharmaceutical composition comprising any one of the rAAV vector of the invention.
  • the pharmaceutical composition is formulated for intravenous administration (e.g., to the blood) or intrathecal administration (e.g., to spinal fluid).
  • Another aspect of the invention provides a cell infected with any one of the rAAV vector of the invention.
  • Another aspect of the invention provides a recombinant AAV virus comprising any one of the rAAV vector of the invention, wherein the serotype or pseudotype of the recombinant AAV virus is for preferential infection of a neuronal cell or a cell in neuronal tissue.
  • Another aspect of the invention provides a method of treating a (genetic) disease or disorder in a subject, the method comprising administering the recombinant AAV virus of the invention into the subject.
  • the genetic disease or disorder is a neuronal disease or disorder, and/or wherein the recombinant AAV virus preferentially infects a neuronal cell or a cell in neuronal tissue of the subject, thereby treating the neuronal disease or disorder.
  • the rAAV virus is administered to the subject intravenously or intrathecally.
  • Another aspect of the invention provides a method of producing any one of the rAAV vector of the invention, the method comprising introducing any one of the rAAV vector of the invention into a packaging cell line that constitutively or inducibly provides rep/cap proteins in trans.
  • the rep/cap proteins comprise one or more AAV9 capsid proteins.
  • FIG. 1 is a schematic drawing (not to scale) showing a generic structure of the subject rAAV vector, as well as an intended mode of action for the vector.
  • the Gene of Interest (GOI) is put under the control of its native promoter. Transcription from the native promoter is further enhanced by the presence of a powerful enhancer, such as a viral enhancer like CMV enhancer, to facilitate the expression of the GOI in the native cells such GOI is normally expressed.
  • the entire recombinant sequence is flanked by two AAV ITR sequences, such as AAV9 ITR sequences for the preferred neuronal cell tropism.
  • FIG. 2 is a gel image confirming the construction of rAAV viral vectors comprising a number of representative GOI's that are defective in neuronal diseases/disorders. Also shown is a rAAV vector encoding a GFP reporter gene, which can be switched out by any of the GOI's.
  • FIG. 3 is a gel image confirming the construction of additional rAAV viral vectors comprising a number of representative GOI' s that are defective in neuronal diseases/disorders.
  • the invention described herein provides methods and compositions for using recombinant viral vectors, such as AAV-based viral vectors or lentiviral vectors to express any gene-of-interest (GOI) under the control of its native promoter so that the GOI is preferentially and exclusively expressed in the cells the GOI is intended to be expressed.
  • the GOI is further under the transcriptional control of a powerful heterologous enhancer, such as a viral enhancer like CMV enhancer, which further boosts the transcription and expression of the GOI. Therefore, after a single intravenous treatment, the protein product of interest is expressed at high levels only in the endogenous/native cell types, thereby improving therapeutic safety and efficacy.
  • the invention described herein provides a recombinant adeno-associated viral (rAAV) vector, comprising: (1) a polynucleotide comprising a gene of interest (GOI), under the control of the native/endogenous promoter of said GOI; (2) a heterologous enhancer that enhances transcription of the GOI from the native/endogenous promoter; and, (3) a pair of inverted terminal repeat (ITR) flanking the polynucleotide.
  • a adeno-associated viral vector comprising: (1) a polynucleotide comprising a gene of interest (GOI), under the control of the native/endogenous promoter of said GOI; (2) a heterologous enhancer that enhances transcription of the GOI from the native/endogenous promoter; and, (3) a pair of inverted terminal repeat (ITR) flanking the polynucleotide.
  • a adeno-associated viral vector comprising: (1) a polynucleotide
  • the GOI is a neuronal gene.
  • the neuronal gene is defective (e.g., loss-of-function) in a neuronal disease or condition.
  • the neuronal disease or condition is a CNS disease or condition.
  • the CNS disease or condition is: Ceroid-Lipofuscinosis, Fucosidosis, Giant Axonal Neuropathy, Globoid cell leukodystrophy, Mucolipidiosis Type IV, Neuronal Ceroid Lipofusinoses, Niemann-Pick Disease, Sandhoff Disease, Sanfilippo syndrome, Tay-Sachs Disease, Hunter Syndrome, Sialidosis, Multiple Sulfatase Deficiency, Childhood Epilepsy, CMT Peripheral Neuropathy, Neuronal Ceroid Lipofusinoses, Aspartylglycosaminuria, Mucopolysaccharidoses, Parkinsons Disease/Symptoms, GM1-Gangliosidosis, Charcot-Marie-Tooth Type 1A, Retts Syndrome, Dannon Disease, Mucopolysaccharidoses (due to GNS, HGSNAT, NAGLU, or ARSB), Sly Syndrome, Biotin basal ganglia disease, Pelizaeus-M
  • the GOI is: CLN3, FUCA1, GAN, GALC, MCOLN1, PPT1, SMPD1, HEXB, SGSH, HEXA, IDS, NEU1, SUMF1, GAT1/SLCA1, FIG. 4 , CLN5, AGA, IDUA, GDNF, GLB1, PMP22/MFN2, MECP2, LAMP2, NAGLU, ARSB, GUSB, SLC19A3, PLP1, TPP1/CLN2, ACY2/ASPA, MANBA, LIPA, CTNS, GNS, HGSNAT, NEUROD1, SLC17A5, CLN6, CLN8, or GM2A.
  • the rAAV has a tropism for neuronal cells.
  • the rAAV serotype is, AAV2.5, rAAVrh.39, rAAVrh.43, rAAV7, rAAVrh.8 or rAAVrh.10.
  • the rAAV serotype is AAV9. In certain embodiments, the rAAV has a pseudotype of xxx. In certain embodiments, the polynucleotide further comprises a 5′-UTR coding region, and a 3′-UTR coding region.
  • the heterologous enhancer is a viral enhancer.
  • the viral enhancer is CMV enhancer.
  • the viral enhancer is SV40 enhancer.
  • Another aspect of the invention provides a pharmaceutical composition comprising the rAAV vector of the invention.
  • the pharmaceutical composition is formulated for intravenous administration.
  • Another aspect of the invention provides a cell infected with the rAAV vector of the invention.
  • Another aspect of the invention provides a recombinant AAV virus comprising the rAAV vector of the invention, wherein the serotype or pseudotype of the recombinant AAV virus is for preferential infection of a neuronal cell.
  • Another aspect of the invention provides a method of treating a (genetic) disease or disorder in a subject, the method comprising introducing the recombinant AAV virus of the invention into the subject.
  • the genetic disease or disorder is a neuronal disease or disorder
  • the recombinant AAV virus preferentially infects a neuronal cell of the subject, and treats the neuronal disease or disorder.
  • Another aspect of the invention provides a method of producing the rAAV vector of the invention, comprising introducing the rAAV vector of the invention into a packaging cell line that constitutively or inducibly provides rep/cap proteins in trans.
  • the recombinant AAV vector and recombinant AAV virus of the invention, together with the pharmaceutical composition comprising the same, can be used to treat a number of diseases and conditions, particularly neuronal diseases or indications treatable by gene therapy.
  • Diseases or conditions treatable by the AAV vector/virus/method of the invention are typically characterized by lacking a functional neuronal protein or enzyme, which neuronal diseases or indications can be alleviated (if not cured), treated, or prevented (e.g., at least the onset is delayed), by providing functional versions of the proteins or enzymes.
  • the externally provided functional versions of the proteins or enzymes may be identical in sequence to the wild-type proteins or enzymes that are lacking in a host having such diseases or conditions, or may be a variant of the wild-type proteins or enzymes.
  • the neuronal diseases or conditions can be treated by introducing into the host the subject AAV virus that will infect target host (neuronal) tissues, and stably express the encoded GOI that is defective in the diseases or conditions throughout the life span of the host.
  • the subject AAV virus may only need to be introduced into the host once, or can be introduced into the host multiple times as needed.
  • polynucleotide sequences of the invention encoding several genes-of-interest (GOI) are listed below. These polynucleotide sequences can be part of the subject AAV vector sequences. These sequences are for illustrative purpose only and are non-limiting.
  • GAN Giant Axonal Neuropathy
  • Giant axonal neuropathy is a severe, slowly progressive neurodegenerative disorder characterized by progressive motor and sensory peripheral neuropathy, central nervous system involvement (including pyramidal and cerebellar signs), and characteristic kinky hair in most cases.
  • GAN is inherited as an autosomal recessive trait, and is associated with more than 50 different causative mutations in GAN gene (16q24.1), encoding the ubiquitously expressed cytoskeletal protein gigaxonin.
  • the GOI is GAN that encodes gigaxonin
  • the disease or condition treatable by the subject AAV vector/virus is Giant Axonal Neuropathy (GAN).
  • GAN Giant Axonal Neuropathy
  • the composition and methods of the invention can be used to produce gigaxonin expressed permanently from AAV vectors stably maintained with the host's genome as extra chromosomal material, in native neuronal cells that normally express GAN, thus effectively treating giant axonal neuropathy.
  • a representative polynucleotide of the invention that can be encoded by the AAV vector of the invention is described below.
  • double underlined sequences represent proprietary viral enhancer sequences (SEQ ID NO: 1, see below); bold sequences are native promoter sequences for GAN; italic sequences are coding sequence for the GOI (GAN in this case); the subscripted sequences immediately 5′ and 3′ to the GOI coding sequence are 5′-UTR and 3′-UTR sequences, respectively.
  • the proprietary enhancer sequences (SEQ ID NO: 1) is listed below.
  • the GAN protein sequence is:
  • Conservative changes to the protein sequence above e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative residue changes, or up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% conservative residue changes
  • a functional equivalent version of the protein e.g., that retains at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% of the wild-type enzyme activity
  • nucleotide sequence encoding the protein and variants thereof such as in SEQ ID NO: 2
  • nucleotide sequence changes that do not result in sequence change of the encoded protein in SEQ ID NO: 3 due to, for example, codon degeneracy are within the scope of the invention.
  • codon optimization of any of the polynucleotide sequence for optimal expression in a specific type of host cell is also within the scope of the invention (see further details below).
  • Neuronal ceroid lipofuscinosis refers to a family of at least eight genetically separate neurodegenerative lysosomal storage diseases that result from excessive accumulation of lipopigments (lipofuscin) made up of fats and proteins in the body's tissues.
  • lipopigments lipopigments
  • the most commonly identified mutations are in the CLN3 gene, located on the short arm of chromosome 16 (16p12.1), found in childhood or juvenile NCLs (JNCLs).
  • JNCL is an autosomal recessive disorder.
  • the normal function of CLN3 gene is not presently known, but it is known to be a transmembrane protein.
  • Studies of the yeast CLN3 ortholog which encodes Battenin have suggested that the protein may play a role in lysosomal pH homeostasis.
  • recent studies have also implied the protein's role in cathepsin D deficiency; the overexpression of the defective protein appears to have significant effects on cathepsin D processing, with implications suggesting that accumulation of ATP synthase subunit C would result.
  • the GOI is CLN3, and the disease or condition treatable by the subject AAV vector/virus is JNCL.
  • the composition and methods of the invention can be used to produce CLN3 gene product expressed permanently from AAV vectors stably maintained with the host's genome as extra chromosomal material, in native neuronal cells that normally express CLN3, thus effectively treating JNCL.
  • a representative polynucleotide of the invention that can be encoded by the AAV vector of the invention is described below.
  • the proprietary enhancer sequence is in SEQ ID NO: 1.
  • Native CLN3 promoter is known in the art.
  • the coding sequence for CLN3 and the 5′ and 3′ UTR sequences are listed below using the same nomenclature.
  • the CLN3 protein sequence is:
  • nucleotide sequence encoding the protein and variants thereof such as in SEQ ID NO: 4
  • nucleotide sequence changes that do not result in sequence change of the encoded protein in SEQ ID NO: 5 due to, for example, codon degeneracy are within the scope of the invention.
  • codon optimization of any of the polynucleotide sequence for optimal expression in a specific type of host cell is also within the scope of the invention.
  • a representative (non-limiting) list of the treatable diseases and disorders, along with the corresponding GOI's, are provides in the table below.
  • the FUCA1 protein sequence is:
  • the GALC protein sequence is:
  • the MCOLN1 protein sequence is:
  • the PPT1 protein sequence is:
  • the SMPD1 protein sequence is:
  • the HEXB protein sequence is:
  • the SGSH protein sequence is:
  • the HEXA protein sequence is:
  • the proprietary viral enhancer sequences, native promoter sequences, coding sequence for HEXA and the 5′ and 3′ UTR sequences are listed below using the same nomenclature.
  • the IDS protein sequence is:
  • the NEU1 protein sequence is:
  • the SUMF1 protein sequence is:
  • the GAT1 protein sequence is:
  • the FIG. 4 protein sequence is:
  • FIG. 4 DNA Sequence
  • the CLN5 protein sequence is:
  • the AGA protein sequence is:
  • the IDUA protein sequence is:
  • the GDNF protein sequence is:
  • the GLB 1 protein sequence is:
  • the PMP22/MFN2 protein sequence is:
  • the MECP2 protein sequence is:
  • the LAMP2 protein sequence is:
  • the NAGLU protein sequence is:
  • the ARSB protein sequence is:
  • the GUSB protein sequence is:
  • the SLC19A3 protein sequence is:
  • the PLP1 protein sequence is:
  • the TPP1/CLN2 protein sequence is:
  • the ACY2/ASPA protein sequence is:
  • the MANBA protein sequence is:
  • the LIPA protein sequence is:
  • CTNS protein sequence is:
  • the GNS protein sequence is:
  • the HGSNAT protein sequence is:
  • the NeuroD1 protein sequence is:
  • the SLC17A5 protein sequence is:
  • the CLN6 protein sequence is:
  • the CLN8 protein sequence is:
  • the GM2A protein sequence is:
  • the HYAL1 protein sequence is:
  • any of the GOI described herein are codon optimized for manufacturing, optimal expression in a host cell, such as in a human cell. Further Codon optimization of GOI can be done based on codon optimization table publically available, such as the Codon and Codon Pair Usage Tables (CoCoPUTs) maintained at the George Washington University (GWU).
  • the CoCoPUTs is derived from all available GenBank and RefSeq data source.
  • the GOI's are codon optimized for expression in neuronal tissues or specific cell types in neuronal tissues, such as neurons, depending on the target cell type the GOI's are to be expressed.
  • TissueCoCoPUTs which are human tissue-specific codon and codon pair usage tables derived from human genomic codon usage information and tissue-specific transcriptomic data.
  • the TissueCoCoPUTs maintained by the GWU website represent 52 human tissues.
  • Transcriptomic data are derived from the Genotype Tissue Expression (GTEx) Portal.
  • the viral vectors (such as AAV) of the invention are delivered to neuronal target cells by intravenous injection or direct intraparenchymal injection or direct intracranial injection. Such delivery means effectively bypass the BBB, and are common means for the administration of gene therapeutics to the brain.
  • the neuronal target cells intended to receive gene therapy through intraparenchymal injection are clustered in a focal location, and are this particularly suitable for focal delivery of the viral vector.
  • the viral vector (such as AAV) of the invention is delivered locally or compartmentally, such as direct intraocular injection, or injection in the cochlea.
  • the viral vectors of the invention are delivered to neuronal target cells by intravenous injection.
  • the neuronal target cells intended to receive gene therapy through intravenous injection are globally located in the CNS, or in multiple CNS regions.
  • Such global delivery across the BBB is achieved by using selected AAV serotypes (such as AAV9) that have been shown to deliver genes across the BBB after intravenous injection.
  • the viral vector (such as AAV) of the invention is delivered to achieve widespread delivery for diseases of extensive pathology including glioblastoma multiforme (GBM), lysosomal storage diseases (LSDs), Alzheimer's disease, Parkinson's disease and Canavan's disease.
  • the viral vectors are delivered intracerebroventricularly, intrathecally, intranasally, or systemicaly (e.g., intravenous and intra-arterial injections).
  • the systemic delivery route for gene delivery vehicles is very promising, and has the potential to be a conduit to the entire CNS (every cell in the brain is a maximum distance of 40 ⁇ m from an endothelial cell), provided that the issue of BBB blockage and the filtration systems of the spleen and liver are adequately dealt with.
  • the viral vectors (such as AAV) of the invention are delivered to neuronal target cells by intravascular, intrathecal, and/or cerebrospinal fluid (CSF) administration.
  • CSF cerebrospinal fluid
  • Many AAV capsids such as AAV9, AAV2.5, rAAVrh.39, rAAVrh.43, rAAV7, rAAVrh.8 and rAAVrh.10, have remarkable CNS tropism after vascular (Foust et al., Nat Biotechnol. 27:59-65, 2009; Gray et al., Gene Ther. 20:450-459, 2013; Zhang et al., Mol Ther.
  • the viral vectors (such as AAV) of the invention are delivered to neuronal target cells by direct intracranial injection.
  • a large range of AAV capsids including pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5, hu.32, hu.37, pi.2, hu.11, rh.8, hu.48R3, bb2, cy5, rh20, rh39, rh43, AAV7, AAV8, and AAV9, have been demonstrated for their CNS gene transfer properties by direct intracranial injection (Burger et al., Mol Ther. 10:302-317, 2004; Cearley et al., Mol Ther.
  • Vectors based on distinct AAV serotypes or pseudotypes can be chosen for specific applications in the nervous system, depending on their preferential or global tropism within the different regions of the brain and target neuronal tissues.
  • the viral vector is AAV9.
  • AAV9 The vast majority (>90%) of cells in the CNS transduced by intravenously injected AAV9 using strong promoters like CBA are astrocytes and neurons. The distribution of vector is widespread with transduced cells detected in all areas of the brain from the olfactory bulb to cerebellum.
  • delivery of the viral vectors of the invention across the BBB is further enhanced by a variety of techniques including: intra-nasal administration and/or intracranial NSC transplantation that bypasses the BBB; transiently permeating the BBB using osmotic manipulation; magnetic resonance imaging guided focused ultrasound (MRIgFUS) to selectively open the BBB and allow therapeutics to pass into the parenchyma; convection enhanced delivery (Debinski and Tatter, Curr Opin Mol Ther. 12:647-653, 2010); introduction into a fluid space, e.g., vasculature, brain ventricles or intrathecal space in the spinal cord, such that cells lining the space produce the deficient protein (Chen et al., Nat Med.
  • MRIgFUS magnetic resonance imaging guided focused ultrasound
  • the viral vector (such as AAV) of the invention comprises a targeting peptide that bind selectively to the brain endothelial cells or a brain endothelial cell receptor (such as the low-density lipoprotein (LDL) and other related receptors that can be targeted by Apolipoprotein E-derived peptides) to target the viral vector to the brain vasculature.
  • a targeting peptide that bind selectively to the brain endothelial cells or a brain endothelial cell receptor (such as the low-density lipoprotein (LDL) and other related receptors that can be targeted by Apolipoprotein E-derived peptides) to target the viral vector to the brain vasculature.
  • LDL low-density lipoprotein
  • Another aspect of the invention provides a method of producing the rAAV vector of the invention, comprising introducing the rAAV vector of the invention into a packaging cell line that constitutively or inducibly provides rep/cap proteins in trans.
  • the packaging cell line is a HEK293 cell line, a HeLa cell, or an A549 cell.
  • the subject rAAV vector can be produced using any of many art-recognized approach.
  • the rAAV vector is produced based on the helper-virus-free transient transfection method with all cis and trans components (e.g., vector plasmid and packaging plasmids, along with helper genes isolated from adenovirus) in host cells such as 293 cells.
  • the rAAV vector is produced using recombinant herpes simplex virus (rHSV)-based AAV production system, which utilizes rHSV vectors to bring the AAV vector and the Rep and Cap genes into the cells.
  • rHSV herpes simplex virus
  • the rAAV vector is produced based on baculovirus system which requires simultaneous infection of insect cells with several baculovirus vectors to deliver the rAAV vector cassette and the Rep and Cap genes.
  • the rAAV vector is produced based on the AAV producer cell lines derived from HeLa or A549, which stably harbored AAV Rep/cap genes.
  • the AAV vector cassette can either be stably integrated in the host genome or be introduced by an adenovirus that contained the cassette.

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