AAV-5 pseudotype vectors for gene therapy of neurological diseases

Abstract

The present invention relates to methods of treating diseases affecting motor function (e.g., motor function affected by diseases, brain and / or spinal cord injuries) using an adeno-associated virus (AAV) gene therapy vector comprising an adeno-associated virus 5 (AAV5) capsid protein and a gene product of interest flanked by an AAV ITR. In particular, the gene therapy vectors of the invention are administered by injection into cerebrospinal fluid (CSF), preferably by lumbar injection and / or injection into the cerebellar medullary pool.

Description

Field of the Invention

[0001] The present invention relates to the fields of virology and gene therapy. In particular, the present invention relates to a method for treating or preventing neurological diseases in mammals, preferably humans. Background of the Invention

[0003] The central nervous system (CNS) is a complex system of vertebrates, including the brain and spinal cord. The peripheral nervous system is the part of the nervous system outside the brain and spinal cord. The spinal cord conducts sensory information from the peripheral nervous system to the brain and conducts motor information from the brain to various effectors.

[0004] Gene therapy methods using viral vectors have been studied for delivering therapeutic agents to treat various CNS-related diseases. Adeno-associated virus (AAV) vectors have many advantages, including low toxicity and immunogenicity and long-term transgene expression in the CNS (Kaplitt et al (1994) Nat Genet 8:148-154; Bartlett et al. (1998) Hum Gene Ther 9:1181-1186; Passini et al. (2002) J Neurosci 22:6437-6446). Therefore, further research on AAV for CNS gene therapy has been carried out.

[0005] Many CNS diseases, such as amyotrophic lateral sclerosis (ALS, also known as motor neuron disease (MND)), affect cortical and spinal motor neurons that are widely distributed in the CNS. It is often seen that viral vectors injected into the CNS transduce cells near the injection site, but do not transduce cells in the required extensive areas. In addition, injecting into the brain is a surgically invasive method subject to safety considerations. Some studies have been conducted to evaluate gene therapy methods using lumbar puncture. The disadvantages reported in the art are that genes transferred by lumbar puncture cannot transduce neuronal tissue, but only transduce meningeal fibroblasts, unless injected intraparenchymally (Finegold (1999) Hum Gene Ther 10:1251-1257; Milligan (2005) Eur J Neurosci 21:2136-2148; Milligan (2005) Mol Pain 1:9; Milligan (2006) Pain 126:294-308), resulting in transgene expression of less than 2 weeks (Milligan (2005) Eur J Neurosci 21:2136-2148; Milligan (2005) Mol Pain 1:9; Lin (2002) Neurosci Lett 317:1-4; Lin (2002) Gene Ther 9:1247-1253), requiring multiple injections (Milligan (2006) Pain 126:294-308) or requiring pretreatment (Vulchanova (2010) Molecular Pain 6:31).

[0006] Storek and colleagues (Storek (2008) PNAS 105(3):1055-1060) revealed that they could not detect transgene expression using conventional single-stranded rAAV2, so they examined various modifications of rAAV vectors: pseudotyping with capsids of different serotypes and double-stranded self-complementary rAAV. In particular, Storek et al. revealed that self-complementary AAV8 is effective and selectively transduces primary sensory neurons in the dorsal root ganglion when administered intrathecally, but does not transduce in the brain, and long-term gene expression is established after single-vector administration.

[0007] Accordingly, there is still a need in the art for gene transfer methods that can be used to treat or prevent neurological diseases in mammals. There is a particular need for gene transfer methods that transduce neuronal tissue without one or more of the above disadvantages. Summary of the Invention

[0009] In a first aspect, the present invention relates to an adeno-associated virus (AAV) gene therapy vector for use as a medicament in a mammalian subject, preferably a human, wherein the gene therapy vector comprises an AAV serotype 5 capsid protein and a gene product of interest flanked by AAV ITRs, and wherein the gene therapy vector is administered by lumbar intrathecal administration.

[0010] In a preferred embodiment, the lumbar intrathecal administration is at a site selected from L4-L5, L3-L4, L1-L2 and L2-L3.

[0011] In a preferred embodiment, the AAV gene therapy vector is a single-stranded AAV gene therapy vector or a monomeric duplex vector.

[0012] In a preferred embodiment, the gene product of interest is used for treating or preventing a disorder selected from the group consisting of: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), pain, Huntington's disease, Alzheimer's disease, Tay-Sachs disease, Friedreich ataxia, ataxia telangiectasia, spinocerebellar ataxia types 1, 2, and 3, Niemann-Pick disease types A, B, and C, dopa-responsive dystonia, Fragile X syndrome, Krabbe disease, glycogen storage disease type 2 (Pompe), primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, giant axonal neuropathy, multiple system atrophy (MSA), proximal myotonic myopathy, neuronal ceroid lipofuscinosis (Batten disease), and cancer.

[0013] In a preferred embodiment, the AAV ITR is an AAV serotype 2 ITR.

[0014] In a preferred embodiment, the gene product of interest is selected from: Aspartylglucosaminidase, α-Galactosidase A, Palmitoyl Protein Thioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein, Multiple gene products, Cysteine transporter, Acid ceramidase, Acid α-L-fucosidase, Protective protein / cathepsin A, Acid β-glucosidase, or glucocerebrosidase, Acid β-galactosidase, Iduronate-2-sulfatase, α-L-Iduronidase, Galactocerebrosidase, Acid α-mannosidase, Acid β-mannosidase, Arylsulfatase B, Arylsulfatase A, N-Acetylgalactosamine-6-sulfate sulfatase, Acid β-galactosidase, N-Acetylglucosamine-1-phosphotransferase, Acid sphingomyelinase, NPC-1, Acid α-glucosidase, β-Hexosaminidase B, Heparan N-sulfatase, α-N-Acetylglucosaminidase,Acetyl-CoA: α-glucosaminide N-acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, α-N-Acetylgalactosaminidase, α-N-Acetylgalactosaminidase, α-Neuramidase, β-Glucuronidase, β-Hexosaminidase A, Acid Lipase, neurotrophic factors such as Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), Neurotrophin-4 / 5 (NT-4 / 5), Brain derived neurotrophic factor (BDNF), Cerebral Dopamine Neurotrophic factor (CDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), growth factor Insuline-like growth factor (IGF-1) and miRNA for downregulating faulty gene.,

[0015] In a preferred embodiment, the gene product of interest is operably linked to an expression control element comprising a promoter that produces sufficient expression of the gene product of interest to obtain a therapeutic effect, wherein the promoter is preferably selected from: cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK), CAG promoter (a combination of cytomegalovirus early enhancer element and chicken β-actin promoter), glial fibrillary acidic protein (GFAP) promoter, synapsin-1 promoter, neuron-specific enolase (NSE), and inducible promoters such as gene switch or tet-operator-derived promoters.,

[0016] In a preferred embodiment, the gene therapy vector is further administered into the cisterna magna before, simultaneously with, or after intrathecal administration in the lumbar spine.

[0017] In a preferred embodiment, 2×10 13 –2×10 15 、more preferably 8×10 13 –6×10 14 genomic copies per kilogram of body weight are administered to the subject.

[0018] In a preferred embodiment, the subject does not receive intravenous mannitol pretreatment before intralumbar administration of the AAV gene therapy vector.

[0019] In a preferred embodiment, the AAV gene therapy vector is not a self-complementary gene vector.

[0020] Definition

[0021] "Nucleic acid construct" is defined as a nucleic acid molecule that is isolated from a naturally occurring gene or has been modified to contain nucleic acid segments that are combined or juxtaposed in a manner that does not occur naturally. The nucleic acid molecule is represented by a nucleotide sequence. Optionally, the nucleotide sequence present in the nucleic acid construct is operably linked to one or more control sequences that direct the production or expression of a peptide or polypeptide in a cell or subject.

[0022] The term "homologous" when used to denote the relationship between a specified (recombinant) nucleic acid or polypeptide molecule and a specified host organism or host cell is to be understood to mean that the nucleic acid or polypeptide molecule is in fact produced by a host cell or organism of the same species. The term "heterologous" may be used to denote that the nucleic acid or polypeptide molecule is in fact produced by a host cell or organism of a different species.

[0023] "Expression control sequence" refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence operably linked thereto. An expression control sequence is "operably linked" to a nucleotide sequence when it controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, an expression control sequence can include a promoter, an enhancer, an internal ribosome entry site (IRES), a transcription terminator, a start codon preceding a protein-coding gene, an intron splicing signal, and a stop codon. The term "expression control sequence" encompasses at least sequences whose presence is designed to affect expression and can also include additional advantageous elements. For example, a leader sequence and a fusion ligand sequence are expression control sequences. The term can also include the design of such nucleic acid sequences whereby unwanted potential start codons in and out of frame are removed from the sequence. It can also include the design of such nucleic acid sequences whereby unwanted potential splice sites are removed. It includes sequences that direct the addition of a polyA tail or polyadenylation sequence (pA), i.e., a string of adenine residues at the 3'-end of an mRNA, which can be referred to as a polyA sequence. It can also be designed to enhance mRNA stability. Expression control sequences that affect transcriptional and translational stability and are suitable for insect cells, such as promoters and sequences that effect translation such as Kozak sequences, are well known to those skilled in the art. An expression control sequence can have the property of regulating the nucleotide sequence operably linked thereto, thereby achieving a lower or higher level of expression.

[0024] As used herein, the term "promoter" or "transcription regulatory sequence" is a nucleic acid fragment having the function of controlling the transcription of one or more coding sequences, located upstream in the transcriptional direction of the transcription start site of the coding sequence, and structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the amount of transcription from the promoter, including, for example, attenuators or enhancers and silencers. A "constitutive promoter" is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulatable, e.g., by the application of a chemical inducer. A "tissue-specific promoter" is a promoter that is active only in a particular type of tissue or cell.

[0025] "3'UTR" or "3' untranslated sequence" (also referred to as 3' untranslated region or 3' end) refers to a nucleic acid sequence found downstream of the gene coding sequence, which contains, for example, a transcription termination site and a polyadenylation signal (such as AAUAAA or its variants) in most but not all eukaryotic mRNAs. After transcription termination, the mRNA transcript can be cleaved downstream of the polyadenylation signal and a poly(A) tail can be added, which is involved in the transport of the mRNA to the cytoplasm (where translation occurs).

[0026] "Vector" is a nucleic acid molecule (typically DNA or RNA) that transfers a loaded nucleic acid sequence (typically DNA or RNA) into a host cell. Three common types of vectors include plasmids, bacteriophages, and viruses. Preferably, the vector is a virus. Vectors containing a promoter and a cloning site that can be operably linked to a polynucleotide are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or alter the cloned 5' and / or 3' untranslated portions to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the start codon to enhance expression.

[0027] "Viral vector" refers to a vector that contains some or all of the following: viral genes encoding gene products, control sequences, and viral packaging sequences. "Parvovirus vector" is defined as a recombinant parvovirus or parvovirus particle that contains a polynucleotide that is delivered to a host cell in vivo, ex vivo, or in vitro. Exemplary parvovirus vectors include, for example, adeno-associated virus vectors. In this article, a parvovirus vector construct refers to a polynucleotide that contains the viral genome or a portion thereof and a transgene.

[0028] As used herein, the terms "promoter" or "transcription regulatory sequence" refer to a nucleic acid fragment having the function of controlling the transcription of one or more coding sequences, located upstream in the transcriptional direction of the transcription start site of the coding sequence, and structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the amount of transcription from the promoter, including, for example, attenuators or enhancers, and silencers. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer. A "tissue-specific promoter" is a promoter that is active only in a particular type of tissue or cell.

[0029] A promoter can be any suitable promoter sequence that exhibits transcriptional activity in a cell, including mutant, truncated, and hybrid promoters, which can be derived from genes encoding extracellular or intracellular polypeptides that are homologous (native) or heterologous (foreign) to the cell.

[0030] A promoter can be a promoter that is naturally associated with the coding sequence to be expressed. A promoter can also be a constitutive or inducible promoter that is foreign to the coding sequence to be expressed. Suitable promoters for use in mammalian cells are, for example, those described in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Suitable promoters for use in yeast include, for example, glycolytic promoters.

[0031] As used herein, the term "flanking" with respect to a sequence that is additional element sequences means the presence of one or more flanking elements upstream and / or downstream, i.e., at the 5' and / or 3' ends, relative to the sequence. The term "flanking" does not mean that the sequences must be contiguous. For example, there can be intervening sequences between the nucleic acid encoding the transgene and the flanking elements. "Flanking" is the sequence of two other elements (e.g., ITRs), meaning that one element is located at the 5' end of the sequence and the other is located at the 3' end of the sequence; however, intervening sequences can be present therebetween. In a preferred embodiment, the nucleotide sequence of (i) has parvovirus inverted terminal repeat nucleotide sequences on each side.

[0032] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or isochemically modified, non-natural, or derivatized nucleobases. "Oligonucleotide" generally refers to a polynucleotide of single-stranded or double-stranded DNA that is approximately 5 to approximately 100 nucleotides in length. However, for the purposes of the present invention, there is no upper limit on the length of the oligonucleotide. Oligonucleotides are also referred to as oligomers or oligonucleic acids and can be isolated from genes or chemically synthesized by methods known in the art.

[0033] As used herein, the term "treatment" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, completely or partially preventing a disease or its symptoms, and / or can be therapeutic, partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment in a mammal, particularly a human, including:

[0034] (a) preventing the occurrence of a disease in a subject that may be susceptible to the disease but has not been diagnosed as having the disease;

[0035] (b) inhibiting the disease, i.e., arresting its development; and

[0036] (c) alleviating the disease, i.e., causing the disease to regress.

[0037] "Sequence identity" and "sequence similarity" can be determined by comparing two peptide or two nucleotide sequences according to the lengths of the two sequences, using a global or local alignment algorithm. Sequences of similar length are preferably compared using a global alignment algorithm (e.g., Needleman Wunsch), optimally with a full-length sequence alignment, while sequences of significantly different lengths are preferably compared using a local alignment algorithm (e.g., Smith Waterman). Then when the sequences (when optimally aligned by a program such as GAP or BESTFIT using default parameters, for example) exhibit at least a certain minimum percentage sequence identity (as defined below), the sequences can be said to be "substantially identical" or "substantially similar". GAP uses the Needleman and Wunsch global alignment algorithm to compare two sequences of full length (entire length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, global alignment is suitable for determining sequence identity. Generally, using GAP default parameters, the gap creation penalty = 50 (nucleotides) / 8 (proteins), and the gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and scoring for percentage sequence identity can be determined using a computer program, such as the GCG Wisconsin Package, Version 10.3, obtained from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open-source software such as the program "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) of EmbossWIN version 2.10.0, which uses the same parameters as the GAP program described above, or using default setting parameters (for both "needle" and "water" and for both protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; the default scoring matrix is Blossum62 for proteins and DNAFull for DNA). When the sequences are significantly different in overall length, local alignment is preferred, such as those methods using the Smith Waterman algorithm. Alternatively, the percentage similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. DETAILED DESCRIPTION OF THE INVENTION

[0039] To date, gene delivery to the CNS has been hampered by size and the complexity of the CNS. After a single injection into the brain parenchyma, transgene expression is local and mainly remains confined to the injected area. Using CSF as a method of delivering viral vectors, a larger area can be reached. Current studies have shown that the goal of achieving transduction in a larger area of the CNS can be accomplished using AAV-5 vectors. This treatment is very well tolerated as no adverse clinical signs or significant neuronal loss have been observed. Both neurons and glial cells are transduced using the ubiquitous CAG promoter and no transgene expression is observed outside the CNS. Using cell-specific promoters such as the GFAP promoter or the synapsin-1 promoter, expression can be directed to specific populations. We have shown that areas of the cortex, cerebellum, and subventricular zone show transgene expression in neurons and glial cells. In the spinal cord, motor neuron transduction is widespread and transduction of neurons in the dorsal root ganglia is also widespread. These results show that CNS-mediated delivery methods can be used to deliver genes for gene therapy approaches. Indications that can benefit therefrom are: motor neuron diseases, sensory-related indications, and a variety of other neurological indications.

[0040] In a first aspect, the invention relates to an adeno-associated virus (AAV) gene therapy vector for use as a medicament in a mammalian subject. Preferably, the gene therapy vector comprises an AAV serotype 5 capsid protein and a gene product of interest flanked by AAV ITRs. More preferably, the gene therapy vector is administered into the cerebrospinal fluid (CSF), preferably by intrathecal administration, and / or into the cisterna magna, even more preferably by lumbar administration, and / or into the cisterna magna. The invention relates to an AAV gene therapy vector for use as a medicament as described above. That is, the invention provides a method of treating a human or animal body by treating with the AAV gene therapy vector of the invention. The invention further relates to an AAV gene therapy vector as described above for treating a disorder such as a motor neuron disease, a sensory-related indication, and a variety of other neurological disorders. Thus, the invention relates to the use of the AAV gene therapy vector of the invention for the preparation of a medicament for use in a method of treating a disease as further defined herein.

[0041] Viruses of the Parvoviridae family are small DNA animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which affect insects. Members of the Parvovirinae subfamily are referred to herein as parvoviruses and include the Dependovirus genus. As can be deduced from its genus name, members of the Dependovirus genus are unique in that they typically require co-infection with a helper virus such as adenovirus or herpesvirus to be effectively infectious in cell culture. The Dependovirus genus includes AAV, which typically infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Further information on other members of the Parvovirinae and Densovirinae subfamilies is described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996).

[0042] The genomic structures of all known AAV serotypes are very similar. The AAV genome is a linear single-stranded DNA molecule that is less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeat (ITR) sequences flank the unique coding nucleotide sequences of the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2, and -3) make up the capsid. The terminal 145 nt are self-complementary and form a T-shaped hairpin structure, which can form an energetically stable intramolecular duplex. These hairpin structures function as the viral DNA replication origin and serve as primers for the cellular DNA polymerase complex. After infection of mammalian cells with wild-type (wt) AAV, the Rep genes (i.e., Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and both of these Rep proteins have the function of replicating in the viral genome. Splicing events in the Rep ORF result in the expression of actually four proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that the unspliced mRNAs encoding the Rep78 and Rep52 proteins are sufficient for AAV vector production in mammalian cells. In insect cells, the Rep78 and Rep52 proteins are also sufficient for AAV vector production.

[0043] "Recombinant parvovirus or AAV vector" (or rAAV vector) as used herein refers to a vector that contains one or more polynucleotide sequences of interest, gene products of interest, genes of interest or "transgenes", flanked by at least one parvovirus or AAV inverted terminal repeat (ITR). Such rAAV vectors can replicate and package into infectious virus particles when present in insect host cells that express AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g., in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), the rAAV vector is typically referred to as a "pro-vector", which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions. Preferably, the gene product of interest is flanked by AAV ITRs on each side. Any AAV ITR can be used in the constructs of the present invention, including the ITRs of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8 and / or AAV9. Most preferably, the ITR of AAV2 is used. Preferred ITR sequences for use in the preferred nucleic acid constructs of the present invention are, for example, the sequences shown in SEQ ID NO:1 (left or upstream ITR) and SEQ ID NO:2 (right or downstream ITR).

[0044] AAV can infect many mammalian cells. See, for example, Tratschin et al. (1985, Mol. Cell Biol. 5 :3251-3260) and Grimm et al. (1999, Hum. Gene Ther. 10 :2445-2450). However, AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al., Arthritis Res, 3:1 (2001), and the tropicity of AAV varies between serotypes. See, for example, Davidson et al. (2000, Proc. Natl. Acad. Sci. USA, 97 :3428-3432), which discuss the differences in tropism and transduction efficiency of AAV2, AAV4 and AAV5 for mammalian CNS cells, and Goncalves, 2005, Virol J. 2 (1):43, which discuss methods for modifying AAV tropism.

[0045] The AAV gene therapy vectors used in the present invention can be produced in mammalian cells or in insect cells. Both methods are described in the art. For example, Grimm et al. (2003 Molecular Therapy 7(6):839-850) disclose a method for producing AAV vectors in a helper virus-free and light-controllable manner, which is based on the transfection of only two plasmids into 293T cells. They disclose a method for producing a hybrid AAV vector containing AAV2 ITR and AAV5 capsid proteins. This reference is incorporated herein by reference in its entirety. Further information can also be found in Blits et al. (2010) (Journal of Neuroscience methods 185(2):257-263). The terms "hybrid" and "pseudotyped" are used interchangeably herein to describe vectors in which the Rep protein, ITR, and / or capsid protein are from different serotypes. For example, the ITR and Rep protein are of the AAV2 serotype, and the capsid protein is of the AAV5 serotype. The term "chimeric" as used herein describes a single gene such as a capsid, which is composed of at least two sequences derived from different serotypes.

[0046] AAV5 can be produced, for example, in mammalian cells, according to the following methods, but not limited to the following: The vector genome contains a transgene expression cassette flanked by two inverted terminal repeats (ITRs) derived from the AAV serotype 2. The total length of the viral vector genome can be the wild-type genome size not exceeding 4.7 kB to retain efficient packaging efficiency. A single capsid is composed of 60 viral proteins of VP1 (62 kDa), VP2 (73 kDa), or VP3 (87 kDa) in a ratio of 1:1:10. The production method of the AAV vector is based on the transfection of two plasmids into human embryonic kidney production cells (HEK293) in a roller bottle (850 cm 2 surface area) by Ca(PO4)2, followed by purification of the capsidated vector genome by filtration and chromatography techniques. The first plasmid is a viral vector plasmid containing an expression construct flanked by AAV2 ITRs. The second plasmid is a packaging plasmid encoding the AAV rep 2 and cap 5 genes of the desired serotype and the adenovirus early helper genes E2A, VA, E4 (pDP5; nucleotide sequence as shown in SEQ ID NO:3). The genome of the production cell line contains adenovirus E1 to provide helper functions. After co-transfection with the two plasmids in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FCS), the cells are incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) for 3 days to allow vector production. The average vector production in a roller bottle yields 3×103 yield per vector genome / cell or 4×10 11 yield per vector genome / roller bottle (quantified by qPCR). Subsequently, the cell culture was lysed with buffer containing Triton-X-100 and cell debris was removed by low speed centrifugation. The clarified bulk was purified by AVB agarose affinity chromatography, concentrated and diafiltered using a 400 kDa hollow fiber module (e.g., from Spectrum Laboratories) and formulated in PBS / 5% sucrose.

[0047] Alternatively, the AAV gene therapy vectors used in the present invention can be produced in insect cells as previously described by Urabe et al. (Journal of Virology 2006 80(4):1874-1885). Modifications in the previously disclosed Rep and VP1, VP2, and VP3 sequences can also be used in the present invention, such as those described in International Publications WO 2007 / 046703, WO 2007 / 148971, WO 2009 / 014445, WO 2009 / 104964, and / or WO 2011 / 112089.

[0048] The AAV ITR and Rep sequences that can be used in the present invention to generate rAAV vectors in insect cells can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences that are significantly homologous at the amino acid and nucleic acid levels. This provides the same set of genetic functions to produce virions that are substantially physiologically and functionally equivalent. A review of the genomic sequences and genomic similarities of various AAV serotypes can be found, for example, in GenBank accession number U89790; GenBank accession number J01901; GenBank accession number AF043303; GenBank accession number AF085716; Chiorini et al. (1997, J. Vir. 71:6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chiorini et al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74:8635-47). rAAV serotypes 1, 2, 3, 4, and 5 are preferred sources of AAV nucleotide sequences for use in the present invention. Preferably, the AAV ITR sequences for use in the present invention are derived from AAV1, AAV2, and / or AAV5. More preferably, the ITR sequence for use in the present invention is the AAV2 ITR. Similarly, the Rep (Rep78 / 68 and Rep52 / 40) coding sequences are preferably derived from AAV1, AAV2, and / or AAV5, more preferably AAV2.

[0049] In most serotypes, the AAV Rep and ITR sequences are particularly conserved. The Rep78 proteins of various AAV serotypes are, for example, more than 89% identical, and the overall nucleotide sequence identity at the genomic level between AAV2, AAV3A, AAV3B, and AAV6 is approximately 82% (Bantel-Schaal et al., 1999, J. Virol., 73(2):939-947). In addition, it is known that in the production of AAV particles in mammals, the Rep sequences and ITRs of many AAV serotypes effectively cross-complement (i.e., functionally substitute for) the corresponding sequences of other serotypes. US2003148506 reports that in insect cells, AAV Rep and ITR sequences also effectively cross-complement other AAV Rep and ITR sequences.

[0050] It is known that AAV VP proteins determine the tropism of AAV virions. The VP protein coding sequences are significantly less conserved compared to the Rep proteins and genes in different AAV serotypes. The sequences encoding the viral proteins (VPs) VP1, VP2, and VP3 capsid proteins used in the present invention are derived from AAV5. Most preferably, VP1, VP2, and VP3 are AAV5 VP1, VP2, and VP3. Alternatively, VP1, VP2, and VP3 are wild-type AAV5 sequences, as shown in SEQ ID NO:3 (nucleotide sequence) and SEQ ID NO:4 (amino acid sequence). The ability of the Rep and ITR sequences to cross-complement the corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles that contain the capsid proteins of one serotype (e.g., AAV5) and the ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of the present invention. Herein, the pseudotyped rAAV particles can be referred to as "x / y" type, where "x" represents the source of the ITR and "y" represents the serotype of the capsid protein. For example, 2 / 5 rAAV particles have ITRs from AAV2 and capsid proteins from AAV5.

[0051] Modified "AAV" sequences can also be used in the present invention, for example, to produce rAAV vectors in insect cells. Such modified sequences include, for example, sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or higher nucleotide and / or amino acid sequence identity to the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR, Rep, or VP (e.g., sequences having about 75% to about 99% nucleotide sequence identity), and can be used to replace the wild-type AAV ITR, Rep, or VP sequences.

[0052] Although similar to other AAV serotypes in many respects, AAV5 differs from other human and simian AAV serotypes more than other known human and simian serotypes. Given this, the production of rAAV5 in insect cells can be different from the production of other serotypes. In cases where the method of the present invention is used to produce rAAV5, preferably one or more constructs comprise (collectively in more than one construct): a nucleotide sequence comprising AAV5 ITR, a nucleotide sequence comprising an AAV5 Rep coding sequence (i.e., a nucleotide sequence comprising AAV5 Rep78) and / or an AAV5 Cap coding sequence. Such ITR, Rep, and Cap sequences can be modified as desired to obtain efficient production of rAAV5 or pseudotyped rAAV5 vectors in insect cells. For example, the start codon of the Rep sequence can be modified, the VP splice site can be modified or eliminated, and / or the VP1 start codon can be modified to improve the production of rAAV5 vectors in insect cells, as disclosed in, for example, WO 2007 / 046703, WO 2007 / 148971, and / or WO2009 / 014445. The present invention also includes chimeric AAV5 capsids, wherein, for example, part or all of the VP1 of AAV5 is replaced by VP1 derived from AAV2, and VP2 and 3 are derived from AAV5 (Urabe et al., 2006; WO2000 / 028004). In such chimeric AAV5 capsids, at least VP3 should be AAV5 or derived from AAV5, while one or both of VP1 and VP2 can be a different AAV serotype or derived from a different AAV serotype.

[0053] Preferred adenoviral vectors are modified to reduce the host response, as reviewed by Russell (2000, J. Gen. Virol. 81 :2573-2604), or as described in US20080008690 and Zaldumbide and Hoeben (Gene Therapy 2008:239-246).

[0054] Preferably, the Rep78 protein having the nucleic acid sequence of SEQ ID NO:5 and / or the amino acid sequence of SEQ ID NO:6 is used in the present invention, and the Rep52 protein having the nucleic acid sequence of SEQ ID NO:7 is used in the present invention.

[0055] "Intrathecal" administration refers to intrathecal injection, i.e., administration within the cerebrospinal fluid at any level of the cerebrospinal axis, including injection into the ventricles (see also "Route of Administration". Data Standards Manual. Food and Drug Administration. Retrieved 11 March 2011). This is achieved by injection through the spinal meninges into the subarachnoid space, thereby reaching the CSF. This method of administration can be used for, e.g., spinal anesthesia, chemotherapy, or pain management. This route is also used to introduce drugs against certain infections, especially after neurosurgery. Substances administered intrathecally avoid the need to cross the blood-brain barrier. Drugs administered intrathecally usually do not contain any preservatives or other potentially harmful inactive ingredients, which are sometimes found in drugs administered intravenously. The intrathecal delivery method is considered less invasive than methods of infusion into the central nervous system (CNS) tissue itself, as infusion into the CNS requires complex brain surgery. Intrathecal delivery can be performed without the need for a specialized brain injection center.

[0056] The neck region of the spine is called the cervical spine. This region consists of seven vertebrae, abbreviated as C1 to C7 (from top to bottom). These vertebrae protect the brainstem and spinal cord, support the skull, and allow for a wide range of head movements. The first cervical vertebra (C1) is called the atlas. The atlas is ring-shaped and supports the skull. C2 is called the axis. It is ring-shaped and has a blunt tooth-like structure (called the dens or odontoid process) that projects into the atlas. The atlas and axis together allow the head to rotate. The other cervical vertebrae (C3 to C7) are box-like in shape with small spinous processes (finger-like projections) that project from the back of the vertebrae. Below the last cervical vertebra are the 12 thoracic vertebrae. These thoracic vertebrae are abbreviated as T1 to T12 (from top to bottom). T1 is the smallest thoracic vertebra and T12 is the largest. The thoracic vertebrae are larger than the cervical vertebrae and have longer spinous processes. In addition to the longer spinous processes, the attachment of the ribs increases the strength of the thoracic vertebrae. These structures make the thoracic vertebrae more stable than the cervical or lumbar vertebrae. Furthermore, the thoracic cage and connecting systems limit the range of motion of the thoracic vertebrae and protect many important organs. The lumbar spine has 5 vertebrae, abbreviated as L1 to L5 (the largest). The size and shape of each lumbar vertebra are designed to bear most of the body weight. Each structural element of the lumbar vertebrae is larger, wider, and broader compared to the similar elements in the neck and chest regions. The lumbar spine has a greater range of motion than the thoracic spine, but less than the cervical spine. The lumbar zygapophyseal joints allow for significant flexion and extension movements, but limit rotation. The sacrum is located behind the pelvis. Five bones abbreviated as S1 to S5 fuse to form a triangle and make up the sacrum. The sacrum is between the two hip bones that connect the spine to the pelvis. The lowest lumbar vertebra (L5) connects (moves) with the sacrum. Below the sacrum are 5 additional bones that fuse together to form the coccyx (tailbone).

[0057] In a preferred embodiment of the present invention, the gene therapy vector is administered by lumbar injection. More preferably, intrathecal lumbar administration is at a site selected from: L4–L5, L3–L4, L1–L2, and L2–L3. Most preferably, the gene therapy vector is administered by intrathecal injection between L4 and L5. In a preferred embodiment, the gene therapy vector is administered only by lumbar administration, i.e., not at other sites.

[0058] Alternatively or in combination with a previously preferred embodiment of the present invention, in a preferred embodiment of the present invention, the gene therapy vector is administered into the cisterna magna. In a preferred embodiment, the gene therapy vector is administered only into the cisterna magna, i.e., not at other sites. More preferably, administration into the cisterna magna is carried out in combination with intrathecal administration, or in other words, before, simultaneously with, or after intrathecal administration.

[0059] In another preferred embodiment, administration into the cisterna magna is carried out in combination with lumbar administration, whereby preferably administration into the cisterna magna is before, simultaneously with, or after lumbar administration. Preferably in this embodiment, lumbar administration is in the intrathecal region.

[0060] In the most preferred embodiment, the gene therapy vector is administered into the cisterna magna only in combination with lumbar administration, i.e., not at other sites except the cisterna magna and the lumbar region. Preferably in this embodiment, lumbar administration is in the intrathecal region.

[0061] The "cisterna magna" is one of the three principal openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. The openings are collectively referred to as cisterns. The cisterna magna is located between the cerebellum and the dorsal surface of the medulla oblongata. Cerebrospinal fluid produced in the fourth ventricle flows into the cisterna magna through the lateral and median apertures.

[0062] As used herein, combined administration or administration "before, simultaneously with, or after" intrathecal administration means that administration into the cisterna magna and intrathecal administration occur within a time interval preferably less than 2 weeks, 1 week, 4 days, 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, or 5 minutes.

[0063] Recombinant AAV vectors can have single-stranded DNA or double-stranded DNA. The single-stranded DNA (ssDNA) AAV genome must be converted to double-stranded DNA (dsDNA) before encoding transgene expression. This step can be avoided by using self-complementary vectors or monomeric double-stranded vectors that package inverted repeat vector genomes that fold into dsDNA without DNA synthesis or base pairing between multiple vector genomes, thereby increasing the efficacy of AAV-mediated gene transfer. For a review of self-complementary AAV vectors, see, for example, McCarty, D.M. Mol. Ther. (2008) 16:1648-1656. The so-called self-complementary AAV vectors (WO2001 / 092551) contain at least one modified ITR, preferably modified by deletion of the terminal resolution site (trs), and are different in design from monomeric double-stranded AAV vectors, which can be produced using only intact AAV ITRs (WO2011 / 122950). In a preferred embodiment, self-complementary AAV vectors are not within the scope of the present invention. In contrast, the monomeric double-stranded vectors as described above are different from self-complementary vector sequences and can be advantageously used in the present invention because the vector genomes involved are half (1 / 2) or smaller than the wild-type AAV vector size (i.e., 4.8 kb) in addition to the intact ITR sequence. This has the advantage that multiple repeats (tandem arrays of 2, 4, 8 or more expression units) can be established in the AAV vector, resulting in monomeric, dimeric, tetrameric double-stranded vectors. Examples of expression units in such vectors can be vectors containing shRNA or miRNA. In the present invention, it has been shown that widespread transduction of the brain tissue can be obtained after lumbar injection using single-stranded AAV gene therapy vectors. Thus, in a preferred embodiment of the present invention, the AAV gene therapy vector is a single-stranded AAV gene therapy vector.

[0064] The AAV gene therapy vector of the present invention achieves widespread transduction in the central nervous system after intrathecal administration in the lumbar spine. Using a ubiquitous promoter (CAG promoter), neurons and glial cells are transduced in the cortex, cerebellum, and subventricular zone. In addition, in the spinal cord, motor neuron transduction is widespread, and neuron transduction in the dorsal root ganglia is also widespread. Thus, the therapeutic method of the present invention can be used to treat severe CNS diseases. In a preferred embodiment, the AAV gene therapy vector of the present invention comprises a gene product of interest, which can be used to treat or prevent disorders selected from the following: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), pain, lysosomal storage diseases (LSDs), Huntington's disease, Alzheimer's disease, Tay-Sachs disease, Friedreich's ataxia, ataxia-telangiectasia, spinocerebellar ataxia types 1, 2, and 3, Niemann-Pick diseases types A, B, and C, dopa-responsive dystonia, fragile X syndrome, Krabbe disease, glycogen storage disease type 2 (Pompe), primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, giant axonal neuropathy, multiple system atrophy (MSA), proximal myotonic myopathy, neuronal ceroid lipofuscinosis (Batten disease), and various forms of CNS cancer, such as primary CNS lymphoma, metastatic or secondary brain tumors, and primary spinal tumors. CNS cancers include, for example, gliomas, astrocytomas, oligodendrogliomas, ependymomas, meningiomas, medulloblastomas, gangliogliomas, schwannomas, craniopharyngiomas, chordomas, non-Hodgkin CNS lymphoma.

[0065] A group of metabolic diseases called lysosomal storage diseases (LSDs) includes more than 40 genetic disorders, many of which involve genetic defects in various lysosomal hydrolases. Representative lysosomal storage diseases and the associated defective enzymes are listed in Table 1.

[0066] Table 1

[0067]

[0068]

[0069] In another preferred embodiment, the gene product of interest is used to treat or prevent disorders associated with motor neurons and / or neurons in the DRG. These disorders include, but are not limited to, pain, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and pseudobulbar palsy.

[0070] In a particularly preferred embodiment, the AAV gene therapy vector of the invention comprises a gene product of interest useful for treating or preventing a disorder selected from: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's disease, multiple system atrophy (MSA), and lysosomal storage diseases selected from the group consisting of: Fabry disease, juvenile Batten disease (CNL3), Gaucher disease types 1, 2 and 3, Hunter, Pompe, Sanfilippo A and Sanfilippo B.

[0071] Accordingly, in a preferred embodiment, the gene product of interest is selected from: aspartylglucosaminidase, alpha-galactosidase A, palmitoylprotein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, multi-gene product, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase, protective protein / cathepsin A, acid beta-glucosidase, or glucocerebrosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid beta-galactosidase, N-acetylglucosamine-1-phosphate transferase, acid sphingomyelinase, NPC-1, acid alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, alpha-N-acetylgalactosaminidase, alpha-Neuramidase, beta-glucuronidase, beta-hexosaminidase A, acid lipase, neurotrophic factors such as nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), brain-derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and growth factor insulin-like growth factor (IGF-1). In addition, miRNA sequences can also be used to downregulate the expression of certain genes, for example in the treatment of Huntington's disease.

[0072] Most of the diseases mentioned in this article, except for Huntington's disease, can be treated by providing the correct form of the defective gene. However, Huntington's disease requires downregulation of a gene, namely the Huntington gene located on human chromosome 4 (also known as HTT, HD, and IT15). Subjects with Huntington's disease carry an HTT gene with more than 36 trinucleotide CAG repeats at the 5' end of the gene. Developing an RNAi-based treatment for Huntington's disease involves, for example, knocking down all HTT (mutant and wild-type genes) targeting exon 1. Other methods are, for example, allele-specific silencing of only mutant HTT targeting the CAG repeats in HTT exon 1 and allele-specific inhibition of mutant HTT expression targeting SNPs in exon 67, for example.

[0073] In a preferred embodiment, the gene product of interest is operably linked to an expression control element comprising a promoter that produces sufficient expression of the gene product of interest to obtain a therapeutic effect. In eukaryotic cells, the transgenic expression level is mainly determined by the transcriptional promoter in the transgenic expression cassette. Promoters that exhibit long-term activity and are tissue- and even cell-specific are used in some embodiments. Non-limiting examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet., 8:148-164), the CMV / human β3-globin promoter (Mandel et al. (1998) J. Neurosci., 18:4271-4284), the phosphoglycerate kinase (PGK) promoter, the CAG promoter (a combination of the cytomegalovirus early enhancer element and the chicken β-actin promoter) (Klein et al. (2008) Mol Ther 16(1):89-96; Shevtsova et al (2004) Exp Physiol 90:53-59), the GFAP promoter (Xu et al. (2001) Gene Ther., 8:1323-1332; Lee et al. (2008) Glia 56:481-493), the synapsin-1 promoter (Kuegler et al. (2001) Mol Cell Neurosci 17:78-96; Drinkut et al. (2012) Molecular Therapy 20:534-543), the 1.8-kb neuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol., 150:183-194), the chicken β-actin (CBA) promoter (Miyazaki (1989) Gene, 79:269-277), and the β-glucuronidase (GUSB) promoter (Shipley et al. (1991) Genetics, 10:1009-1018). To prolong expression, other regulatory elements can be additionally operably linked to the transgene, such as the woodchuck hepatitis virus regulatory element (WPRE) (Donello et al. (1998) J. Virol., 72, 5085-5092) or the bovine growth hormone (BGH) polyadenylation site. Drinkut and colleagues used the glial fibrillary acidic protein (GFAP) promoter to express GDNF and demonstrated that it was more beneficial than expression from a neuron-specific promoter (Drinkut et al., 2012, supra).Alternatively, in addition to using a specific promoter, the use of regulatable expression elements such as gene switch systems (Maddalena et al. (201) Molecular Therapy–Nucleic Acids 2:e106) or derivatives of the tetracycline operon (Gossen and Bujard (1992) PNAS 89:5547-5551) can achieve regulated expression.

[0074] In a preferred embodiment of the invention, the AAV gene therapy vector comprises an AAV5 capsid protein, AAV2 ITRs, and a gene product of interest located between the ITRs. Preferably, the gene therapy vector is administered by lumbar injection, optionally in combination with cisterna magna injection. Preferred gene products and diseases (for treatment or prevention) treated using such a preferred AAV gene therapy vector are selected from: GDNF or IGF-1 for treating ALS, GDNF or IGF-1 for treating SMA, miRNA for downregulating the defective survival motor neuron (SMN) gene to treat SMA, miRNA for achieving allele-specific downregulation or downregulation of both alleles of HTT to treat Huntington's disease, GDNF for treating Huntington's disease, GDNF for treating MSA, alpha-galactosidase A for treating Fabry disease, lysosomal transmembrane protein for treating juvenile Batten disease (CNL3), acid beta-glucosidase or glucocerebrosidase for treating Gaucher disease types 1, 2, and 3, iduronate-2-sulfatase for treating Hunter disease, acid alpha-glucosidase for treating Pompe disease, heparan N-sulfatase for treating Sanfilippo A, and alpha-N-acetylglucosaminidase for treating Sanfilippo B.

[0075] A therapeutically effective amount of the AAV gene therapy vector or pharmaceutical composition of the invention can be administered to a patient in need thereof.

[0076] The AAV gene therapy vector of the invention is typically included in a pharmaceutical composition, optionally in combination with a pharmaceutical carrier, diluent, and / or adjuvant. Such compositions include an effective amount of a nucleic acid, nucleic acid construct, parvovirus virion, or pharmaceutical composition sufficient to provide the desired therapeutic or prophylactic effect, and a pharmaceutically acceptable carrier or excipient. "Effective amount" includes a therapeutically effective amount or a prophylactically effective amount.

[0077] "Therapeutically effective amount" means an amount that is effective, at such dosage and for such time as are necessary, to achieve the desired therapeutic result, such as alleviation of the symptoms of a disease. The therapeutically effective amount of a nucleic acid, nucleic acid construct, parvovirus virion or pharmaceutical composition can vary according to various factors, such as the individual's disease state, age, sex and weight, and the ability of the nucleic acid, nucleic acid construct, parvovirus virion or pharmaceutical composition to elicit the desired response in the individual. The dosing regimen can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also typically an amount in which any toxic or detrimental effects of the nucleic acid, nucleic acid construct, parvovirus virion or pharmaceutical composition are outweighed by the therapeutic benefit.

[0078] "Prophylactically effective amount" means an amount that is effective, at such dosage and for such time as are necessary, to achieve the desired prophylactic result, such as preventing or inhibiting various conditions. Prophylactic doses can be used in a subject before the onset of a disease or in the early stages of a disease, and the prophylactically effective amount can be more or less than the therapeutically effective amount in some cases.

[0079] In certain embodiments, the therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition can range from 1×10 13 to 1×10 16 genomic copies (gc) / kg, preferably 2×10 13 to 2×10 15 , more preferably 8×10 13 to 6×10 14 , even more preferably about 2×10 14 gc / kg of subject body weight. Note that the dosage value can vary according to the severity of the condition being alleviated. For any particular subject, it can be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. The dosage ranges described herein are only exemplary and do not limit the dosage ranges that can be selected by a medical practitioner.

[0080] Preferably, the volume infused into the CSF is less than about 20 cc in children and less than about 30 cc in adults. In non-human primates, 6 cc can be safely delivered into the CSF with a total volume of 20 - 25 cc. Alternatively, at least half of the infusion volume is aspirated and replaced with the gene therapy vector of the present invention to achieve a replacement of up to about 50% of the volume.

[0081] In addition, the AAV gene therapy vectors of the present invention can be administered in a concentrated form by standard methods known in the art, including but not limited to density gradient centrifugation (e.g., CsCl, iodixanol), dialysis, ultrafiltration, ion exchange (IEX) chromatography (e.g., anion, cation), gel filtration, affinity chromatography, tangential flow filtration (TFF), spin filtration columns (e.g., Centricon). Exogenous virus or helper virus clearance can also be performed to improve the quality of the AAV gene therapy vectors for use in the present invention, as described, for example, in WO 2013 / 036118.

[0082] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Such media and formulations are well known in the art for the use of pharmaceutical active substances. Except insofar as any conventional media or formulation is incompatible with the active compound and / or the site of injection, its use in the pharmaceutical compositions of the present invention is included in the present invention.

[0083] Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the present invention. Guidance on the co-administration of additional therapeutic agents can be found, for example, in the Compendium of Pharmaceutical and Specialties (CPS) of the Canadian Pharmacists Association.

[0084] For example, Vulchanova (2010 Molecular Pain 6:31) showed that administration of viral vectors by direct lumbar puncture has not been reported to result in appreciable transduction of spinal cord and DRG neurons. Vulchanova et al. therefore suggested pretreatment with mannitol to enhance the penetration of AAV5 and AAV8 vectors into the spinal cord parenchyma and into the cell bodies of DRG neurons. In particular, in mice, an AAV vector at a lumbar injection dose of approximately 4×10 12 vector genomes / kg requires intravenous pretreatment with mannitol prior to intrathecal lumbar administration of the vector to obtain expression of green fluorescent protein (GFP) in the spinal cord and to select subsets of DRG neurons.

[0085] Pretreatment with mannitol has drawbacks, and mannitol is known to cause side effects. In particular, side effects frequently reported during or after mannitol infusion include: pulmonary congestion, water and electrolyte disorders, acidosis, electrolyte loss, dry mouth, thirst, marked polyuria, urinary retention, edema, headache, blurred vision, convulsions, nausea, dizziness, vomiting, rhinitis, arm pain, skin necrosis, thrombophlebitis, shivering, vertigo, urticaria, dehydration, hypotension, tachycardia, fever, and angina-like chest pain (Mannitol IV FDA Prescribing Information).

[0086] In a preferred embodiment of the present invention, the subject does not receive pretreatment prior to administration of the AAV gene therapy vector. In particular, the subject does not receive pretreatment with a substance that controls intracranial pressure and / or can disrupt the intercellular tight junctions between endothelial cells of the CNS microvasculature and / or facilitate the delivery of drugs such as chemotherapeutic agents to the CNS. More preferably, the subject is not pretreated with an osmotic agent prior to administration of the AAV gene therapy vector. Osmotic agents include, but are not limited to, urea, glycerol, sugars or sugar alcohols such as polyols, for example mannitol and hypertonic saline. In particular, the subject is not pretreated with mannitol, such as intravenous administration of mannitol, prior to lumbar intrathecal administration of the AAV gene therapy vector.

[0087] In a preferred embodiment of the present invention, mammalian subjects include, but are not limited to, mice, rats, monkeys, livestock, sport animals, pets, and humans. More preferably, the mammalian subject is a human.

[0088] In another aspect, the present invention relates to a method for producing recombinant parvovirus (e.g., rAAV) virions (comprising a recombinant parvovirus (rAAV) vector as described above) in insect cells. Preferably, the method comprises the steps of: (a) culturing insect cells as defined herein under conditions for producing a recombinant parvovirus (e.g., rAAV) vector; and (b) recovering the recombinant parvovirus (e.g., rAAV) vector. It is understood that the recombinant parvovirus (rAAV) vector produced in the method is preferably an infectious parvovirus or AAV virion that comprises the recombinant parvovirus (rAAV) vector nucleic acid. The growth conditions of insect cells in culture and the production of heterologous products in insect cells are well known in the art, as described, for example, in the references on molecular engineering of insect cells mentioned above. Preferred methods and constructs for producing the rAAV virions of the present invention are disclosed, for example, in WO2007 / 046703 and WO2007 / 148971.

[0089] Preferably, the method for generating recombinant parvovirus virions further comprises an affinity purification step of using an anti-AAV antibody, preferably a fixed antibody, for the virions of a recombinant parvovirus (rAAV) vector. The anti-AAV antibody is preferably a monoclonal antibody. Particularly suitable antibodies are, for example, single-chain camel antibodies or fragments thereof obtainable from camels or llamas (see, for example, Muyldermans, 2001, Biotechnol. 74 : 277-302). The antibody used for affinity purification of rAAV is preferably an antibody that specifically binds to an epitope on the AAV capsid protein, so preferably the epitope is an epitope present on the capsid proteins of more than one AAV serotype. For example, the antibody can be generated or selected based on specific binding to the AAV2 capsid protein and also specifically binding to the AAV1, AAV3, and AAV5 capsid proteins.

[0090] In this specification and its claims, the verb "comprising" and its variations are used in their non-limiting sense, meaning including the matter following that word, but not excluding matters not specifically mentioned. In addition, the indefinite article "a" used in relation to an element does not exclude the possibility of there being more than one element, unless the context clearly requires one and only one of the said elements. The indefinite article "a" thus generally means "at least one".

[0091] All patents and references cited in this specification are incorporated by reference in their entirety.

[0092] The following examples are provided only to illustrate the invention and are not intended to limit the scope of the invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 : Schematic diagram of the AAV vector genome. The expression cassette encoding the GOI (gene of interest or gene product of interest) is under the control of the CAG promoter, preceded by a Kozak sequence (gccaccatg…), and polyadenylated by the BGH polyadenylation signal. In the examples of the present invention, the GOI is green fluorescent protein (GFP). Flanking this expression cassette are two non-coding ITR sequences. Using a recombinant baculovirus for production, this expression cassette is packaged into the AAV-5 capsid.

[0094] Figure 2 : Summary of brain transduction. Representative sections A-E were taken from the brain and stained for GFP. Expression was observed throughout the brain.

[0095] Figure 3 : Parietal cortex at higher magnification. Neurons and glial cells were observed to be transduced several layers deep.

[0096] Figure 4 : Occipital cortex at higher magnification. Several layers of neurons and glial cells can be observed to be transduced deeply.

[0097] Figure 5 : Cerebellum at higher magnification. Many cells are GFP-positive.

[0098] Figure 6 : Transduction in the cervical spinal cord. Sensory fibers terminating in the gray matter and speckled ascending fibers (a) can be seen. In addition, GFP-positive motor neuron fibers (b) from motor neuron aggregates can be observed (c). In (d), some glial cells are also GFP-stained positive.

[0099] Figure 7 : Transduction in the thoracic spinal cord. Ascending sensory fibers (a) can be seen in the spinal column. In addition, GFP-positive motor neuron fibers (b) from motor neuron aggregates can be observed (c). In (d), some fibers that can be either descending or ascending are observed.

[0100] Figure 8 : Transduction in the lumbar spinal cord. Sensory fibers terminating in the gray matter (a) are observed. In addition, GFP-positive motor neurons in motor neuron aggregates are present in the dorsolateral part of the spinal cord. These motor neurons send fibers through the ventral part of the spinal cord into the ventral root (d).

[0101] Figure 9 : GFP-stained section of DRG, counterstained with hematoxylin. Transduction along the dorsal root ganglia of the spinal cord can be seen. DRGs from cervical (A, B), thoracic (C, D), and lumbar (E, F) levels are transduced. At higher magnification (right panels, B, D, F), fibers in the root are visible through their GFP expression. Almost all DRG neurons are transduced.

[0102] Figure 10 : Double staining of the cerebellum for GFP and NeuN (neuronal marker). Bergmann glial cells (Bc) show GFP expression, and Purkinje cells (Pc) can also be observed based on their morphology.

[0103] Figure 11 : Double staining of the cortex for GFP and NeuN. Arrows point to neurons expressing GFP in the cortex.

[0104] Figure 12 : Double staining of the cortex for GFP and GFAP (astrocyte marker). Arrows point to astrocytes expressing GFP. Triangles point to cells with neuronal characteristics.

[0105] Figure 13: Hematoxylin and eosin staining of brain and cerebellum sections. Upper left is an overview of the cerebellum. Upper right is the molecular layer of the cerebellum at higher magnification. Finely arranged are large and round neurons (also seen in the inset), Purkinje cells. Generally, no obvious infiltration or irregularity was observed, indicating that the treatment was well tolerated.

[0106] Figure 14 : GFP-stained lumbar spinal cord sections show dose-dependent transduction of motor neurons in the lumbar spinal cord by the presence of large neurons in the ventral horn. 5E13 (A), 5E14 (C), or 8E14 (E) genome copies (GC) were injected into the CSF of the animals. The ventral horn at higher magnification is shown, where motor neurons were located and depicted (B, D, and F). Example

[0107] Example 1

[0108] In this experiment, an AAV-5 vector encoding enhanced green fluorescent protein (GFP) under the control of the CAG promoter was intrathecally infused into the CSF of non-human primates. Transgene expression was detected 4 weeks after injection. GFP expression was observed in motor neurons and dorsal root ganglia (DRG) at all spinal cord levels. In addition, in the cerebellum, many Bergmann glial cells as well as some Purkinje cells were transduced. In the brain, the cortex also expressed GFP in neurons and glial cells. Cells along the subventricular zone (SVZ) were also transduced. Overall, these results suggest that using this injection route, most CNS regions are covered and gene therapy for the CNS becomes a realistic option.

[0109] Example 1.1: Materials and Methods

[0110] 1.1.1. Preparation of the vector

[0111] The expression cassette of rAAV serotype 5 (rAAV-5) contains the human cDNA, enhanced green fluorescent protein (EGFP) cDNA gene. Expression is under the control of the CAG promoter, which is a combination of the cytomegalovirus (CMV) early enhancer element and the chicken β-actin promoter. In front of GFP is the Kozak sequence, and it is polyadenylated by the bovine growth hormone polyadenylation (BGHpA) signal. The complete expression cassette is flanked by two non-coding inverted terminal repeats of AAV-2 (see Figure 1)。The recombinant AAV-5 vector was prepared using a baculovirus expression system as previously described (Urabe et al., 2002, Unzu et al., 2011, reviewed by Kotin, 2011). Briefly, SF9 insect cells were infected with three recombinant baculoviruses, one encoding REP for replication and packaging, one encoding CAP-5 of the AAV-5 capsid, and one with an expression cassette. Purification was performed using AVB Sepharose high performance affinity media (GE Healthcare, Piscataway, NJ). The vector was titrated by Q-PCR method using a primer-probe combination specific for the transgene, and the titer was expressed as genome copies / ml (GC / ml). The titer of the vector was 1.4E14 GC / ml.

[0112] 1.1.2. Animals and tissue collection

[0113] Approximately 4 kg male cynomolgus macaques (Macaca fascicularis) were used in this study. Daily health observations were recorded and body weights were measured regularly to assess the health of the animals. The veterinarian reported no abnormal symptoms during the study. All procedures were approved by the Institutional Animal Care and Use Committee at Valley Biosystems (Sacramento, CA, USA). Before the start of the procedures, the animals were tested negative for the presence of AAV neutralizing antibodies (see below). For the procedures described, the animals were anesthetized with a mixture of ketamine (Ketaset, 7 mg / kg) and dexmedetomidine hydrochloride (Dexdomitor, 0.015 mg / kg). After the surgery, all animals received an intramuscular injection of atipamezole hydrochloride to recover from anesthesia (Antisedan, 0.15 mg / kg). One month after the administration of AAV-5, the animals were deeply anesthetized and perfused through the heart with phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) in PBS. Brains and organs were collected and processed for histological analysis.

[0114] Briefly, the brain and the cervical, thoracic, and lumbar spinal cords were sectioned coronally into 6-mm blocks, fixed overnight in 4% PFA-PBS, and cryoprotected in 30% (w / v) sucrose the next day. For histological staining, the brain blocks were sectioned into 40-μm serial sections using a sliding microtome (Thermo Scientific Microm HM 450; Thermo Fisher Scientific, Waltham, MA). The sections were then placed in cryoprotectant solution until use.

[0115] 1.1.3. AAV Delivery

[0116] After induction of anesthesia, the animal's head was placed in a stereotaxic frame, flexed in the prone position, and the neck and back were cleaned with polyvinylpyrrolidone iodine and ethanol. A 3-ml syringe with a 1-inch 23-gauge injection needle attached was mounted on a micromanipulator, and the needle was manually maneuvered into the cisterna magna. A similar lumbar dural puncture was performed between the L4 and L5 spinal cord levels. The puncture was confirmed by aspirating a small volume of cerebrospinal fluid (CSF) into the syringe, and then the syringe was placed against the micromanipulator. The three-way valve on the syringe was then adjusted, and a 3-ml vector was injected at a flow rate of 0.5 ml / minute using a pump (3500 Medfusion; Strategic Applications, Libertyville, IL). After completion of the injection, the cannula was flushed with 0.2 ml of saline. Finally, a small volume of CSF was aspirated to confirm that the needle was still in the CM. After confirmation, the needle was slowly removed. Thus, each animal received 3 ml in the cisterna magna and 3 ml by lumbar injection. A total of 6 ml of 1.4E14 GC / ml of AAV-5-CAG-GFP was injected.

[0117] Similarly, animals could be treated with the same amount and dose of AAV-5-CAG-GFP, administered only by lumbar injection at the L4-L5 spinal cord level (without injection into the cisterna magna). The following amounts of AAV-5-CAG-GFP were administered by lumbar injection: 5E13 (normal dose), 5E14, and 8E14 GC (high dose).

[0118] The normal administration dose of AAV is approximately 1E12 GC and 5E13 GC. For example, Vulchanova (as described above) used 1E11 GC AAV-5 for mice (20 g), which is equivalent to 5E12 / kg. For a 5-kg monkey, 2.5E13 GC was used. In addition, Gray et al (2013, Gene Therapy, 20, 450–459) used 2E12 GC / 6-kg animals. Thus, a dose of 5.0E13 GC can be considered a normal dose in the art, and 5.0E14 and 8.4E14 are considered high doses.

[0119] Since lumbar infusion of blue dye results in blue staining of the cisterna puncture, no difference in outcome was expected if the gene therapy vector was administered only lumbar.

[0120] 1.1.4. Immunohistochemistry

[0121] Forty-lm coronal sections of four 6-mm brain specimens blocks from the prefrontal cortex to the cerebellum and spinal cord were subjected to chromogenic and immunofluorescent staining. Chromogenic GFP staining was performed as previously described (Hadaczek et al., 2009). Briefly, sections were washed in PBS, endogenous peroxidase activity was blocked with 1% H2O2–30% ethanol in PBS, and rinsed in PBST (PBS plus 0.1% Tween 20). Sections were then incubated in Background Sniper blocking solution (BS966G; Biocare Medical, Concord, CA), and incubated with the primary antibody of GFP (rabbit anti-GFP, 1:3000 dilution; Millipore, Bedford, MA) in Da Vinci Green diluent (PD900; Biocare Medical) at 4 °C up to 24 h. The next day, after washing in PBST, sections were incubated in Rabbit Mach 3 Polymer HRP (RP531L; Biocare Medical) at room temperature for GFP staining and developed with 3,3￠-diaminobenzidine (DAB) (DAB peroxidase substrate kit, SK-4100; Vector Laboratories, Burlingame, CA). Sections were then mounted on slides, dehydrated, and covered with Shandon-Mount (cat. No 1900333; Thermo Fisher Scientific).

[0122] To determine the phenotype of GFP-positive cells, double immunofluorescence staining was performed. Brain sections were washed with PBST, blocked in 20% normal horse serum (NHS; Jackson ImmunoResearch, West Grove, PA) for 60 minutes, and then incubated overnight at 4°C with anti-GFP (rabbit or mouse, 1:200 dilution; Millipore) and antibodies specific for microglia (antibody-Iba1, rabbit polyclonal antibody, 1:500 dilution; Biocare Medical), neurons (anti-NeuN, mouse monoclonal antibody, 1:300 dilution; Millipore), and fibrous astrocytes (anti-glial fibrillary acidic protein [GFAP], mouse monoclonal antibody, 1:500 dilution; Dako, Carpinteria, CA). All antibodies were diluted in Da VinciGreen diluent (PD900; Biocare Medical). After incubation with the primary antibodies, the sections were washed in PBST; incubated for 2 hours at room temperature with a mixture in PBST of the appropriate secondary antibodies (FITC-conjugated anti-rabbit antibody [1:200 dilution; Jackson ImmunoResearch] or Alexa 488-conjugated anti-mouse antibody [1:500 dilution; Molecular Probes / Invitrogen, Carlsbad, CA] and Alexa 555-conjugated anti-mouse antibody [1:500 dilution; Molecular Probes / Invitrogen] or Alexa 555-conjugated anti-rabbit antibody [1:500 dilution; Molecular Probes / Invitrogen]); washed in PBS; and wet-mounted on frozen slides. The sections were covered with a hard matrix containing 4′,6-diamidino-2-phenylindole (DAPI) to identify all cell nuclei (Vectashield H-1200; Vector Laboratories).

[0123] 1.1.5. Anti-AAV5 antibody titer in NHP

[0124] Before administration of the vector, the antibody titer against the AAV capsid was determined by ELISA on blood samples. Briefly, an AAV capsid solution at 2E10 GC / ml in 1 mM carbonate buffer was dispensed into 96-well titration plates and incubated overnight at 4 °C. The next day, the plates were washed and blocked with a 5% non-fat milk solution in PBST. Serum was diluted in the range of 1:50 to 1:6400 and incubated for 1 h at room temperature. The wells were then washed with PBST and incubated with a horseradish peroxidase (HRP)-conjugated anti-monkey secondary antibody (Sigma-Aldrich, St. Louis, MO) for 1 h at room temperature. The cells were washed again in PBST and then developed with 3,3-,5,5-tetramethylbenzidine (TMB). The reaction was terminated by addition of hydrochloric acid, and absorbance was read at 450 nm on a plate reader (Bevan et al., 2011).

[0125] 1.1.6 Biodistribution of AAV

[0126] Before administration: Blood samples were collected from adult cynomolgus macaques (Macaca fasciularis) for screening and analysis of serum anti-AAV-5 antibody titers, serum clinical chemistry, and hematological evaluations.

[0127] CSF injection: Animals were immobilized with ketamine (10 mg / kg) IM and medetomidine (15 μg / kg) IM. A spinal puncture needle was placed at the lumbar region, and a single dose (5 mL, over 5 min) of AAV5-CAG-GFP was injected into the CSF of the animals. Three different doses (5E14, 5E13, and 5E12 GC) were administered to these animals.

[0128] Tissue harvest: Animals injected with CSF were euthanized 8 weeks after injection. Each animal was deeply anesthetized with ketamine (10 - 15 mg / kg, IM) and medetomidine (15 μg / kg). An euthanasia solution was administered to the animals. After confirming deep anesthesia (i.e., no toe pinch response, corneal reflex, and jaw tone), a thoracotomy was performed and a liver sample was taken. The brain and spinal cord were harvested, and tissue was sectioned with a tissue chopper for molecular (Q-PCR) analysis. The remaining tissue was preserved in paraformaldehyde for histological evaluation.

[0129] Q-PCR analysis: The tissues were analyzed by standard Q-PCR methods using primers specific for the GFP gene. The results are shown in Table 2.

[0130] 1.2. Results

[0131] The procedures were well tolerated by the animals. The veterinarians did not observe weight loss or note any clinical symptoms.

[0132] Three different doses of AAV were tested: 5E13 GC, 5E14 GC, and 8.4E14 GC. 5E13 GC can be considered a regular dose, while 5E14 GC and 8.4E14 GC are high doses.

[0133] As Figure 14 shown, administration of the regular dose (5E13 GC) of AAV5A resulted in little to no transduction, confirming the consensus in the art that AAV5 only inadequately transduces neuronal cells after intrathecal administration. Surprisingly, higher doses of AAV5 resulted in significant motor neuron transduction. Figure 14 It is shown that transduction of motor neurons increases when higher doses are used. In the dorsal horn, some fibers were also observed, suggesting transduction of DRG neurons. However, the major transduced cell type was motor neurons.

[0134] Further, the presence of the AAV genome was determined in the spinal cord, brain, and liver. In particular, the presence of AAV in the liver indicates that AAV5 leaks from the CNS into the periphery. It has been well known that AAV vectors can leak into the periphery when administered into the CNS. For example, Haurigot et al (2013, J. Of Clin. Invest., 123:3254–3271) used AAV9 for systemic modulation by intracranial injection, which also had an effect on the liver. Interestingly, in contrast to, for example, AAV9, most AAV5 particles remained within the spinal cord and disseminated to the brain. The liver was not invaded by AAV5 (Table 2).

[0135] Table 2: Presence of AAV particles after CSF injection. Where +++ indicates a huge amount of AAV genome present, ++ indicates a large amount of AAV genome present, + indicates genome present, and – indicates no AAV genome detected.

[0136]

[0137] The following observations were obtained from experiments conducted with 8.4E14 GC: GFP expression was observed throughout the CNS including the brain, cerebellum, and spinal cord. Expression was observed in both neurons and glial cells. In particular, GFP expression was mainly observed in motor neurons. Vulchanova et al also observed transduction of neuronal cells with AAV5 after pretreatment with mannitol. However, Vulchanova et al observed a higher transduction efficiency of DRG neurons compared to motor neurons (and their neurites extending into the dorsal horn of the spinal cord). Thus, the ratio of transduced motor neurons / DRG favors DRG with mannitol and motor neurons with high-dose AAV5, making administration of high-dose AAV5 particularly suitable for treating and / or preventing motor neuron-related disorders.

[0138] An overview of some brain sections showed good expression throughout the brain, mainly in the regions adjacent to the ventricular wall (see Figure 2 ). Expression in the parietal cortex showed expression throughout several cortical layers. Some astrocytes were also transduced ( Figure 3 ). The same was true in the occipital cortex ( Figure 4 ). In the cerebellum, the sulci and gyri of the cerebellar structure were clearly shown by the transduced cells ( Figure 5 ). In this part of the brain, both neurons and glial cells were observed to be transduced.

[0139] In the spinal cord, many (if not all) motor neurons along the entire length of the spinal cord were transduced ( Figure 6 , 7 and 8). A similar situation was observed in the neurons in the DRG ( Figure 9 ). It was also highly transduced to express GFP. To identify the cells expressing the transgene, double staining of neurons and astrocytes was performed. Using this specific CAG promoter, both neurons and astrocytes were transduced ( Figure 10 , 11 and 12). To examine whether neuronal loss had occurred, H&E staining was performed on cerebellar sections ( Figure 13 ). Based on the presence of the remaining Purkinje cell layer, no neurotoxicity was observed using this special method.

[0140] 1.3. Discussion

[0141] Current delivery of genes to the CNS is hindered by its size and the complexity of the CNS. After a single injection into the brain, the expression of the transgene is local and mainly limited to the injection site. Using CSF as a method for delivering viral vectors, a larger area can be reached. This study shows that using the AAV-5 vector at a relatively high dose of more than 1E14 GC / kg, this goal of achieving transduction in a larger area of the CNS may be realized. The treatment was well tolerated as no clinical symptoms or significant neuronal loss were observed. Both neurons and glial cells were transduced using this specific CAG promoter. Using cell-specific promoters, such as the GFAP promoter or the synapsin-1 promoter, expression in specific populations can be directed. We revealed that the cortex, cerebellum, and subventricular zone showed expression of the transgene in both neurons and glial cells. In the spinal cord, motor neuron transduction was prevalent, and transduction of neurons in the dorsal root ganglia was also prevalent. These results show that the CNS-mediated delivery method can be used to deliver genes for gene therapy methods. Indications that can benefit from this are: motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), or sensory-related indications such as pain. Other neurological indications that can benefit from this method are, for example, Huntington's disease, Alzheimer's disease, Tay-Sachs disease, Friedreich's ataxia, ataxia-telangiectasia, spinocerebellar ataxia (types 1, 2, and 3), Niemann-Pick disease (types A, B, and C), dopa-responsive dystonia, fragile X syndrome, Krabbe disease, glycogen storage disease type 2 (Pompe), primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, giant axonal neuropathy, multiple system atrophy (MSA), proximal myotonic myopathy, and neuronal ceroid lipofuscinosis (Batten disease).

[0142] Most of the relevant genetic defects are known and can be incorporated into the AAV(-5) vector. Using this delivery system as a therapeutic agent, it is conceivable that it can reverse the genotype and thus hopefully also reverse the phenotype.

[0143] In addition, intrathecal delivery methods are considered less invasive than infusion into the CNS itself, as brain surgery requires complex surgical procedures. The intrathecal method has the additional value that it can be performed without the need for a specialized brain injection center.

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Claims

1. An adeno-associated virus (AAV) gene therapy vector for use as a medicament in a mammalian subject, wherein the gene therapy vector comprises an AAV serotype 5 capsid protein and a gene product of interest flanked by AAV ITRs, and wherein the gene therapy vector is administered by lumbar intrathecal administration.

2. The AAV gene therapy vector of claim 1, wherein the lumbar intrathecal administration is at a site selected from L4–L5, L3–L4, L1–L2 and L2–L3.

3. The AAV gene therapy vector of claim 1 or 2, wherein the AAV gene therapy vector is a single-stranded AAV gene therapy vector or a monomeric double-stranded vector.

4. The AAV gene therapy vector of any one of the preceding claims, wherein the gene product of interest is for the treatment or prevention of a disorder selected from: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), pain, Huntington's disease, Alzheimer's disease, Tay-Sachs disease, Friedreich's ataxia, ataxia telangiectasia, spinocerebellar ataxia types 1, 2 and 3, Niemann-Pick disease types A, B and C, dopa-responsive dystonia, fragile X syndrome, Krabbe disease, glycogen storage disease type 2 (Pompe), primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, giant axonal neuropathy, multiple system atrophy (MSA), proximal myotonic myopathy, neuronal ceroid lipofuscinosis (Batten disease) and cancer.

5. The AAV gene therapy vector of any one of the preceding claims, wherein the AAV ITR is an AAV serotype 2 ITR.

6. The AAV gene therapy vector of any one of the preceding claims, wherein the gene product of interest is selected from: aspartylglucosaminidase, alpha-galactosidase A, palmitoylprotein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, multi-gene product, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase, protective protein / cathepsin A, acid beta-glucosidase, or glucocerebrosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid beta-galactosidase, N-acetylglucosamine-1-phosphate transferase, acid sphingomyelinase, NPC-1, acid alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosidase, alpha-N-acetylgalactosidase, alpha-Neuramidase, beta-glucuronidase, beta-hexosaminidase A, acid lipase, neurotrophic factors such as nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), brain-derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), growth factor insulin-like growth factor (IGF-1) and miRNA for downregulating defective genes.

7. The AAV gene therapy vector of any one of the preceding claims, wherein the gene product of interest is for treating or preventing a disorder associated with motor neurons and / or neurons in the DRG.

8. The AAV gene therapy vector of any one of the preceding claims, wherein the gene product of interest is operably linked to an expression control element comprising a promoter that produces sufficient expression of the gene product of interest to obtain a therapeutic effect, wherein the promoter is preferably selected from: cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK), CAG promoter (a combination of a cytomegalovirus early enhancer element and a chicken beta-actin promoter), glial fibrillary acidic protein (GFAP) promoter, synapsin-1 promoter, neuron-specific enolase (NSE) and inducible promoters such as gene switch or tet-operator-derived promoters.

9. The AAV gene therapy vector of any one of the preceding claims, wherein the gene therapy vector is further administered into the cisterna magna before, simultaneously with, or after intrathecal administration at the lumbar spine.

10. The AAV gene therapy vector of any one of the preceding claims, wherein 2×10 13 –2×10 15 genomic copies / kg body weight are administered to the subject.

11. The AAV gene therapy vector of claim 10, wherein 8×10 13 –6×10 14 genomic copies / kg body weight are administered to the subject.

12. The AAV gene therapy vector of any one of the preceding claims, wherein the subject does not receive pretreatment with a substance that controls intracranial pressure and / or can disrupt the intercellular tight junctions between endothelial cells of the CNS microvasculature and / or facilitate the delivery of drugs such as chemotherapeutic drugs to the CNS.

13. The AAV gene therapy vector of any one of the preceding claims, wherein the subject does not receive intravenous mannitol pretreatment prior to intrathecal administration of the AAV gene therapy vector via the lumbar spine.

14. The AAV gene therapy vector of any one of the preceding claims, wherein the mammalian subject is a human.

15. The AAV gene therapy vector of any one of the preceding claims, wherein the AAV gene therapy vector is not a self-complementary gene vector.

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