Enhanced delivery of viral particles to striatum and cortex

Abstract

To provide medicaments for delivering recombinant adeno-associated virus (rAAV) particles to the central nervous system of a mammal (e.g., a human).SOLUTION: Provided is a medicament containing a recombinant adeno-associated virus (rAAV) particle containing an AAV serotype 2 (AAV2) capsid, which is used for delivering the rAAV particle to a central nervous system of a mammal, the rAAV particle being administered to each hemisphere of a striatum at a rate greater than 1 μL / min to 5 μL / min by convection-enhanced delivery (CED), the rAAV particle comprising an rAAV vector comprising a heterologous nucleic acid encoding a therapeutic polypeptide, and the heterologous nucleic acid being expressed in at least the striatum and at least an occipital cortex and / or layer IV of a cerebral cortex of the mammal.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 114,544, filed February 10, 2015, and U.S. Provisional Application No. 62 / 220,997, filed September 19, 2015, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 159792012740SEQLIST.txt, Date Recorded: February 4, 2016, Size: 1 KB).

[0003] The present invention relates to the delivery of AAV gene therapy vectors to the brain, for example the striatum and / or cortex. [Background technology]

[0004] Adeno-associated virus (AAV)-based vectors are becoming the preferred vector system for neurological gene therapy with an excellent safety record established in multiple clinical trials (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). Effective treatment of neurological disorders has been hindered by problems associated with the delivery of AAV vectors to affected cell populations. This delivery problem has been particularly difficult to solve for disorders involving the cerebral cortex. Simple injection is only effective within a radius of 1 to 3 mm as it relies on diffusion, which does not effectively distribute AAV vectors. An alternative method, convection-enhanced delivery (CED) (Non-Patent Document 4), has been used clinically in gene therapy for Parkinson's disease (AAV2-hAADC) (Non-Patent Document 5). The basic principle of CED involves pumping infusion fluid into the brain parenchyma under sufficient pressure to overcome the hydrostatic pressure of the interstitial fluid, thereby forcing it into intimate contact with the densely vascularized peri-tissue of the brain. The pulsation of these tubes acts as a pump to distribute particles over long distances in the parenchyma (Non-Patent Document 6). To increase the safety and efficacy of CED, reflux-resistant cannulas (Non-Patent Document 7) can be employed along with monitoring of delivery using real-time MRI. Monitoring of delivery allows quantification and control of abnormal events such as cannula reflux and leakage of infusion fluid into the cavity (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 8). However, there is still a need for improved procedures to achieve widespread expression of AAV vectors in the cortex and / or striatum. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaplitt et al. (2007) Lancet 369:2097-2105 [Non-Patent Document 2] Eberling et al. (2008) Neurology 70:1980-1983 [Non-Patent Document 3] Fiandaca et al. (2009) Neuroimage 47, Supplement 2:T27-35 [Non-Patent Document 4] Nguyen et al. (2003) J. Neurosurg.98:584~590 [Non-Patent Document 5] Fiandaca et al. (2008) Exp. Neurol. 209:51~57 [Non-Patent Document 6] Hadaczek et al. (2006) Hum. Gene Ther. 17:291-302 [Non-Patent Document 7] Krauze et al. (2009) Methods Enzymol.465:349~362 [Non-Patent Document 8] Saito et al. (2011) Journal of Neurosurgery Pediatrics 7:522~526 Summary of the Invention [Means for solving the problem]

[0006] The present invention provides a method for delivering recombinant adeno-associated virus (rAAV) particles to the central nervous system of a mammal, comprising administering rAAV particles to the striatum, wherein the rAAV particles comprise a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal.In some embodiments, the present invention provides a method for delivering rAAV particles to the central nervous system of a mammal, comprising administering rAAV particles to the striatum, wherein the rAAV particles comprise a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal, wherein the rAAV particles comprise an AAV serotype 1 (AAV1) capsid.In some embodiments, the present invention provides a method for delivering rAAV particles to the central nervous system of a mammal, comprising administering rAAV particles to the striatum, wherein the rAAV particles comprise a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal, wherein the rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the mammal is a human.

[0007] In some embodiments, the rAAV particles are administered to at least the putamen and caudate nucleus of the striatum. In some embodiments, the rAAV particles are administered to at least the putamen and caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particles are administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal cortex, occipital cortex, and / or layer IV of the mammal. In some embodiments, the heterologous nucleic acid is expressed in at least the prefrontal association cortical area, the premotor cortex, the primary somatosensory cortical area, the sensory motor cortex, the parietal cortex, the occipital cortex, and / or the primary motor cortex. In some embodiments, the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex. In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra, and / or hippocampus. In some embodiments, the rAAV particles are administered to the caudate nucleus and putamen at a rate of greater than 1 μL / min to about 5 μL / min.

[0008] In some embodiments of the above aspects and embodiments, the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the ITR of AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the ITR of AAV is AAV2 ITR. In some embodiments, the ITR and capsid of rAAV particles are derived from the same AAV serotype. In some embodiments, the ITR and capsid are derived from AAV2. In other embodiments, the ITR and capsid of rAAV viral particles are derived from different AAV serotypes. In some embodiments, the ITR is derived from AAV2 and the capsid is derived from AAV1.

[0009] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is operably linked to a promoter. In some embodiments, the promoter expresses the heterologous nucleic acid in a cell of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in a cell of the brain. In some embodiments, the promoter expresses the heterologous nucleic acid in a neuron and / or a glial cell. In some embodiments, the neuron is a medium spiny neuron of the caudate nucleus, a medium spiny neuron of the putamen, a neuron of the cortical layer IV and / or a neuron of the cortical layer V. In some embodiments, the glial cell is an astrocyte. In some embodiments, the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible. In further embodiments, the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.

[0010] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an enzyme, a neurotrophic factor, a polypeptide that is deficient or mutated in individuals with a CNS-related disorder, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (PSA), or a combination thereof. A;ARSA), N-acetylglucosamine 1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine 6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase , alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuramidase. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.

[0011] In some embodiments of the above aspects and embodiments, the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer. In some embodiments, the disorder is aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten CNL4, Batten CNL5, Batten CNL6, Batten CNL7, Batten CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, The lysosomal storage disease is selected from the group consisting of beta-mannosidosis, Maroteaux-Lamy, metachromatic leukodystrophy, Morquio A, Morquio B, mucolipidosis II / III disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe disease, Sandhoff disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs disease, and Wollman disease.

[0012] In some embodiments of the above aspects and embodiments, the rAAV particles are in the form of a composition. In further embodiments, the composition is a pharmaceutical composition that includes a pharma- ceutical acceptable excipient.

[0013] In some of the above aspects and embodiments, the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, where transfection of the nucleic acids into the host cell generates a host cell capable of producing the rAAV particles. In other embodiments, the rAAV particles are produced by a producer cell line that includes one or more of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

[0014] In some embodiments of the above aspects and embodiments, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are delivered using a CED delivery system. In some embodiments, the CED system includes a cannula. In some embodiments, the cannula is a reflux-resistant cannula or a step-wise cannula. In some embodiments, the CED system includes a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0015] In some embodiments, the present invention provides a method for delivering rAAV particles to the central nervous system of a mammal. The method includes administering a composition comprising rAAV particles to the striatum by CED, the composition being administered to the striatum at a rate of greater than 1 μL / min to about 5 μL / min. In some aspects, the invention provides a method for delivering rAAV particles to the central nervous system of a mammal, the method includes administering a composition comprising rAAV particles to the striatum by CED, the composition comprising rAAV particles and a poloxamer. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the concentration of the poloxamer in the composition ranges from about 0.0001% to about 0.01%. In some embodiments, the concentration of the poloxamer in the composition is about 0.001%. In some embodiments, the composition further comprises sodium chloride, the concentration of the sodium chloride in the composition ranges from about 100 mM to about 250 mM. In some embodiments, the concentration of the sodium chloride in the composition is about 180 mM. In some embodiments, the composition further comprises sodium phosphate, the concentration of sodium phosphate in the composition is in the range of about 5 mM to about 20 mM, and the pH is about 7.0 to about 8.0. In some embodiments, the composition further comprises sodium phosphate, the concentration of sodium phosphate in the composition is about 10 mM, and the pH is about 7.5. In some embodiments, the composition is administered to the caudate nucleus and putamen at a rate of faster than 1 μL / min to about 5 μL / min. In some embodiments, the amount of the composition delivered to the putamen is about twice the volume delivered to the caudate nucleus. In some embodiments, about 20 μL to about 50 μL of the composition is administered to the caudate nucleus of each hemisphere, and about 40 μL to about 100 μL of the composition is administered to the putamen of each hemisphere. In some embodiments, about 30 μL of the composition is administered to the caudate nucleus of each hemisphere, and about 60 μL of the composition is administered to the putamen of each hemisphere.

[0016] In some embodiments, the invention provides a method of treating a CNS disorder in a mammal, comprising administering to the mammal an effective amount of rAAV particles by the methods described above.

[0017] In some aspects, the present invention provides a method for treating Huntington's disease in a mammal, comprising administering an effective amount of rAAV particles to the striatum, wherein the rAAV particles comprise a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal.In some embodiments, the rAAV particles comprise AAV1 capsid or AAV2 capsid.In other aspects, the present invention provides a method for treating Parkinson's disease in a mammal, comprising administering an effective amount of rAAV particles to the striatum, wherein the rAAV particles comprise a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal.In some embodiments, the rAAV particles comprise AAV2 capsid.In some embodiments, the mammal is a human.

[0018] In some embodiments, the rAAV particles are administered to at least the putamen and caudate nucleus of the striatum. In some embodiments, the rAAV particles are administered to at least the putamen and caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particles are administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal cortex, occipital cortex, and / or layer IV of the mammal. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal association cortex region, the premotor cortex, the primary somatosensory cortex region, the sensorimotor cortex, the parietal cortex, the occipital cortex, and / or the primary motor cortex. In some embodiments, the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex. In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra, and / or hippocampus.

[0019] In some embodiments of the above aspects and embodiments, the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the ITR of AAV is the ITR of AAV2. In some embodiments, the ITR and capsid of rAAV particle are derived from the same AAV serotype. In some embodiments, the ITR and capsid are derived from AAV2. In other embodiments, the ITR and capsid of rAAV virus particle are derived from different AAV serotypes. In some embodiments, the ITR is derived from AAV2 and the capsid is derived from AAV1.

[0020] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is operably linked to a promoter. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the brain. In some embodiments, the promoter expresses the heterologous nucleic acid in neurons and / or glial cells. In some embodiments, the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of cortical layer IV and / or neurons of cortical layer V. In some embodiments, the glial cells are astrocytes. In some embodiments, the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible. In further embodiments, the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a terminal resolution sequence mutation.

[0021] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide or the therapeutic nucleic acid inhibits the expression of HTT or inhibits the accumulation of HTT in cells of the CNS of a mammal with Huntington's disease. In some embodiments, the heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme, or a DNAzyme. In some embodiments, the heterologous nucleic acid encodes an miRNA that targets Huntington's disease. In some embodiments, the Huntington's disease-associated mutation.

[0022] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid for treating Huntington's disease. In some embodiments, the therapeutic polypeptide or the therapeutic nucleic acid inhibits the expression of HTT or inhibits the accumulation of HTT in cells of the CNS of a mammal with Huntington's disease. In some embodiments, the heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme, or a DNAzyme. In some embodiments, the heterologous nucleic acid encodes an miRNA that targets Huntington's disease. In some embodiments, the Huntington's disease-associated mutation.

[0023] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid for treating Parkinson's disease. In some embodiments, the therapeutic polypeptide is glial cell-derived growth factor (GDNF), brain-derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), and / or amino acid decarboxylase (AADC).

[0024] In some embodiments of the above aspects and embodiments, the rAAV particles are in the form of a composition. In further embodiments, the composition is a pharmaceutical composition that includes a pharma- ceutical acceptable excipient.

[0025] In some of the above aspects and embodiments, the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, where transfection of the nucleic acids into the host cell generates a host cell capable of producing the rAAV particles. In other embodiments, the rAAV particles are produced by a producer cell line that includes one or more of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

[0026] In some embodiments of the above aspects and embodiments, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are delivered using a CED delivery system. In some embodiments, the CED system includes a cannula. In some embodiments, the cannula is a reflux-resistant cannula or a step-wise cannula. In some embodiments, the CED system includes a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0027] In some aspects, the present invention provides a system for expressing heterologous nucleic acid in mammalian cerebral cortex and striatum, comprising: a) a composition comprising rAAV particle, wherein the rAAV particle comprises a rAAV vector encoding heterologous nucleic acid; and b) a device for delivering the rAAV particle to striatum.In some embodiments, the rAAV particle comprises AAV1 capsid or AAV2 capsid.In some embodiments, the mammal is human.

[0028] In some embodiments of the system of the present invention, the rAAV particles are administered to the putamen and the caudate nucleus of the striatum. In some embodiments, the rAAV particles are administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is administered to at least the frontal cortex of the mammal. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal association cortex region, the premotor cortex, the primary somatosensory cortex region, the sensorimotor cortex, the parietal cortex, the occipital cortex, and / or the primary motor cortex. In some embodiments, the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex. In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, the subthalamic nucleus, the globus pallidus, the substantia nigra, and / or the hippocampus.

[0029] In some embodiments of the inventive system, the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the rAAV particle ITRs and capsid are derived from the same AAV serotype. In some embodiments, the ITRs and capsid are derived from AAV2. In other embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes, hi some embodiments, the ITRs are derived from AAV2 and the capsid is derived from AAV1.

[0030] In some embodiments of the system of the present invention, the heterologous nucleic acid is operably linked to a promoter. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the brain. In some embodiments, the promoter expresses the heterologous nucleic acid in neurons and / or glial cells. In some embodiments, the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of layer IV of the cortex and / or neurons of layer V of the cortex. In some embodiments, the glial cells are astrocytes. In some embodiments, the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible. In further embodiments, the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a terminal resolution sequence mutation.

[0031] In some embodiments of the system of the present invention, the heterologous nucleic acid is a therapeutic polypeptide or The heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an enzyme, a neurotrophic factor, a polypeptide that is deficient or mutated in individuals with CNS-related disorders, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha- D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspar tylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuramidase. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme.In some embodiments, the therapeutic polypeptide or nucleic acid is used to treat a disorder of the CNS.

[0032] In some embodiments of the system of the present invention, the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer. In some embodiments, the disorder is aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten CNL4, Batten CNL5, Batten CNL6, Batten CNL7, Batten CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, The lysosomal storage disease is selected from the group consisting of beta-mannosidosis, Maroteaux-Lamy, metachromatic leukodystrophy, Morquio A, Morquio B, mucolipidosis II / III disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe disease, Sandhoff disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs disease, and Wollman disease.

[0033] In some embodiments, the rAAV of the present invention comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid for treating Huntington's disease. In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid inhibits expression of HTT or inhibits accumulation of HTT in cells of the CNS of a mammal with Huntington's disease. In some embodiments, the heterologous nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense oligonucleotide, or a combination thereof. In some embodiments, the heterologous nucleic acid encodes a miRNA that targets huntingtin. In some embodiments, the huntingtin comprises a mutation associated with Huntington's disease.

[0034] In some embodiments, the rAAV particles of the invention comprise a heterologous nucleic acid encoding a therapeutic polypeptide or nucleic acid for treating Parkinson's disease. In some embodiments, the therapeutic polypeptide is glial cell-derived growth factor (GDNF), brain-derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), and / or amino acid decarboxylase (AADC).

[0035] In some embodiments of the system, the rAAV particles are in the form of a composition, hi further embodiments, the composition is a pharmaceutical composition comprising a pharma- ceutical acceptable excipient.

[0036] In some embodiments of the systems, the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, where transfection of the nucleic acids into the host cell generates a host cell capable of producing the rAAV particles. In other embodiments, the rAAV particles are produced by a producer cell line that includes one or more of a nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

[0037] In some embodiments of the systems of the invention, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are delivered using a CED delivery system. In some embodiments, the CED system includes a cannula. In some embodiments, the cannula is a reflux-resistant cannula or a step-wise cannula. In some embodiments, the CED system includes a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0038] In some aspects, the present invention provides a kit for use in any of the above-mentioned methods, comprising rAAV particles, the kit comprising rAAV vector encoding heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of a mammal.In some embodiments, the rAAV particles comprise AAV serotype 1 (AAV1) capsid.In some embodiments, the rAAV particles comprise AAV serotype 2 (AAV2) capsid.

[0039] In some aspects, the present invention provides a kit for treating Huntington's disease in a mammal, comprising a composition comprising an effective amount of rAAV particles, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal. In some aspects, the present invention provides a kit for treating Parkinson's disease in a mammal, comprising a composition comprising an effective amount of rAAV particles, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal. In some embodiments, the rAAV particles of the kit comprise an AAV serotype 1 (AAV1) capsid or an AAV serotype 2 (AAV2) capsid. In some embodiments, the kit further comprises a device for delivering the rAAV particles to the striatum. In some embodiments, the rAAV particles of the kit are in the form of a composition. In some embodiments, the composition comprises a buffer and / or a pharmaceutical agent. In a further embodiment, the kit comprises instructions for delivering the composition of rAAV particles to the striatum.

[0040] In some embodiments, the present invention provides rAAV particles for use in any of the above-mentioned methods.In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for use in delivering to the central nervous system of a mammal, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for use in delivering to the central nervous system of a mammal, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal, and the rAAV particles further comprising an AAV serotype 1 (AAV1) capsid. In some aspects, the invention provides recombinant adeno-associated virus (rAAV) particles for use in delivering rAAV particles to the central nervous system of a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal, and the rAAV particle further comprises an AAV serotype 2 (AAV2) capsid.

[0041] In some aspects, the present invention provides rAAV particles for use in treating Huntington's disease in mammals, the rAAV particles comprise rAAV vectors encoding heterologous nucleic acids that are expressed in at least the cerebral cortex and striatum of mammals.In some aspects, the present invention provides rAAV particles for use in treating Parkinson's disease in mammals, the rAAV particles comprise rAAV vectors encoding heterologous nucleic acids that are expressed in at least the cerebral cortex and striatum of mammals.In some embodiments, the rAAV particles comprise AAV2 capsid.In some embodiments, the mammal is human.

[0042] In some embodiments, the rAAV particles of the present invention are administered to at least the putamen and caudate nucleus of the striatum. In some embodiments, the rAAV particles are administered to at least the putamen and caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particles are administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal cortex, occipital cortex, and / or layer IV of the mammal. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal association cortex region, the premotor cortex, the primary somatosensory cortex region, the sensorimotor cortex, the parietal cortex, the occipital cortex, and / or the primary motor cortex. In some embodiments, the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex. In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra, and / or hippocampus.

[0043] In some embodiments of the above aspects and embodiments, the rAAV particles of the invention comprise an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector flanks one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the rAAV particle ITRs and capsid are derived from the same AAV serotype. In some embodiments, the ITRs and capsid are derived from AAV2. In other embodiments, the rAAV particle ITRs and capsid are derived from different AAV serotypes. In some embodiments, the ITRs are derived from AAV2 and the capsid is derived from AAV1.

[0044] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is operably linked to a promoter. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the brain. In some embodiments, the promoter expresses the heterologous nucleic acid in neurons and / or glial cells. In some embodiments, the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of cortical layer IV and / or neurons of cortical layer V. In some embodiments, the glial cells are astrocytes. In some embodiments, the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible. In further embodiments, the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a terminal resolution sequence mutation.

[0045] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an enzyme, a neurotrophic factor, a polypeptide that is deficient or mutated in individuals with CNS-related disorders, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingolipidase (ASSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), N-acetylglucosamine-1-phosphotransferase (N ... Gomielinase (ASM), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (L IPA), aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuramidase. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.

[0046] In some embodiments of the above aspects and embodiments, the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer. In some embodiments, the disorder is aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten CNL4, Batten CNL5, Batten CNL6, Batten CNL7, Batten CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, The lysosomal storage disease is selected from the group consisting of beta-mannosidosis, Maroteaux-Lamy, metachromatic leukodystrophy, Morquio A, Morquio B, mucolipidosis II / III disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe disease, Sandhoff disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs disease, and Wollman disease.

[0047] In some embodiments of the above aspects and embodiments, the rAAV particles are in the form of a composition. In further embodiments, the composition is a pharmaceutical composition that includes a pharma- ceutical acceptable excipient.

[0048] In some of the above aspects and embodiments, the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, where transfection of the nucleic acids into the host cell generates a host cell capable of producing the rAAV particles. In other embodiments, the rAAV particles are produced by a producer cell line that includes one or more of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

[0049] In some embodiments of the above aspects and embodiments, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are delivered using a CED delivery system. In some embodiments, the CED system includes a cannula. In some embodiments, the cannula is a reflux-resistant cannula or a step-wise cannula. In some embodiments, the CED system includes a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0050] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 shows rhesus macaque body weights measured immediately prior to surgery (black) and at the time of necropsy (grey) in animals administered AAV1 and AAV2 vectors generated by the triple transfection (TT) and producer cell line (PCL) process. [Diagram 2]Figures 2A-2D show representative brain sections stained for GFP 30 days after infusion of AAV1-GFP(TT) into the caudate and putamen of rhesus monkeys. Sections in Figures 2A-2D span the brain in a rostral to caudal direction. Sections from three representative animals are displayed in each panel. [Diagram 3] Figures 3A-3D show representative brain sections demonstrating cortical expression of GFP in the frontal cortex (Figures 3A and 3B) and occipital cortex (Figures 3C and 3D), in both astrocytes (Figures 3A and 3C) and cortical neurons (Figures 3B and 3D), following infusion of AAV1-GFP(TT) into the caudate nucleus and putamen of rhesus monkeys. [Figure 4] Figures 4A-4D show representative brain sections stained for GFP 30 days after infusion of AAV2-GFP(TT) into the caudate and putamen of rhesus monkeys. Sections in Figures 4A-4D span the brain in a rostral to caudal direction. Each panel shows sections from three representative animals. [Diagram 5] Figures 5A-5D show representative brain sections stained for GFP 30 days after infusion of AAV1-GFP generated by producer cell line (PCL) (Figures 5A and 5B) or triple transfection (TT) (Figures 5C and 5D) processes into the caudate and putamen of rhesus monkeys. [Figure 6] Figures 6A-6D show representative brain sections stained for GFP 30 days after infusion of AAV2-GFP generated by producer cell line (PCL) (Figures 6A and 6B) or triple transfection (TT) (Figures 6C and 6D) processes into the caudate and putamen of rhesus monkeys. [Figure 7-1]Distribution of GFP in AAV1-eGFP and AAV2-eGFP-infused non-human primate (NHP) brains. AAV1-eGFP and AAV2-eGFP vectors were infused into the bilateral striatum of nine Rhesus macaques. Four weeks after surgery, brains were processed for immunohistochemistry (IHC) for GFP. Each column shows representative GFP-stained brain sections from four study groups infused with AAV1-eGFP (triple transfection; TT); AAV1-eGFP (producer cell line; PCL); AAV2-eGFP (TT); AAV2-eGFP (PCL). Representative sections show various coronal planes of the brain to demonstrate the distribution of GFP expression throughout the brain from the frontal cortex, striatum (site of infusion), midbrain to the occipital cortex. All groups showed robust GFP signals at the injection sites (putamen and caudate nucleus), as well as widespread transport to cortical regions and substantia nigra. Based on IHC staining, the coverage of GFP expression in both the target structure (striatum) and cortical regions was calculated for each monkey and is summarized in Table 7. [Figure 7-2] Continued from Figure 7-1. [Figure 8] Figure 1 shows the ratio of primary area of ​​transduction (PAT) to vector distribution (Vd). Primary areas of GFP expression in the striatum were depicted on scans from GFP stained sections and divided by the values ​​obtained from matching MRI scans with gadolinium signals. A ratio of >1.0 indicates that the extent of GFP expression exceeds the boundaries of the gadolinium signal after infusion. Results from monkeys infused with AAV vectors showed that AAV1 spread better into the brain parenchyma than AAV2 (1.21±0.1 vs. 0.74±0.04; p<0.007 using an unpaired two-tailed t-test). [Figure 9-1]Figures 9A-9H show GFP expression in the brains of NHPs transduced with AAV1-eGFP and AAV2-eGFP. Figure 9A: Higher magnification image (40x) of the caudate nucleus, a target structure transduced with AAV1-eGFP (TT) in subject #1. Dark brown GFP+ neurons stained with DAB are visualized against a densely stained network of positive neuronal fibers. Such a robust signal was detected in all monkeys injected with AAV1-eGFP vectors produced by both TT and PCL methods. Figure 9B: Fragment of the prefrontal cortex of subject #1 (Figure 7) demonstrating mass transport of the vector AAV1-eGFP from the injection site (striatum) to cortical regions. Based on the morphology of the GFP+ cells, both neurons and astrocytes were detected in the cortex. Figure 9C: Higher magnification image (40x) of the frame shown in Figure 9B, showing numerous cortical neurons expressing GFP. FIG. 9D: Higher magnification (40x) of the cortex from subject #1 showing GFP+ cells with astrocytic morphology. FIG. 9E: Higher magnification (40x) of the putamen, the target structure transduced with AAV2-eGFP(TT) from subject #6. The dark brown DAB signal indicates the expression of GFP in neurons and their network of densely stained fibers. FIG. 9F: Fragment of the prefrontal cortex from subject #6 (FIG. 7) demonstrating the mass transport of the vector AAV2-eGFP from the striatum (site of injection) to the cortical region. The majority of the GFP-positive cells had neuronal morphology (magnification 2.5x). FIG. 9G: Higher magnification (40x) of the frame shown in FIG. 9F showing numerous cortical neurons expressing GFP. FIG. 9H: Higher magnification (20x) of the internal capsule from subject #6 showing GFP+ cells with astrocytic morphology. [Figure 9-2] Continued from Figure 9-1. [Figure 10-1]Figures 10A-10E show the cellular affinity of AAV1-eGFP and AAV2-eGFP injected into monkey brains. Monkey brain sections were processed for double immunofluorescence staining for GFP and various cell markers to determine the cellular affinity of the injected vectors. Figure 10A: Sections from the caudate nucleus (target structure) from subject #1 stained with an antibody against GFP (green channel for DyLight™ 488 dye; left column) and an antibody against the neuronal marker NeuN (red channel for DyLight™ 549 dye; center column). Merged pictures from both channels (20x magnification; right column) show numerous neurons expressing GFP, demonstrating the neuronal affinity of AAV1-eGFP. Figure 10B: The same staining was performed on sections from the prefrontal cortex of subject #1, showing transduction of neurons in distal brain structures receiving neuronal projections from the striatum, evidence of retrograde transport of AAV1-eGFP. FIG. 10C: Section from the caudate nucleus (target structure) from subject #1 stained with an antibody against GFP (green channel for DyLight™ 488 dye; left column) and an antibody against the astrocyte marker S-100 (red channel for DyLight™ 549 dye; center column). Merged pictures from both channels (20x magnification; right column) show numerous astrocytes expressing GFP, demonstrating that AAV1-eGFP also transduces astrocytes. FIG. 10D: Section from the caudate nucleus (target structure) from subject #6 stained with an antibody against GFP (green channel for DyLight™ 488 dye; left column) and an antibody against the neuronal marker NeuN (red channel for DyLight™ 549 dye; center column). Merged pictures from both channels (20x magnification; right column) show numerous neurons expressing GFP, demonstrating the neuronal affinity of AAV2-eGFP. FIG. 10E: Section from the caudate nucleus (target structure) from subject #3 stained with an antibody against GFP (green channel for DyLight™ 488 dye; left column) and an antibody against the microglial cell marker Iba-1 (red channel for DyLight™ 549 dye; center column).The absence of co-staining with both markers in the merged photographs (20x magnification; right column) indicates that AAV1 does not transduce microglial cells, which was also the case for AAV2 (data not shown). [Figure 10-2] Continued from Figure 10-1. [Figure 11-1] Figures 11A-11C show the efficiency of transduction of neurons in the striatum of NHPs injected with AAV1-eGFP and AAV2-eGFP. Double immunofluorescence staining for GFP and the neuronal marker NeuN of monkey brain sections was performed to calculate the efficiency of transduction of neurons in the striatum (target structure) and cortical regions. For the striatum, the efficiency of transduction was calculated in the primary area of ​​GFP transduction (PAT), where the signal was robust and GFP+ neurons were densely distributed (Figure 11A). Neurons were also detected in areas outside the primary area of ​​GFP transduction (OPAT; Figure 11C). Figure 11B shows a scheme for the technique of counting GFP+ neurons in PAT (inner shaded) and GFP+ neurons in OPAT (outer shaded). Table 8 (PAT) and Table 9 (OPAT) show the data from the individual counts for each monkey and brain structure. [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 12]Figures 12A and 12B show histological findings related to the vector. Independent evaluation of hematoxylin and eosin (H&E) staining of circular fragments from the area of ​​primary transduction (PAT) revealed normal gliosis around the cannulation in all experimental groups. H&E staining also revealed perivascular cellular infiltration in all animals regardless of the vector used. The incidence and severity of perivascular wrapping was increased in the AAV1-injected group, especially when the vector was prepared by the TT method. Figure 12A: H&E stained fragments from subject #3 show numerous perivascular wrappings in the left putamen transduced with AAV1-eGFP (TT). One vessel is magnified (5x) in the lower right corner. Figure 12B: H&E stained fragments from subject #5 show only very little focal perivascular wrapping in the left caudate nucleus transduced with AAV1-eGFP (PCL). Magnify (5x) two to three blood vessels in the lower left corner. [Figure 13] 13A and 13B show quantitative PCR (QPCR) analysis of eGFP mRNA expression in liver, spleen, heart, kidney, and lung samples one month after AAV1-eGFP injection into the caudate and putamen of rhesus monkeys. (FIG. 13A) AAV1 and AAV2-eGFP vectors produced by triple transfection (TT) process. (FIG. 13B) AAV1 and AAV2-eGFP vectors produced by producer cell line (PCL) process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] As discussed in detail herein, the inventors have found that AAV vectors (e.g., AAV1 and AAV2 vectors) efficiently target both striatal and cortical structures in the rhesus monkey brain when delivered to the striatum (e.g., by convection-enhanced delivery, CED).Two different manufacturing platforms were also evaluated in these studies, and these studies demonstrate that AAV produced by triple transfection and producer cell line targets both striatal and cortical structures in the rhesus monkey brain.The intrastriatal delivery of rAAV particles (e.g., AAV1 and AAV2 vectors) produced using both platforms allows transduction of neurons in the striatum as well as neurons located at a considerable distance from the infusion site (e.g., cortical structures). Thus, the present invention provides a method of delivering recombinant adeno-associated virus (rAAV) particles containing an rAAV vector encoding a heterologous nucleic acid to the central nervous system of a mammal by administering the rAAV particles to the striatum, where the heterologous nucleic acid is expressed in at least the cerebral cortex and striatum of the mammal.

[0053] The present invention also provides methods for treating a CNS disorder (e.g., Huntington's disease) in a mammal by administering to the striatum rAAV particles encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum, as well as systems and methods for the expression of heterologous nucleic acids in the cerebral cortex and striatum of a mammal using the rAAV particles described herein. Methods of the invention may also utilize a delivery device (e.g., a CED device) for delivering rAAV particles to the striatum of a mammal, and similarly, systems and kits of the invention may further include a device for delivering rAAV particles to the striatum of a mammal.

[0054] I. General Techniques The techniques and procedures described or referred to herein are generally well recognized and commonly employed by those skilled in the art using conventional techniques, such as those described in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FMA Usubel et al., eds., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J. E. Elis, Academic Press, 1998); Introduction to Cell and Tissue Culture (ed. J. P. Mather and P. E. Oberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J. B. Griffiths, and D. G. Newell, J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (ed. D. M. Weir and CCBlackwell, 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (eds. M. Zanetti and J.D. Capra, Harwood. Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (eds. VT DeVita et al., JBLippincott Company, 2011), which are widely used techniques.

[0055] II. Definition A "vector," as used herein, refers to a recombinant plasmid or virus containing a nucleic acid to be delivered to a host cell, either in vitro or in vivo.

[0056] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term encompasses, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as typically found in RNA or DNA), or may contain modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits, such as phosphoramidates, thus oligodeoxynucleoside phosphoramidates (P-NH 2 ) or a hybrid phosphoramidate-phosphodiester oligomer. In addition, double-stranded polynucleotides can be obtained from single-stranded polynucleotide products of chemical synthesis, either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0057] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, without any minimum length limitation. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and their fragments are included in this definition. The term also encompasses post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, "polypeptide" also refers to proteins that include modifications, such as deletions, additions, and substitutions (generally of a conservative nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, for example, via site-directed mutagenesis, or may be accidental, for example, via PCR amplification, or host mutations that result in proteins or errors.

[0058] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs.

[0059] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) flanked by at least one or two AAV inverted terminal repeats (ITRs). Such rAAV vectors can replicate and be packaged into infectious viral particles when present in a host cell infected with a suitable helper virus (or expressing suitable helper functions) and expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection), the rAAV vector may also be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can also be encapsidated in a variety of ways, including, but not limited to, liposomes, complexed with lipids, and the like. The rAAV vector may be in any of a number of forms, such as a plasmid, a linear artificial chromosome, encapsidated in a viral particle, e.g., an AAV particle, etc. The rAAV vector can be packaged into the capsid of the AAV virus to generate a "recombinant adeno-associated viral particle (rAAV particle)."

[0060] "Heterologous" means derived from an entity having a genotype distinct from that of the remainder of the entity to which it is compared, introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (which can encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or a part thereof) incorporated into a viral vector is a heterologous nucleotide sequence with respect to that vector. Heterologous nucleic acid may refer to a nucleic acid derived from an entity having a genotype distinct from that of the remainder of the entity to which it is compared, introduced or incorporated.

[0061] The term "heterologous nucleic acid" refers to a polynucleotide that can be introduced into cells and transcribed into RNA, and can optionally be translated and / or expressed under appropriate conditions.In some embodiments, transgenes confer desired characteristics to the cells into which they are introduced, or otherwise produce desired therapeutic or diagnostic results.In another embodiment, transgenes can be transcribed into molecules that mediate RNA interference, such as miRNA, siRNA, or shRNA.

[0062] "Chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken β-actin gene (e.g., chicken (Gallus gallus) beta actin, designated Entrez gene number 396526 in GenBank). "Chicken β-actin promoter" as used herein may refer to a promoter containing a cytomegalovirus (CMV) early enhancer element, the promoter and first exon and intron of the chicken β-actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequence described in Miyazaki, J. et al. (1989) Gene 79(2):269-77. The term "CAG promoter" as used herein may be used synonymously. The term "CMV early enhancer / chicken beta actin (CAG) promoter" as used herein may be used synonymously.

[0063] The terms "genome particles (gp)", "genome equivalents", or "genome copies", when used in reference to viral titer, refer to the number of virions containing the DNA genome of a recombinant AAV, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0064] The term "vector genome (vg)" as used herein may refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, e.g., a viral vector. A vector genome may be encapsidated in a viral particle. Depending on the particular viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or double-stranded RNA. A vector genome may include endogenous sequences associated with a particular viral vector and / or any heterologous sequences inserted into a particular viral vector via recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a sequence of interest (e.g., a heterologous nucleic acid), and a polyadenylation sequence. A complete vector genome may include the complete set of polynucleotide sequences of a vector. In some embodiments, the titer of the viral vector nucleic acid can be measured in vg / mL. Suitable methods for measuring the titer are known in the art (e.g., quantitative PCR).

[0065] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0066] The term "transducing unit (tu)", when used in reference to viral titer, refers to the number of infectious recombinant AAV vector particles that result in the production of a functional heterologous nucleic acid product, as measured in the Examples herein, or in a functional assay such as that described, for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0067] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short sequences found at the ends of viral genomes in inverted orientation.

[0068] "AAV inverted terminal repeat (ITR)" sequence is a term well understood in the art and is a sequence of approximately 145 nucleotides present at both ends of a native single-stranded AAV genome. The outermost 125 nucleotides of the ITR may be present in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions (designated A, A', B, B', C, C' and D regions) that are self-complementary, allowing intrastrand base pairing to occur within this portion of the ITR.

[0069] "Terminal resolution sequence" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. The mutated terminal resolution sequence is resistant to cleavage by the AAV rep protein.

[0070] "AAV helper function" refers to the function that allows AAV to replicate and package by host cell.AAV helper function can be provided in any of many forms, including but not limited to helper virus or helper virus gene that helps AAV replication and packaging.Other AAV helper functions are known in the field, such as genotoxic agents.

[0071] A "helper virus" for AAV refers to a virus that allows AAV (which is a defective parvovirus) to replicate and be packaged by a host cell. Helper viruses provide "helper functions" that allow AAV to replicate. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia and baculovirus. Adenoviruses encompass many different subgroups, with adenovirus type 5 (Ad5) of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as ATCC. Herpes family viruses that are also available from depositories such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenoviral helper functions for AAV replication include E1A, E1B, E2A, VA and E4orf6 functions. and Autographa californica nuclear polyhedrosis virus.

[0072] The rAAV preparations are prepared with a ratio of infectious AAV particles to infectious helper virus particles of at least about 10. 2 :1; at least about 10 4 :1, at least about 10 6 :1; or at least about 10 8A preparation is said to be "substantially free" of helper virus if its concentration is 0.1 or higher. In some embodiments, the preparation also does not contain a corresponding amount of helper virus protein (i.e., protein that would be expected to be present as a result of such levels of helper virus if the above-mentioned helper virus particle impurities were present in a disrupted form). Contaminating viral and / or cellular proteins can generally be observed as the presence of Coomassie stained bands in SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2 and VP3).

[0073] "AAV helper functions" refers to functions that allow AAV to replicate and be packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper viruses or helper virus genes that aid in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents. "Helper virus" in reference to AAV refers to a virus that allows AAV (which is a defective parvovirus) to replicate and be packaged by a host cell. Many such helper viruses have been identified, including adenoviruses, herpes viruses, and pox viruses, such as vaccinia. Adenoviruses encompass many different subgroups, with adenovirus type 5 (Ad5) of subgroup C being the most commonly used. Many adenoviruses from human, non-human mammals, and birds are known and available from depositories such as the ATCC. Herpes family viruses also available from depositories such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV).

[0074] "Percent (%) sequence identity" to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to those in the reference polypeptide or nucleic acid sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary. Alignment for the purpose of determining percent amino acid or nucleic acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supplement 30, section 7.7.18, table 7.7.1, and BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine the appropriate parameters for measuring alignment, such as any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in that program's alignment of A and B, and Y is the number of amino acid residues in B that are scored as identical matches by a sequence alignment program in that program's alignment of A and B. % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D (alternatively, it can be expressed as a given nucleic acid sequence C having or containing a particular % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 x fraction W / Z, where W is the number of nucleotides scored as identical matches by a sequence alignment program in the alignment of C and D in that program, and Z is the total number of nucleotides in D. It is understood that if the length of a nucleic acid sequence C is not equal to the length of a nucleic acid sequence D, the % amino acid sequence identity of A to B is not expected to be equal to the % amino acid sequence identity of B to A. For the purposes described herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (alternatively, it can be expressed as a given nucleic acid sequence C having or containing a particular % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 x fraction W / Z, where W is the number of nucleotides scored as identical matches by a sequence alignment program in the alignment of C and D in that program, and Z is the total number of nucleotides in D. It is understood that if the length of a nucleic acid sequence C is not equal to the length of a nucleic acid sequence D, the % nucleic acid sequence identity of C to D is not expected to be equal to the % nucleic acid sequence identity of D to C.

[0075] An "isolated" molecule (eg, a nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0076] An "effective amount" is an amount sufficient to effect a beneficial or desired result, such as a clinical result (e.g., alleviation of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to alleviate, stabilize, or delay the onset of a disease.

[0077] The term "convection-enhanced delivery (CED)" as used herein may refer to delivery of a therapeutic agent to the CNS by infusion at a rate where hydrostatic pressure causes distribution by convection. In some embodiments, the infusion is at a rate greater than 0.5 μL / min. However, any suitable flow rate may be used such that intracranial pressure is maintained at a suitable level that does not damage brain tissue. CED may be achieved, for example, by using a suitable catheter or cannula (e.g., a step-designed anti-reflux cannula) by positioning the tip of the cannula at least near the target CNS tissue (e.g., inserting the tip into the CNS tissue). After the cannula is positioned, it is connected to a pump that delivers the therapeutic agent to the target CNS tissue through the tip of the cannula. A pressure gradient from the tip of the cannula may be maintained during the infusion. In some embodiments, infusion can be monitored by a tracing agent detectable by imaging methods such as intraoperative MRI (iMRI) or another real-time MRI technique, and / or delivered by standard stereotactic injection equipment and techniques (e.g., the ClearPoint® system from MRI Interventions, Memphis, TN).

[0078] The term "poloxamer," as used herein, may refer to a block copolymer composed of a polyoxypropylene chain adjacent to two polyoxyethylene chains. Trade names under which poloxamers may be sold include, but are not limited to, PLURONIC® (BASF), KOLLIPHOR® (BASF), LUTROL® (BASF), and SYNPERONIC® (Croda International).

[0079] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human.

[0080] "Treatment," as used herein, is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, the following: These include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (e.g., not worsening) state of disease, prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, alleviation or palliative care, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0081] The term "prophylactic treatment" as used herein refers to treatment where an individual is known or suspected to have or be at risk of having a disorder, but exhibits no or minimal symptoms of the disorder. An individual receiving prophylactic treatment is treated prior to the onset of symptoms.

[0082] "Huntington's disease (HD)" refers to a progressive brain disorder typically caused by mutations in the HTT gene (aka huntingtin, HD or IT15). Huntington's disease can be characterized by symptoms such as abnormal movements (called chorea), gradual loss of motor function, emotional or psychiatric illness, and increasingly impaired cognition. Most symptoms appear in the third and fourth decades, although younger forms of the disease have also been observed. For further description of HD, see OMIM Accession No. 143100, which is incorporated herein by reference in its entirety.

[0083] "Huntingtin (HTT)" can refer to either the genes or their polypeptide products that are associated with most cases of Huntington's disease. The normal function of huntingtin is not fully understood. However, mutations in the huntingtin gene are known to cause HD. These mutations are typically inherited in an autosomal dominant manner and result in the expansion of trinucleotide CAG repeats in the HTT gene, giving rise to polyglutamine (polyQ) tracts in the Htt protein.

[0084] A "therapeutic" agent (e.g., a therapeutic polypeptide, nucleic acid, transgene, or the like), as used herein, is one that brings about a beneficial or desired clinical outcome, such as the exemplary clinical outcomes described above. As such, a therapeutic agent can be used in treatments as described above.

[0085] Reference herein to a value or parameter "with respect to" encompasses (describes) embodiments directed to that value or parameter per se. For example, a reference "with respect to X" encompasses a description of "X."

[0086] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise specified.

[0087] It is understood that aspects and embodiments of the invention described herein encompass "comprising," "consisting," and / or "consisting essentially of" aspects and embodiments.

[0088] III. Methods for Delivering rAAV Particles In some embodiments, the present invention provides a method for delivering recombinant adeno-associated virus (rAAV) particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal. In a further embodiment, the present invention provides a method for delivering rAAV particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal, wherein the rAAV particles comprise an AAV serotype 1 (AAV1) capsid. In yet additional embodiments, In one embodiment, the present invention provides a method for delivering rAAV particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, the rAAV particles comprising a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal, and the rAAV particles comprising an AAV serotype 2 (AAV2) capsid.In yet another embodiment, the present invention provides a method for treating Huntington's disease in a mammal, comprising administering the rAAV particles to the striatum, the rAAV particles comprising a rAAV vector encoding a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of the mammal.In some embodiments, the mammal is a human.

[0089] Certain aspects of the present disclosure relate to the administration of rAAV particles to one or more regions of the central nervous system (CNS). In some embodiments, the rAAV particles are administered to the striatum. The striatum is known as the region of the brain that receives input from the cerebral cortex (the term "cortex" may be used interchangeably herein) and sends output to the basal ganglia (the striatum is also referred to as the striate nucleus and the neostriatum). As mentioned above, the striatum controls both motor function and emotional control / motivation and has been implicated in many neurological disorders, such as Huntington's disease. Several cell types of interest are located in the striatum, including, but not limited to, spiny projection neurons (also known as medium spiny neurons), GABAergic interneurons, and cholinergic interneurons. Medium spiny neurons make up the majority of striatal neurons. These neurons are GABAergic and express dopamine receptors. Each hemisphere of the brain contains a striatum.

[0090] Important underlying structures of the striatum include the caudate nucleus and putamen. In some embodiments, rAAV particles are administered to the caudate nucleus (the term "caudate" may be used interchangeably herein). The caudate nucleus is known as a structure of the dorsal striatum. The caudate nucleus has been shown to be involved in the control of functions such as directed movement, spatial working memory, memory, goal-directed behavior, emotion, sleep, language, and learning. Each hemisphere of the brain contains a caudate nucleus.

[0091] In some embodiments, rAAV particles are administered to the putamen.Together with the caudate nucleus, the putamen is known as a structure of the dorsal striatum.The putamen includes a portion of the lentiform nucleus and connects the cerebral cortex with the substantia nigra and the globus pallidus.The putamen is highly integrated with many other structures of the brain, and has been shown to be involved in the control of functions such as learning, motor learning, motor performance, motor tasks, and limb movements.Each hemisphere of the brain contains a putamen.

[0092] rAAV particles may be administered to one or more sites in the striatum.In some embodiments, rAAV particles are administered to the putamen and caudate nucleus of the striatum.In some embodiments, rAAV particles are administered to the putamen and caudate nucleus of each hemisphere of the striatum.In some embodiments, rAAV particles are administered to at least one site in the caudate nucleus and two sites in the putamen.

[0093] In some embodiments, rAAV particles are administered to one hemisphere of the brain. In some embodiments, rAAV particles are administered to both hemispheres of the brain. For example, in some embodiments, rAAV particles are administered to the putamen and caudate nucleus of each hemisphere of the striatum. In some embodiments, the composition containing rAAV particles is administered to the striatum of each hemisphere. In other embodiments, the composition containing rAAV particles is administered to the striatum of the left hemisphere or the striatum of the right hemisphere, and / or the putamen of the left hemisphere or the putamen of the right hemisphere. In some embodiments, the composition containing rAAV particles is administered to any combination of the caudate nucleus of the left hemisphere, the caudate nucleus of the right hemisphere, the putamen of the left hemisphere, and the putamen of the right hemisphere.

[0094] In some embodiments, the composition containing the rAAV particles comprises at least one of: Simultaneously or sequentially administered. In some embodiments, multiple injections of the composition containing rAAV particles are spaced apart by about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours or 24 hours or less. In some embodiments, multiple injections of the composition containing rAAV particles are spaced apart by more than about 24 hours.

[0095] Generally, about 1 μL to about 1 mL of the compositions of the present invention can be delivered (e.g., about 100 μL to about 500 μL of the composition). In some embodiments, the amount of the composition delivered to the putamen is greater than the volume delivered to the caudate nucleus. In some embodiments, the amount of the composition delivered to the putamen is about 2 times the volume delivered to the caudate nucleus. In other embodiments, the amount of the composition delivered to the putamen is either about 1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, or 10 times (or any ratio therebetween) the volume delivered to the caudate nucleus. For example, in some embodiments, the ratio of rAAV particles administered to the putamen to the caudate nucleus is at least about 2:1 (e.g., about 30 μL of the composition is administered to the caudate nucleus of each hemisphere and about 60 μL of the composition is administered to the putamen of each hemisphere). In some embodiments, about 20 μL to about 50 μL (or any amount therebetween) of the composition is administered to the caudate nucleus of each hemisphere and about 40 μL to about 100 μL (or any amount therebetween) of the composition is administered to the putamen of each hemisphere. In some embodiments, the volume of the composition administered to the caudate nucleus of each hemisphere is less than about any of the following volumes (in μL): 50, 45, 40, 35, 30, or 25. In some embodiments, the volume of the composition administered to the caudate nucleus of each hemisphere is more than about any of the following volumes (in μL): 20, 25, 30, 35, 40, or 45. That is, the volume of the composition administered to the caudate nucleus of each hemisphere may be any of a range of volumes having an upper limit of 50, 45, 40, 35, 30, or 25 and an independently selected lower limit of 20, 25, 30, 35, 40, or 45, where the lower limit is lower than the upper limit. In some embodiments, the volume of the composition administered to the putamen of each hemisphere is less than approximately any of the following volumes (in μL): 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45. In some embodiments, the volume of the composition administered to the putamen of each hemisphere is more than approximately any of the following volumes (in μL): 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95.That is, the volume of composition administered to the putamen of each hemisphere can be any of a range of volumes having an upper limit of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45 and an independently selected lower limit of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, where the lower limit is lower than the upper limit.

[0096] In some embodiments, the composition is administered to the striatum at a rate of more than 1 μL / min to about 5 μL / min. In some embodiments, the composition is administered to the caudate nucleus and putamen at a rate of more than 1 μL / min to about 5 μL / min. In some embodiments, the composition is administered to the striatum (caudate nucleus and / or putamen) at a rate of more than about 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, the composition is administered at a concentration of from about 1 μL / min to about 10 μL / min, from about 1 μL / min to about 9 μL / min, from about 1 μL / min to about 8 μL / min, from about 1 μL / min to about 7 μL / min, from about 1 μL / min to about 6 μL / min, from about 1 μL / min to about 5 μL / min, from about 1 μL / min to about 4 μL / min, from about 1 μL / min to about 3 μL / min, from about 1 μL / min to about 2 μL / min, from about 2 μL / min to about 10 μL / min, from about 2 μL / min to about 9 μL / min, from about 2 μL / min to about 8 μL / min, from about 2 μL / min to about 7 μL / min, from about 2 μL / min to about 6 μL / min, μL / min, approximately 2 μL / min to approximately 5 μL / min, approximately 2 μL / min to approximately 4 μL / min, approximately 2 μL / min to approximately 3 μL / min, approximately 3 μL / min to approximately 10 μL / min, approximately 3 μL / min to approximately 9 μL / min, approximately 3 μL / min to approximately 8 μL / min, approximately 3 μL / min to approximately 7 μL / min, approximately 3 μL / min to approx. 6 μL / min, approx. 3 μL / min to approx. 5 μL / min, approx. 3 μL / min to approx. 4 μL / min, approx. 4 μL / min to approx. 10 μL / min, approx. 4 μL / min to approx. 9 μL / min, approx. 4 μL / min to approx. 8 μL / min, approx. 4 μL / min to approx. 7 μL / min, approx. 6μL / min, about 4μL / min to about 5μL / min, about 5μL / min to about 10μL / min, about 5μL / min to about 9μL / min, about 5μL / min to about 8μL / min, about 5μL / min ~ approx. 7 μL / min, approx. 5 μL / min ~ approx. 6 μL / min, approx. 6 μL / min ~ approx. 10 μL / min, approx. 6 μL / min ~ approx. 9 μL / min, approx. 6 μL / min ~ approx. 8 μL / min, approx. The composition is administered to the striatum (caudate and / or putamen) at a rate of between 6 μL / min and about 7 μL / min, between about 7 μL / min and about 10 μL / min, between about 7 μL / min and about 9 μL / min, between about 7 μL / min and about 8 μL / min, between about 8 μL / min and about 10 μL / min, between about 8 μL / min and about 9 μL / min, or between about 9 μL / min and about 10 μL / min. In some embodiments, the composition is administered at an incremental (i.e., "step-wise") flow rate throughout delivery.

[0097] In some embodiments, administration of the rAAV particles is performed once. In other embodiments, administration of the rAAV particles is performed more than once. One of skill in the art can determine how many administrations of the rAAV particles to perform based, in part, on, for example, the disorder being treated and / or the patient's response to the treatment.

[0098] In some embodiments, the method includes CNS administration of an effective amount of recombinant viral particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and the striatum. In some embodiments, the viral titer of the rAAV particles is at least about 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50×10 12 In some embodiments, the viral titer of the rAAV particles is about 5×10 12 ~6×10 12 , 6×10 12 ~7×1012 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50×10 12 ~100×10 12 In some embodiments, the viral titer of the rAAV particles is about 5×10 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50×10 9 In some embodiments, the viral titer of the rAAV particles is about 5×10 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 or 50×10 9 ~100×10 9 In some embodiments, the viral titer of the rAAV particles is about 5×10 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25 × 10 9 ~50×10 9 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 , 30×10 10 , 40×10 10 , or 50×10 10 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 infectious units / mL. 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×1010 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50×10 10 ~100×10 10 In some embodiments, the viral titer of the rAAV particles is either infectious units / mL or 100000000000000. , at least about 5 × 10 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 10 10 ~50×10 10 infectious units / mL.

[0099] In some embodiments, the method comprises CNS administration of an effective amount of recombinant viral particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and the striatum. In some embodiments, the dose of viral particles administered to an individual is at least about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 per kg of body weight. 8 ~1×10 13 It is a genome copy.

[0100] In some embodiments, the method comprises CNS administration of an effective amount of recombinant viral particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and the striatum. In some embodiments, the total amount of viral particles administered to the individual is at least about 1×10 9 ~Approx. 1×10 14 In some embodiments, the total amount of viral particles administered to an individual is about 1×10 genome copies. 9 ~Approx. 1×10 14 It is a genome copy.

[0101] The compositions of the invention (e.g., rAAV particles) can be used either alone or in combination with one or more additional therapeutic agents to treat any or all of the disorders described herein. The interval between sequential administrations may be on the order of at least minutes, hours, or days (or alternatively less).

[0102] IV. Expression Constructs In some aspects, the present invention provides a method for delivering rAAV particles to the CNS of a mammal by administering the rAAV particles to the striatum.In some embodiments, the rAAV particles comprise an rAAV vector.The rAAV vector can code for heterologous nucleic acid (e.g., heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum).The rAAV vector is described in more detail below.

[0103] In some embodiments, the rAAV vector encodes a heterologous nucleic acid. In some embodiments, the heterologous nucleic acid may encode a therapeutic polypeptide or a therapeutic nucleic acid. The therapeutic polypeptide or the therapeutic nucleic acid can be used, for example, to alleviate symptoms, prevent or delay progression, and / or provide treatment for a disorder (e.g., a disorder described herein). In some embodiments, the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS, as described in more detail below.

[0104] Heterologous nucleic acid is expressed in one or more regions of interest in CNS.For example, in some embodiments, heterologous nucleic acid is expressed at least in cerebral cortex and striatum.Heterologous nucleic acid is expressed ubiquitously in CNS or in a subset of CNS cells.

[0105] In some embodiments, the heterologous nucleic acid is expressed in the frontal cortex, occipital cortex, and / or layer IV of a mammal. The cerebral cortex is known as the outer layer of the mammalian brain, which is important for language, consciousness, memory, attention, and cognition. The cerebral cortex is further subdivided into many different components and regions due to its wide range of anatomical and complex functions. The cerebral cortex can be subdivided into left and right hemispheres. In addition, the cerebral cortex contains four broad lobes: the frontal cortex, the parietal cortex, the temporal cortex, and the occipital cortex. The frontal cortex may refer to the frontal lobe of the cortex, which is known to provide diverse neurological functions related to task-free memory, social interaction, decision-making, and other complex cognitive functions. The occipital cortex may refer to the occipital lobe of the cortex, which is known to be involved in visual processing. The parietal cortex may refer to the parietal lobe of the cortex, which is known to be involved in visual processing. The medial cortex may refer to the temporal lobe of the cortex, which is known to be involved in language processing, proprioception, and sensory input related to touch. The medial cortex may refer to the temporal lobe of the cortex, which is known to be involved in language, memory, and emotional associations.

[0106] In addition, three general types of cortical regions have been described: sensory, motor, and association. These can be subdivided into five functional subdivisions: primary motor cortex (involved in muscle control), premotor cortex (higher motor areas that command the primary motor cortex), association areas (e.g., parieto-temporal-occipital or frontal; these areas are involved in planning, memory, attention, and other higher cognitive tasks and account for the majority of the human cortex), higher areas (sensory processing), and primary sensory areas (e.g., auditory, visual, and somatosensory). In some embodiments, the heterologous nucleic acid is expressed in frontal association cortical areas, premotor cortex, primary somatosensory cortical areas, sensory motor cortex, parietal cortex, occipital cortex, and / or primary motor cortex.

[0107] In addition, the cerebral cortex can be subdivided (from superficial to deep) into different cortical layers, each containing a characteristic pattern of neuronal connectivity and cell types. These layers can be subdivided into supragranular (layers I-III), infragranular (IV), and infragranular (V and VI) layers. The supragranular layers typically project to other cortical layers, whereas the infragranular layers receive input from the supragranular layers and output to structures outside the cortex (e.g., motor, sensory, and thalamic regions). Layer V contains pyramidal neurons with axons that are connected to subcortical structures such as the basal ganglia. Layer V neurons in the primary motor cortex also form the corticospinal tract, which is important for the control of voluntary movement. Layer IV provides important functions for thalamic and cortical integration, as it receives input from the thalamus and is connected to the rest of the gray column. Characteristic cells of layer IV include stellate cells (e.g., spiny stellate cells) and pyramidal neurons.

[0108] In some embodiments, rAAV particles undergo retrograde or anterograde transport in the cerebral cortex. Retrograde transport refers to the phenomenon in which cargo (e.g., rAAV particles) moves from neurites (e.g., axons) to the cell body. Anterograde transport refers to the movement from the cell body to the cell membrane (e.g., synapses). Retrograde transport of AAV particles is thought to occur via receptor-mediated internalization at the axon terminal, followed by microtubule-mediated transport to the nucleus (see, e.g., Kaspar et al., (2002) Mol. Ther. 5:50-56; Boulis et al., (2003) Neurobiol. Dis. 14:535-541; Kaspar et al., (2003) Science 301:839-842). The striatum is known to contain projections from other brain regions, such as regions of the cortex. Both anterograde and retrograde transport allow rAAV particles to be dispersed throughout the brain, for example, from the cortex to the thalamus (see, e.g., Kells, AP et al. (2009) Proc. Natl. Acad. Sci. 106:2407-2411). Therefore, without wishing to be bound by theory, it is believed that injection of AAV particles into one brain region (e.g., the striatum, caudate, and / or putamen) allows delivery of AAV particles via retrograde transport to other brain regions (e.g., the cortex).

[0109] In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, substantia nigra and / or hippocampus. As mentioned above, mechanisms such as anterograde and / or retrograde transport allow rAAV particles injected into the cerebral cortex and / or striatum to be dispersed to other regions of the brain, especially those connected to the cortex. The thalamus is between the cortex and the midbrain, sending signals (e.g., sensory and motor) from subcortical regions to the cortex, and plays a role in wakefulness and sleep. The thalamus is also connected to the hippocampus, which is part of the limbic system and an important mediator of long-term memory consolidation. The substantia nigra, which is part of the basal ganglia, contains many dopaminergic neurons and is important for movement and reward. CNS disorders such as Parkinson's disease are , is associated with a loss of dopaminergic neurons in the substantia nigra, which in turn supplies the striatum with dopamine, which is important for proper striatal function.

[0110] In some aspects, the invention provides rAAV vectors for use in a method of preventing or treating one or more genetic defects in a mammal (e.g., inherited genetic defects, somatic genetic alterations, and the like), such as genetic defects that cause a deficiency or excess of a polypeptide in a subject, or for treating a subject exhibiting a CNS-related disorder associated with a deficiency of such a polypeptide in cells and tissues, or for reducing the severity or extent of the deficiency. In some embodiments, the method comprises administering to a subject a rAAV vector encoding one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, microRNAs, antisense nucleotides, etc., in a pharma- tically acceptable carrier, in an amount and for a duration sufficient to treat such a CNS-related disorder in a subject having or suspected of having such a disorder.

[0111] The rAAV vector may contain as a transgene a nucleic acid encoding a protein or functional RNA that modulates or treats a CNS-related disorder. The following is a non-limiting list of genes associated with CNS-related disorders: neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line derived growth factor (GDNF), brain derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TM), GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase (SOD1), and amino acid decarboxylase (AADC). For example, a transgene useful in the treatment of Parkinson's disease encodes TH, the rate-limiting enzyme in the synthesis of dopamine; GT, which generates the TII cofactor tetrahydrobiopterin. Transgenes encoding PCII can also be used to treat Parkinson's disease. Transgenes encoding GDNF or BDNF, or AADC, which facilitate the conversion of L-dopa to DA, can also be used to treat Parkinson's disease. For the treatment of ALS, useful transgenes may encode GDNF, BDNF, or CNTF. Similarly, for the treatment of ALS, useful transgenes may encode functional RNA, e.g., shRNA, miRNA, that inhibits the expression of SOD1. For the treatment of ischemia, useful transgenes may encode NAIP or NGF. Certain lysosomal storage diseases (e.g., mucopolysaccharidosis type VII (MPS)) can be treated with GABA. VII)) for the treatment of, transgenes encoding beta-glucuronidase (GUS) may be useful. For the treatment of certain cancers, transgenes encoding prodrug activating genes, such as HSV-thymidine kinase, which convert ganciclovir into toxic nucleotides that disrupt DNA synthesis and cause cell death, may be useful, for example, when administered in combination with a prodrug. For the treatment of pain, transgenes encoding endogenous opioids, such as β-endorphin, may be useful.Other examples of transgenes that can be used in the rAAV vectors of the invention will be apparent to those skilled in the art (see, for example, Costantini LC et al., Gene Therapy (2000) 7, 93-109).

[0112] In some embodiments, the heterologous nucleic acid may encode a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid may include, but is not limited to, an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. As such, the therapeutic nucleic acid may encode an RNA that, when transcribed from the nucleic acid of the vector, can treat a disorder of the invention (e.g., a CNS disorder) by interfering with the translation or transcription of aberrant or excess proteins associated with the disorder of the invention. For example, the nucleic acid of the invention can encode an RNA that treats a disorder by highly specific elimination or reduction of mRNAs encoding aberrant and / or excess proteins. The therapeutic RNA sequence may encode a NA. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (e.g., hammerhead and hairpin ribozymes) that can treat disorders by highly specific elimination or reduction of mRNAs that code for abnormal and / or excess proteins.

[0113] In some embodiments, the heterologous nucleic acid may encode a therapeutic polypeptide. The therapeutic polypeptide may, for example, provide a polypeptide and / or enzyme activity that is absent or present at reduced levels in a cell or organism. Alternatively, the therapeutic polypeptide may provide a polypeptide and / or enzyme activity that indirectly neutralizes an imbalance in a cell or organism. For example, a therapeutic polypeptide for a disorder related to metabolite accumulation caused by a deficiency in a metabolic enzyme or activity may provide the missing metabolic enzyme or activity, or provide an alternative metabolic enzyme or activity that causes a reduction in the metabolic product. Therapeutic polypeptides may also be used to reduce the activity of a polypeptide (e.g., a polypeptide that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated), for example, by acting as a dominant-negative polypeptide.

[0114] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat disorders of the CNS. Without wishing to be bound by theory, it is believed that therapeutic polypeptides or therapeutic nucleic acids can be used to reduce or eliminate expression and / or activity of a polypeptide whose gain of function is associated with the disorder, or to enhance expression and / or activity of a polypeptide to complement a defect associated with the disorder (e.g., a mutation in a gene whose expression exhibits a similar or related activity). Non-limiting examples of CNS disorders of the invention treatable by a therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes that may be targeted or delivered are shown in parentheses for each disorder) include stroke (e.g., caspase-3, Beclin 1, Ask1, PAR1, HIF1α, PUMA, and / or any of the genes described in Fukuda, A. M. and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's disease (mutated HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or any of the genes described in Boison, D. (2010) Epilepsy), and / or any of the genes described in Boison, D. (2010) Epilepsy. 51:1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau, amyloid precursor protein), corticobasal degeneration or CBD (tau), corticobasal ganglionic degeneration or CBGD (tau), frontotemporal dementia or FTD (tau), progressive supranuclear palsy or PSP (tau), multiple system atrophy or MSA (alpha-synuclein), brain cancer (e.g., mutated or overexpressed oncogenes implicated in brain cancer), and lysosomal storage diseases (LSDs). Disorders of the present invention can include disorders involving a large portion of the cortex, e.g., more than one functional area of ​​the cortex, more than one lobe of the cortex, and / or the entire cortex. Other non-limiting examples of disorders of the invention that can be treated by a therapeutic polypeptide or nucleic acid of the invention include traumatic brain injury, disorders related to enzyme dysfunction, psychiatric disorders (such as post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (such as dementia, autism, and depression).Disorders related to enzyme dysfunction include, but are not limited to, leukodystrophies (such as Canavan disease) and any of the lysosomal storage diseases described below.

[0115] In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage diseases. As is generally known in the art, lysosomal storage diseases are rare inherited metabolic disorders characterized by defects in lysosomal function. Such disorders involve the inability to properly bind mucopolysaccharides, glycoproteins, and / or lysosomes, which cause the pathological accumulation of cellular material stored in the lysosomes. It is often caused by a deficiency of enzymes required for protein and / or lipid metabolism. Non-limiting examples of lysosomal storage diseases of the invention treatable by a therapeutic polypeptide or nucleic acid of the invention (exemplary genes that may be targeted or delivered are shown in parentheses for each disorder) include Gaucher disease type 2 or 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis (beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidosis (mannosidases, e.g., alpha-D-mannosidase, MAN2B1), beta-mannosidosis (beta-mannosidase, MANBA), metachromatic leukodystrophy (pseudoarylsulfatase A, ARSA), mucolipidosis II / III disease (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick disease type A (acid sphingomyelinase, AS), M), Niemann-Pick disease type C (Niemann-Pick type C protein, NPC1), Pompe disease (acid alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase beta subunit, HEXB), Sanfilippo disease type A (N-sulfoglucosamine sulfohydrolase, MPS3A), Sanfilippo disease type B (N-alpha-acetylglucosaminidase, NAGLU), Sanfilippo disease type C (heparin acetyl-CoA:ALPHA). These include alpha-glucosaminidase N-acetyltransferase, MPS3C), Sanfilippo disease type D (N-acetylglucosamine-6-sulfatase, GNS), Schindler disease (alpha-N-acetylgalactosaminidase, NAGA), Sly disease (beta-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alpha subunit, HEXA), and Wollman disease (lysosomal acid lipase, LIPA).

[0116] Table 1 below lists additional lysosomal storage diseases, as well as the defective enzymes associated with each disease. In some embodiments, the diseases listed in the table below are treated with therapeutic polypeptides or therapeutic nucleic acids of the invention that complement or otherwise compensate for the corresponding enzyme deficiency.

[0117] [Table 1]

[0118] [Table 2]

[0119] As such, in some embodiments, the therapeutic polypeptide is selected from the group consisting of caspase-3, beclin 1, Ask1, PAR1, HIF1α, PUMA, SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), nephrectomycin, and the like. Mann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), or lysosomal acid lipase (LIPA). A therapeutic polypeptide can increase or decrease the function of a target polypeptide in a subject (e.g., a therapeutic polypeptide can supply a function lost in a lysosomal storage disease or reduce the levels of alpha-synuclein in MSA, e.g., by blocking its function or dysfunction).In some embodiments, the therapeutic nucleic acid is selected from the group consisting of caspase-3, beclin 1, Ask1, PAR1, HIF1α, PUMA, SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (AS). M), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), or lysosomal acid. Lipase (LIPA). The therapeutic nucleic acid can increase or decrease the function of the target polypeptide in the subject (e.g., the therapeutic nucleic acid can provide the lost function in lysosomal storage disease, e.g., by RNAi, or reduce the level of alpha-synuclein in MSA).

[0120] An exemplary disease in which AAV expression in the cortex and striatum may be useful is Huntington's disease (HD). Huntington's disease is caused by a CAG repeat expansion mutation that encodes an expanded polyglutamine (polyQ) repeat in the mutant huntingtin protein (mHTT). HD is an autosomal dominant disease resulting from a mutation in a single allele, making it a particularly attractive target for DNA and RNA-based therapies. AAV vectors provide an ideal delivery system for nucleic acid therapeutics, allowing long-lasting and continuous expression of these huntingtin-reducing molecules in the brain. It is anticipated that delivery to both the striatum and cortex may be required to achieve maximal clinical efficacy in HD. Postmortem analysis of HD patient brains has revealed widespread loss of medium spiny neurons in the striatum in addition to loss of pyramidal neurons in the cerebral cortex and hippocampus. Recently, it has been shown that using a conditional HD transgenic mouse model that genetically reduces mHTT expression in neuronal populations in the striatum and cortex results in significantly higher efficacy than reducing mHTT at either site alone (Wang et al., (2014) Nature Medicine 20:536-541). Collectively, this evidence suggests that delivery of gene therapy agents to both striatal and cortical regions may be ideal for maximum therapeutic efficacy.

[0121] The use of gene therapy vectors to deliver biologics to key brain regions involved in the pathogenesis of Huntington's disease continues to be a challenge, due in large part to the physical constraints of effectively delivering vectors specifically to the striatum and cerebral cortex.Although multiple direct infusions can be effective in small animal brains, the composition and volume of brain tissue increases in primates, making it more difficult to achieve widespread striatal and cortical delivery via single-site infusion.Therefore, our discovery that striatal administration can achieve widespread rAAV distribution, including the cortex and striatum, has utility in the treatment of Huntington's disease.

[0122] Thus, certain aspects of the present invention relate to a method for treating Huntington's disease in a mammal, comprising administering rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal. HD is characterized by progressive symptoms related to gross movement and motor control, cognition, and mental health. The exact nature and extent of symptoms vary between individuals, but generally symptoms progress over time. In most cases, symptoms begin to emerge between the ages of 30 and 40 with an insidious breakdown in motor skills, cognition, and personality. Over time, these progress to jerky, uncontrollable movements and loss of muscle control, dementia, and psychiatric disorders such as depression, aggression, anxiety, and obsessive-compulsive behavior. Death typically occurs 10-15 years after symptom onset. Fewer than 10% of HD cases involve the early-onset form of the disease, which is characterized by faster disease progression. Approximately 1 in 10,000 Americans is thought to have HD.

[0123] Most cases of HD are associated with the expansion of trinucleotide CAG repeats in the HTT gene. The number of CAG repeats in the HTT gene strongly correlates with HD manifestation. For example, individuals with 35 or fewer repeats typically do not develop HD, whereas individuals with 27-35 repeats have a higher risk of having offspring with HD. Individuals with 36 to 40-42 repeats show incomplete penetrance of HD, whereas individuals with more than 40-42 repeats show complete penetrance. Early-onset HD cases may be associated with a CAG repeat size of 60 or more.

[0124] The polyQ-expanded Htt protein resulting from this CAG repeat expansion is associated with cellular aggregates or inclusions, instability in protein homeostasis, and transcriptional dysregulation. These toxic phenotypes can involve many parts of the body, but most typically they are associated with neuronal cell death. HD patients often show cortical thinning and a significant progressive loss of striatal neurons. The striatum is considered the most vulnerable area of ​​the brain to HD (especially the striatal medium spiny neurons), with early effects seen in the putamen and caudate nucleus. Cell death in striatal spiny neurons, increased astrocyte numbers, and microglial activation have been observed in the brains of HD patients. HD may also affect specific regions of the hippocampus, cerebral cortex, thalamus, hypothalamus, and cerebellum.

[0125] Animal models of HD can be used to test possible therapeutic strategies, such as the compositions and methods disclosed herein. Mouse models of HD are known in the art. These include mouse models with mutant HTT fragments, such as R6 / 1 and N171-82Q HD mice (Harper et al., (2005) Proc. Natl. Acad. Sci. USA 102:5820-5825; Rodriguez-Lebron et al., (2005) Mol. Ther. 12:618-633; Machida et al., (2006) Biochem. Biophys. Res. Commun. 343:190-197). Another example of the mouse HD model described herein is the YAC128 mouse model. This model harbors a yeast artificial chromosome (YAC) expressing a mutant human HTT gene with 128 CAG repeats, and YAC128 mice show prominent and widespread accumulation of Htt aggregates in the striatum by 12 months of age (Slow et al., (2003) Hum. Mol. Genet. 12:1555-1567; Pouladi et al., (2012) Hum. Mol. Genet. 21:2219-2232).

[0126] Other animal models of HD can also be used. For example, transgenic rat (von Horsten, S. et al. (2003) Hum. Mol. Genet. 12:617-24) and rhesus monkey (Yang, SH et al. (2008) Nature 453:921-4) models have been described. Non-genetic models are also known. These mostly involve the use of excitotoxic compounds (e.g. quinolinic acid or kainic acid) or mitochondrial toxins (e.g. 3-nitropropionic acid and malonic acid) to induce cell death of striatal neurons in rodents or non-human primates (for further description and references, see Ramaswamy, S. et al. (2007) ILAR J. 48:356-73).

[0127] In some aspects, the present invention provides a method for alleviating symptoms of HD, comprising administering rAAV particles to the striatum, comprising rAAV vectors encoding heterologous nucleic acids that are expressed at least in the cerebral cortex and striatum.In some embodiments, symptoms of HD include, but are not limited to, chorea, rigidity, uncontrollable body movements, loss of muscle control, impaired coordination, restlessness, slow eye movements, abnormal gestures, unsteadiness, ataxic gait, abnormal facial expressions, speech disorders, difficulty chewing and / or swallowing, sleep disorders, seizures, dementia, cognitive disorders (e.g., reduced ability to plan, abstract thinking, flexibility, rule acquisition, interpersonal sensitivity, self-control, attention, learning, and memory), depression, anxiety, personality changes, aggression, compulsive behavior, obsessive behavior, hypersexuality, psychosis, apathy, irritability, suicidal ideation, weight loss, muscle atrophy, heart failure, decreased glucose tolerance, testicular atrophy, and osteoporosis.

[0128] In some embodiments, the present invention provides a method for preventing or delaying the progression of HD. Autosomal dominant HD is a genetic disease that can be genotyped. For example, the number of CAG repeats in HTT can be determined by PCR-based repeat sizing. This type of diagnosis can be performed at any stage of life, through direct testing of young individuals or adults (e.g., upon the appearance of clinical symptoms), prenatal screening or prenatal exclusion testing (e.g., by chorionic villus sampling or amniocentesis), or preimplantation screening of embryos. In addition, HD can be diagnosed by imaging the brain to look for shrinkage of the caudate nucleus and / or putamen and / or enlargement of the syrinx. These symptoms, in combination with a family history and / or clinical symptoms of HD, may indicate HD.

[0129] Means for determining alleviation of symptoms of HD are known in the art. For example, the Unified Huntington's Disease Rating Scale (UHDSR) is used. The UHDRS can be used to assess motor function, cognitive function, behavioral abnormalities, and functional ability (see, e.g., Huntington Study Group (1996) Movement Disorders 11:136-42). This scale is a modified version of the HD Activities of Daily Living Scale (HD Activities of Daily Living Scale). The HD Scale was developed to provide a uniform, comprehensive test of various aspects of disease pathology, incorporating elements from tests such as the HD Chorea Severity Scale (HDS Scale), Marsden and Quinn's Chorea Severity Scale (DSS Scale), Physical Disability and Independence Scale (PDI Scale), HD Motor Rating Scale (HDMRS), HD Functional Capacity Scale (HDFCS), and the quantitated neurological exam (QNE). Other tests useful for determining remission of HD symptoms include, but are not limited to, the Montreal Cognitive Assessment, brain imaging (e.g., MRI), Category Fluency Test, Trail Making Test, Map Search, Stroop Word Reading Test, Speeded Tapping Task, and Symbol Digit Modalities Test.

[0130] In some embodiments of the present invention, the method is used to treat humans with HD. As mentioned above, HD is inherited in an autosomal dominant manner and is caused by the expansion of CAG repeats in the HTT gene. The rAAV particle may contain heterologous nucleic acid, for example, encoding a therapeutic polypeptide or nucleic acid that targets HTT. Early-onset HD is most often inherited paternally. Huntington's disease-like phenotypes have also been shown to be correlated with other loci, such as HDL1, PRNP, HDL2, HDL3, and HDL4. It is believed that other loci may modify the manifestation of HD symptoms, such as mutations in GRIN2A, GRIN2B, MSX1, GRIK2, and APOE genes.

[0131] In some embodiments, recombinant viral particles are delivered by injecting viral particles into the striatum.Intrastriatal administration is to deliver recombinant viral particles to the area of ​​the brain, striatum (including putamen and caudate nucleus), which is highly affected by HD.In addition, without wishing to be bound by theory, it is believed that recombinant viral particles (e.g., rAAV particles) injected into the striatum can also be distributed (e.g., via retrograde transport) to other areas of the brain, such as, but not limited to, projection fields (e.g., cerebral cortex). In some embodiments, the recombinant viral particles are delivered by convection-enhanced delivery (eg, convection-enhanced delivery to the striatum).

[0132] In some embodiments, the transgene (eg, a heterologous nucleic acid described herein) is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the GUSB promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter and the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline responsive promoter (TRE), the HBV promoter, the hAAT promoter, the LSP promoter, the chimeric liver specific promoter (LSP), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken beta-actin / rabbit beta-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9), and the elongation factor 1-alpha promoter (EF1-alpha) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene, 1991, 108(2):193-9). Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter may be a constitutive, inducible or repressible promoter. In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid encoding a heterologous nucleic acid of the present disclosure operably linked to a CBA promoter. In some embodiments, the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter.

[0133] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter [Invitrogen].

[0134] Inducible promoters allow for the control of gene expression and may be controlled by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, e.g., acute phase of cells, a particular differentiation state, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters controlled by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repression system (Gos Examples of such systems include the tetracycline-inducible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995). Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997). Yet other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a particular differentiation state of a cell, or that are regulated only in replicating cells.

[0135] In another embodiment, it is expected that the promoter native to the transgene, or a fragment thereof, is used. When it is desired that the expression of the transgene mimics the native expression, the native promoter may be preferred. When the expression of the transgene must be controlled temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus, the native promoter may be used. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic the native expression.

[0136] In some embodiments, the regulatory sequence provides tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: neuronal, e.g., the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In some embodiments, the tissue-specific promoter is a promoter of a gene selected from neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adaptor molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art. In some embodiments, the promoter is chicken beta-actin promoter.

[0137] In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the CNS. As such, in some embodiments, the therapeutic polypeptide or therapeutic nucleic acid of the present invention can be used to treat disorders of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in cells of the brain. The cells of the brain can refer to any brain cell known in the art, including, but not limited to, neurons (e.g., sensory neurons, motor neurons, interneurons, dopaminergic neurons, medium spiny neurons, cholinergic neurons, GABAergic neurons, pyramidal neurons, etc.), glial cells (e.g., microglia, astroglia, astrocytes, oligodendrocytes, ependymal cells, radial glia, etc.), brain parenchymal cells, microglia, ependymal cells, and / or Purkinje cells. In some embodiments, the promoter expresses the heterologous nucleic acid in neurons and / or glial cells. In some embodiments, the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of cortical layer IV and / or neurons of cortical layer V.

[0138] A variety of promoters for expressing transcripts (e.g., heterologous transgenes) in CNS cells, brain cells, neurons, and glial cells are known in the art and are described herein. Such promoters may include control sequences that are typically linked to a selected gene or heterologous control sequences. Often, useful heterologous control sequences include sequences derived from sequences encoding mammalian or viral genes. Examples include Examples of promoters include, but are not limited to, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (AdMLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, can also be used. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA). CNS-specific and inducible promoters may also be used. Examples of CNS-specific promoters include, but are not limited to, promoters isolated from CNS-specific genes, such as myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Examples of inducible promoters include DNA response elements for ecdysone, tetracycline, metallothionein, and hypoxia, among others.

[0139] The present invention anticipates the use of recombinant virus genome to introduce or package one or more nucleic acid sequences encoding heterologous nucleic acid into AAV viral particles.Recombinant virus genome can include any element for establishing the expression of heterologous transgene, such as promoter, heterologous nucleic acid, ITR, ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal, and / or replication origin.In some embodiments, rAAV vector comprises one or more of enhancer, splice donor / splice acceptor pair, matrix attachment site, or polyadenylation signal.

[0140] In some embodiments, administration of an effective amount of rAAV particles containing a vector encoding a therapeutic nucleic acid or polypeptide transduces cells (e.g., CNS cells, brain cells, neurons, and / or glial cells) at or near the site of administration (e.g., striatum and / or cortex) or more distal to the site of administration. In some embodiments, more than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% of the neurons are transduced. In some embodiments, about 5% to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30% to about 50% of the neurons are transduced. Methods for identifying neurons that have been transduced by recombinant viral particles expressing miRNAs are known in the art; for example, immunohistochemistry, RNA detection (e.g., qPCR, Northern blotting, RNA-seq, in situ hybridization, etc.) or the use of co-expressed markers such as enhanced green fluorescent protein can be used to detect expression.

[0141] In some embodiments, the present invention provides a virus particle comprising recombinant self-complementary genome (e.g., self-complementary rAAV vector). AAV virus particle with self-complementary vector genome and the method of using self-complementary AAV genome are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z. et al. (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. It is expected that the rAAV comprising self-complementary genome will rapidly form a double-stranded DNA molecule by its partially complementary sequence (e.g., the complementary coding and non-coding strands of heterologous nucleic acid). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of that nucleic acid, wherein the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length.

[0142] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., the right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 1). The mutated ITR comprises a deletion of the D region that includes a terminal resolution sequence. As a result, during replication of the AAV viral genome, the rep protein is not expected to cleave the viral genome at the mutated ITR, and as such, a recombinant viral genome is expected to be packaged in the viral capsid that includes the following in 5' to 3' order: AAV ITR, the first heterologous polynucleotide sequence including the control sequence, the mutated AAV ITR, the first heterologous polynucleotide, and the second heterologous polynucleotide in the reverse orientation to the third AAV ITR.

[0143] V. Viral Particles and Methods for Producing Viral Particles rAAV viral particles The present invention provides a method and system for administering rAAV particles. In some embodiments, the rAAV particle comprises a rAAV vector. In some embodiments, the viral particle is a recombinant AAV particle that comprises a nucleic acid that comprises a heterologous nucleic acid flanked by one or two AAV inverted terminal repeats (ITRs). The nucleic acid is encapsidated in the AAV particle. The AAV particle also comprises a capsid protein. In some embodiments, the nucleic acid comprises a coding sequence of interest (e.g., a heterologous nucleic acid) operably linked to the component in the transcriptional direction, a control sequence including a transcription start and termination sequence, thereby forming an expression cassette. The expression cassette has at least one functional AAV ITR sequence at the 5' and 3' ends. By "functional AAV ITR sequence" is meant that the ITR sequence functions for the purpose of rescue, replication and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To carry out some aspects of the invention, a recombinant vector contains at least all of the sequences of AAV essential for encapsidation and the physical structure for infection by rAAV. The ITRs of AAV for use in the vectors of the invention may not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), may be altered by nucleotide insertion, deletion or substitution, or may be derived from any of a number of AAV serotypes. More than 40 AAV serotypes are known to date, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered within the scope of the present invention.In some embodiments, the rAAV vector is a vector derived from an AAV serotype, such as, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV. In some embodiments, the nucleic acid in the ITR of AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV. DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR or the like. In certain embodiments, the nucleic acid in the AAV comprises the ITR of AAV2.

[0144] In some embodiments, the vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may code for green fluorescent protein. In some embodiments, the stuffer nucleic acid may be located between the promoter and the nucleic acid that codes for RNAi.

[0145] In further embodiments, the rAAV particles are selected from the group consisting of AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid, such as ShH10, as described in U.S. Pregrant Publication No. 2012 / 0164106), AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAVrh8R ... capsid, AAV9 capsid (e.g., a wild-type AAV9 capsid or a modified AAV9 capsid described in U.S. Pregrant Publication No. 2013 / 0323226), AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid mutants, heparin-binding capsid mutants, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, AAV DJ capsids (e.g., AAV-DJ / 8 capsid, AAV-DJ / 9 capsid, or any other capsid described in U.S. Pregrant Publication No. 2012 / 0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 capsid, or AAV capsids described in U.S. Patent No. 8,283,151 or International Publication No. WO / 2003 / 042397. In some embodiments, the mutant capsid protein maintains the ability to form an AAV capsid. In some embodiments, the rAAV particles comprise an AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832). In further embodiments, the rAAV particles comprise capsid proteins of an AAV serotype from clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381). In some embodiments, the rAAV particles comprise an AAV1 capsid protein or a mutant thereof. In other embodiments, the rAAV particles comprise an AAV2 capsid protein or a mutant thereof.In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV particles comprise an AAV serotype 1 (AAV1) capsid. In some embodiments, the rAAV particles comprise an AAV serotype 2 (AAV2) capsid.

[0146] Different AAV serotypes are used to optimize transduction of specific target cells or target specific cell types within a specific target tissue (e.g., CNS tissue). rAAV particles may contain viral proteins and viral nucleic acids derived from the same or different serotypes (e.g., mixed serotypes). For example, in some embodiments, rAAV particles may contain AAV1 capsid proteins and at least one AAV2 ITR, or rAAV particles may contain AAV2 capsid proteins and at least one AAV1 ITR. Any combination of AAV serotypes for rAAV particle production is provided herein as if each combination were explicitly stated herein. In some embodiments, the present invention provides rAAV particles comprising the rAAV vector (e.g., expression cassette comprising heterologous nucleic acid) of the present disclosure flanked by AAV1 capsid and at least one AAV2 ITR. In some embodiments, the present invention provides rAAV particles comprising AAV2 capsid. In some embodiments, the ITRs and the capsid are derived from AAV2. In some embodiments, the ITRs are derived from AAV2 and the capsid is derived from AAV1.

[0147] Production of AAV particles Numerous methods are known in the art for producing rAAV vectors, including transfection, production of stable cell lines, and production systems of infectious hybrid viruses, such as adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J. E. et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. All rAAV production cultures for producing rAAV viral particles require 1) a suitable host cell, such as a human-derived cell line, such as HeLa, A549, or 293 cells, or an insect-derived cell line, such as SF-9 in the case of baculovirus production systems; 2) suitable helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) nucleic acid flanked by at least one AAV ITR sequence (e.g., therapeutic nucleic acid); and 5) suitable media and media components that support rAAV production. In some embodiments, the AAV rep and cap gene products may be from any AAV serotype. Generally, but not necessarily, the AAV rep gene product has the same serotype as the ITRs of the rAAV vector genome, so long as the rep gene product functions to replicate and package the rAAV genome. Suitable media known in the art can be used to produce rAAV vectors, including, but not limited to, media produced by Hyclone Laboratories and JRH, such as modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and the like, as described in U.S. Patent No. 6,566,118, and custom formulations such as Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in the production of recombinant AAV vectors.In some embodiments, the helper functions of AAV are provided by adenovirus or HSV, hi some embodiments, the helper functions of AAV are provided by baculovirus and the host cell is an insect cell (e.g., a Spodoptera frugiperda (Sf9) cell).

[0148] In some embodiments, rAAV particles can be produced by a triple transfection method, such as the exemplary triple transfection method shown below. Briefly, a plasmid containing rep and capsid genes can be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells) along with a helper adenovirus plasmid, and the virus can be harvested and optionally purified. As such, in some embodiments, rAAV particles are produced by triple transfection of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, where transfection of the nucleic acids into the host cell produces a host cell capable of producing rAAV particles.

[0149] In some embodiments, rAAV particles can be produced by producer cell line methods, such as the exemplary producer cell line methods set forth below (see also referenced in Martin et al. (2013) Human Gene Therapy Methods 24:253-269). Briefly, a cell line (e.g., a HeLa cell line) can be stably transfected with a plasmid containing the rep gene, the capsid gene, and a promoter-heterologous nucleic acid sequence. The cell line can be screened to select a lead clone for rAAV production, which can then be expanded into a production bioreactor and infected with adenovirus (e.g., wild-type adenovirus) as a helper to initiate rAAV production. The cell line can then be transfected with a plasmid containing the rep gene, the capsid gene, and a promoter-heterologous nucleic acid sequence ... The virus may be harvested, the adenovirus may be inactivated (e.g., by heat) and / or removed, and the rAAV particles may be purified. As such, in some embodiments, rAAV particles were produced by a producer cell line that contains one or more of nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

[0150] In some aspects, a method for producing any of the rAAV particles disclosed herein is provided, comprising: (a) culturing a host cell under conditions in which rAAV particles are produced, the host cell comprising: (i) one or more AAV packaging genes, each of which encodes an AAV replication and / or encapsidation protein; (ii) a rAAV provector comprising a nucleic acid encoding a heterologous nucleic acid as described herein, flanked by at least one AAV ITR; and (iii) AAV helper functions; and (b) recovering the rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2R471B, AAV2R471C, AAV2R471D, AAV2R471E, AAV2R471F ... DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR, etc. In some embodiments, the encapsidation protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1 serotype capsid protein or mutants thereof. In some embodiments, the encapsidation protein is an AAV5 capsid protein, such as an AAV5 capsid protein with a tyrosine capsid mutation. In some embodiments, the encapsidation protein is an AAV5 capsid protein, such as an AAV5 capsid protein with a tyrosine capsid mutation, and the ITRs are AAV2 ITRs. In further embodiments, the rAAV particles comprise capsid proteins of an AAV serotype from clades A-F. In some embodiments, the rAAV particles comprise an AAV1 capsid and a recombinant genome comprising an AAV2 ITR, a mutated AAV2 ITR, and a nucleic acid encoding a therapeutic transgene / nucleic acid. In some embodiments, the ITR of AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR.In certain embodiments, the ITR of AAV is AAV2 ITR.

[0151] Suitable rAAV production culture media of the invention may be supplemented with serum or serum-derived recombinant proteins at levels between 0.5% and 20% (v / v or w / v). Alternatively, as known in the art, rAAV vectors may be produced in serum-free conditions, which may also be referred to as animal-derived product-free media. One of skill in the art will recognize that commercially available or custom media designed to support rAAV vector production may also be supplemented with one or more cell culture components known in the art, such as, but not limited to, glucose, vitamins, amino acids, and / or growth factors, to increase the titer of rAAV in the production culture.

[0152] rAAV production cultures can be grown under a variety of conditions (wide ranges of temperatures, different lengths of time, and the like) suitable for the particular host cell being utilized. As known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, such as spinner flasks, stirred-tank bioreactors, and disposable systems such as the Wavebag system.

[0153] The rAAV vector particles of the invention may be recovered from the rAAV production culture by lysing the host cells of the production culture, or by recovering spent medium from the production culture when the cells are cultured under conditions known in the art that cause release of rAAV particles from intact cells into the medium, as described in more detail in U.S. Patent No. 6,566,118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0154] In further embodiments, the rAAV particles are purified. The term "purified" as used herein encompasses preparations of rAAV particles that are devoid of at least some of the other elements that may be present when the rAAV particles are naturally present or initially prepared from. Thus, for example, isolated rAAV particles can be prepared using purification techniques to enrich them from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as by the proportion of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or infectivity, or enrichment can be measured with respect to a second potentially interfering substance present in the source mixture, such as a contaminant, such as a contaminant from the production culture, or a contaminant from the manufacturing process, such as a helper virus, a medium component, etc.

[0155] In some embodiments, the harvest from the rAAV production culture is clarified to remove host cell debris. In some embodiments, the harvest from the production culture is clarified by filtration through a series of deep filters, such as Millipore Millistak+HC Pod filters of grade DOHC, Millipore Millistak+HC Pod filters of grade A1HC, and Opticap XL1O Millipore Express SHC hydrophilic membrane filters, which are 0.2 μm filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation, or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0156] In some embodiments, the harvest of the rAAV production culture is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is carried out under standard conditions known in the art, e.g., at a final concentration of 1-2.5 units / ml of Benzonase®, at a temperature ranging from ambient to 37° C., for a period of 30 minutes to several hours.

[0157] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size-exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method The method includes all steps in the order as described below. Methods for purifying rAAV particles can be found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and WO2010 / 148143.

[0158] In some embodiments, the rAAV particle is in the form of a pharmaceutical composition.The pharmaceutical composition can be suitable for any mode of administration described herein.The pharmaceutical composition of recombinant viral particles containing a nucleic acid encoding a therapeutic transgene / nucleic acid can be introduced into the CNS (e.g., the striatum and / or cerebral cortex).

[0159] In some embodiments, the rAAV particles are in the form of a pharmaceutical composition that includes a pharma- ceutically acceptable excipient. As is well known in the art, a pharma- ceutically acceptable excipient is a relatively inert substance that facilitates administration of a pharmacologically effective substance, and can be provided as a liquid solution or suspension, as an emulsion, or as a solid form suitable for dissolving or suspending in a liquid prior to use. For example, an excipient can provide form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting agents and emulsifying agents, salts for modifying osmolarity, encapsulating agents, pH buffers, and buffers. Such excipients include any pharmaceutical substance suitable for direct delivery to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. These may include, for example, pharma- ceutically acceptable salts, such as salts of inorganic acids, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. A thorough discussion of pharma-ceutically acceptable excipients can be found in REMINGTON'S PHARMACEUTICAL FORMULATIONS, vol. SCIENCES (Mack Pub. Co., NJ 1991).

[0160] In some embodiments, the administered composition includes rAAV particles and a poloxamer. The term "poloxamer" can encompass many compounds, since different lengths of polyoxypropylene and polyoxyethylene chains can be used in combination. For example, poloxamers include HO(C 2 H 4 O) n (C 3 H 6 O) m (C 2 H 4 O) nH, where n (ie, the polyoxyethylene chain length) has a value of about 60 to about 150 and m (ie, the polyoxypropylene chain length) has a value of about 25 to about 60.

[0161] In some embodiments, the poloxamer is poloxamer 188 (e.g., CAS number 9003-11-6). Poloxamers can be described by a numbering system that specifies their approximate molecular weight and percentage of polyoxyethylene content. These values ​​often refer to average values ​​in a poloxamer composition rather than absolute values ​​for each poloxamer molecule in the composition. According to this approach, multiplying the top two digits by 100 gives the approximate molecular weight of the polyoxypropylene block, and multiplying the top three digits by 10 gives the weight percentage of the polyoxyethylene block. For example, poloxamer 188 may refer to a poloxamer with n having a value of about 80 and m having a value of about 27, as in the formula depicted above. Poloxamer 188 may have an average molecular weight of about 7680 to about 9510 g / mol.

[0162] Poloxamers sold under trade names such as PLURONIC® can be named according to different schemes. A letter can be used to indicate the physical state (e.g., F for solid, P for paste, or L for liquid). Two digits or Three-digit numbers can be used to indicate chemical properties. The first or second digits are multiplied by 300 to obtain the approximate molecular weight of the polyoxypropylene block, and the third digit is multiplied by 10 to obtain the weight percentage of the polyoxyethylene block. For example, PLURONIC® or LUTROL® F68 may refer to a solid poloxamer in which n is about 80 and m is about 27, as in the formula depicted above. Thus, in some embodiments, poloxamer 188 can be PLURONIC® F68 or LUTROL® F68.

[0163] In some embodiments, the concentration of poloxamer in the composition is in the range of about 0.0001% to about 0.01%. In some embodiments, the concentration of poloxamer in the composition is less than about any of the following percentages: 0.01, 0.005, 0.001, or 0.0005. In some embodiments, the concentration of poloxamer in the composition is greater than about any of the following percentages: 0.0001, 0.0005, 0.001, or 0.005. That is, the concentration of poloxamer in the composition may be any of a range of percentages with an upper limit of 0.01, 0.005, 0.001, or 0.0005 and an independently selected lower limit of 0.0001, 0.0005, 0.001, or 0.005, where the lower limit is less than the upper limit. In certain embodiments, the concentration of poloxamer in the composition is about 0.001%.

[0164] In some embodiments, the composition further comprises sodium chloride. In some embodiments, the concentration of sodium chloride in the composition is in the range of about 100 mM to about 250 mM. In some embodiments, the concentration of sodium chloride in the composition is less than about any of the following concentrations (in mM): 250, 225, 200, 175, 150, or 125. In some embodiments, the concentration of sodium chloride in the composition is greater than about any of the following concentrations (in mM): 100, 125, 150, 175, 200, or 225. That is, the concentration of sodium chloride in the composition may be any of a range of concentrations (in mM) having an upper limit of 250, 225, 200, 175, 150, or 125 and an independently selected lower limit of 100, 125, 150, 175, 200, or 225, where the lower limit is less than the upper limit. In certain embodiments, the concentration of sodium chloride in the composition is about 180 mM.

[0165] In some embodiments, the composition further comprises sodium phosphate.Sodium phosphate may refer to any single type of sodium phosphate (e.g., sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, etc.), or may refer to sodium phosphate buffer, a mixture of sodium phosphate monobasic and sodium phosphate dibasic solutions.The formulation of sodium phosphate buffer over a range of pH can be found in various standard molecular biology protocols, such as Promega Protocols&Applications Guide, "Buffers for Biochemical Reactions", Appendix B Part C.

[0166] In some embodiments, the concentration of sodium phosphate in the composition ranges from about 5 mM to about 20 mM. In some embodiments, the concentration of sodium phosphate in the composition is less than about any of the following concentrations (in mM): 20, 15, or 10. In some embodiments, the concentration of sodium phosphate in the composition is greater than about any of the following concentrations (in mM): 5, 10, or 15. That is, the concentration of sodium phosphate in the composition may be any of a range of concentrations (in mM) having an upper limit of 20, 15, or 10 and an independently selected lower limit of 5, 10, or 15, where the lower limit is less than the upper limit. In certain embodiments, the concentration of sodium phosphate in the composition is about 10 mM.

[0167] In some embodiments, the pH of the sodium phosphate in the composition is about 7.0 to about 8.0. For example, in some embodiments, the pH of the sodium phosphate in the composition is about 7.0, about 7.2, about 7.4, about 7.5, about 7.6, about 7.8, or about 8.0. In certain embodiments, the pH of the sodium phosphate in the composition is about 7.5. Any of the pH values ​​of sodium phosphate described herein can be combined with any of the concentration values ​​of sodium phosphate described above. For example, in some embodiments, the concentration of sodium phosphate in the composition is about 10 mM and the pH is about 7.5.

[0168] In some embodiments, a pharmaceutical composition comprising the rAAV particles described herein and a pharma- ceutical acceptable carrier is suitable for administration to a human. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and 1570-1580). In some embodiments, the pharmaceutical composition further comprises a poloxamer (e.g., poloxamer 188, e.g., PLURONIC® or LUTROL® F68). In some embodiments, a pharmaceutical composition comprising the rAAV particles described herein and a pharma- ceutical acceptable carrier is suitable for injection into the CNS of a mammal.

[0169] Such pharma-ceutically acceptable carriers may be sterile liquids, such as water and oils, such as oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions may further comprise additional components, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, and the like. The pharmaceutical compositions described herein may be packaged in single unit dosage forms or multi-dose dosage forms. The compositions are generally formulated as sterile, substantially isotonic solutions.

[0170] VI. Systems for delivering rAAV particles Also provided is a system for expressing heterologous nucleic acid in mammalian cerebral cortex and striatum, comprising: (a) a composition comprising rAAV particles, wherein the rAAV particles comprises a rAAV vector encoding a heterologous nucleic acid; and (b) a device for delivering rAAV particles to striatum.The system and device of the present invention can be used to deliver any of the rAAV particles described herein to mammalian CNS (e.g., striatum).As described above, the rAAV particles delivered to striatum can be used to introduce the rAAV vector encoding a heterologous nucleic acid for expression in cerebral cortex and striatum.

[0171] In some embodiments, rAAV particles are delivered by convection-enhanced delivery (CED). CED is based on pumping an infusion fluid (e.g., a composition containing rAAV particles) into the CNS under pressure to overcome the hydrostatic pressure of the interstitial fluid, thereby bringing the infusion fluid into contact with perivascular tissue of the CNS, which is utilized to distribute the infusion fluid via convection, like a pump, enhancing the extent of its delivery (see, e.g., Hadaczek et al. (2006) Hum. Genet. 1999:1311-1323). Ther. 17:291-302; Bankiewicz et al., (2000) Exp. Neurol. 164:2-14; Sanftner, LM et al., (2005) Exp. Neurol. 194(2):476-483; Forsayeth, JR et al., (2006) Mol. Ther. 14(4):571-577; U.S. Patent No. 6,953,575; U.S. Patent Application Publication No. 2002 / 0141980; U.S. Patent Application Publication No. 2007 / 0259031; WO 99 / 61066; and WO 2010 / 088560).

[0172] As described herein, the advantage of using CED is that it enhances distribution of infused fluid throughout the brain. CED can provide improved delivery at injection sites in the brain (e.g., striatum, caudate nucleus, and / or putamen). In addition, delivery to other regions of the brain (e.g., cerebral cortex, frontal cortex, frontal association cortical regions, premotor cortex, primary somatosensory cortical regions, and / or primary motor cortex) can also be achieved via CED. Without wishing to be bound by theory, it is also believed that recombinant viral particles (e.g., rAAV particles) injected into the striatum can be distributed (e.g., via retrograde transport) to other regions of the brain, including, but not limited to, projection fields (e.g., cortex).

[0173] In some embodiments, the rAAV particles are delivered using a CED delivery system. AAV particles can be delivered by CED (see, e.g., WO99 / 61066). As described herein, CED can be achieved using any of the systems described herein. Devices for CED (e.g., for delivering compositions that include rAAV particles) are known in the art and generally employ pumps (e.g., osmotic and / or infusion pumps as described below) and injection devices (e.g., catheters, cannulas, etc.). Optionally, imaging techniques may be used to guide the injection device and / or monitor the delivery of the infusion fluid (e.g., compositions that include rAAV particles). The injection device can be inserted into the CNS tissue of the subject. Those skilled in the art can determine suitable coordinates to define the location of the injection device in the target CNS tissue. In some embodiments, defining the location is achieved by an anatomical map obtained, for example, by CT and / or MRI imaging of the subject's brain, to guide the injection device to the target CNS tissue. In some embodiments, iMRI and / or real-time imaging of the delivery can be performed. In some embodiments, the device is used to administer rAAV particles to a mammal according to the methods of the invention.

[0174] In some embodiments, intraoperative magnetic resonance imaging (iMRI) and / or real-time imaging of delivery can be performed. In some embodiments, the device is used to administer rAAV particles to a mammal according to the methods of the present invention. iMRI is known in the art as a technique for MRI-based imaging of patients during surgery, helping to confirm the success of the surgery (e.g., to deliver rAAV particles to the CNS) and reduce the risk of injury to other parts of the tissue (for further description, see, e.g., Fiandaca et al., (2009) Neuroimage 47, Suppl 2:T27-35). In some embodiments, a tracing agent (e.g., an MRI contrast enhancing agent) may be delivered with the infusion fluid (e.g., a composition including rAAV particles) to provide real-time monitoring of the tissue distribution of the infusion fluid. See, e.g., Fiandaca et al., (2009) Neuroimage 47, Suppl 2:T27-35; U.S. Pregrant Publication No. 2007 / 0259031; and U.S. Patent No. 7,922,999. The use of a tracing agent can signal interruptions in delivery. Other tracing and imaging means known in the art can also be used to track infusate distribution.

[0175] In some embodiments, rAAV particles can be administered by standard stereotaxic injection using devices and methods known in the art for delivering rAAV particles. Generally, these methods can use an injection device, a planning system for transforming the area of ​​tissue targeted for delivery to a series of coordinates (e.g., parameters along the left-right axis, the dorsoventral axis, and the anterior-posterior axis), and a device for stereotaxic localization according to the planned coordinates (including a stereotaxic device, and optionally a probe and structure for fixing the head in position in alignment with the coordinate system). A non-limiting example of a system that may be useful for MRI-guided surgery and / or stereotaxic injection is the ClearPoint® system (MRI Interventions, Memphis, TN).

[0176] In some embodiments, the device for convection-enhanced delivery includes a pump (e.g., an osmotic pump and / or an infusion pump). Osmotic and / or infusion pumps are commercially available (e.g., from ALZET Corp., Hamilton Corp., ALZA Inc. Palo Alto, CA). The pump system may be implantable. Exemplary pump systems can be found, for example, in U.S. Patent Nos. 7,351,239; 7,341,577; 6,042,579; 5,735,815; and 4,692,147. In some embodiments, the pump is a manual pump. Exemplary devices for CED, such as reflux-resistant and stepwise cannulas, can be found in WO99 / 61066 and WO2006 / 042090, the entirety of which is incorporated herein by reference.

[0177] In some embodiments, the device for convection-enhanced delivery includes a reflux-resistant cannula (e.g., a cannula with an anti-reflux step design). Further description and exemplary reflux-resistant cannulas can be found, for example, in Krauze et al. (2009) Methods Enzymol. 465:349-362; US Pre-grant Publication No. 2006 / 0135945; US Pre-grant Publication No. 2007 / 0088295; and PCT / US08 / 64011. In some embodiments, only one cannula is used. In other embodiments, more than one cannula is used. In some embodiments, the device for convection-enhanced delivery includes a reflux-resistant cannula coupled with a pump that creates sufficient pressure to cause the infusion fluid to flow through the cannula at a controlled rate to the target tissue. Any suitable flow rate can be used so that intracranial pressure is maintained at a suitable level that does not damage the brain tissue.

[0178] In some embodiments, the cannula is a step cannula. For example, as described in WO2006 / 042090, the step cannula has multiple steps (for example, 4 in FIG. 1 of WO2006 / 042090). The step closest to the distal end of the cannula is the first step to enter the target tissue, and therefore, the number of steps that enter the target tissue (for example, the striatum) is expected to depend on the penetration depth required to reach that target in the subject. For delivery to the brain, the operator can easily determine the appropriate penetration depth considering both the size of the subject to be treated and the location in the brain to be targeted.

[0179] The cannula is connected to the pump via a system of tubing that extends through the lumen of the cannula, through which the infusion fluid is delivered. In embodiments that contain tubing, the tubing is flush with the distal end of the cannula. Alternatively, the tubing extends from the distal end of the cannula, and in such embodiments, the amount that the tubing extends can vary depending on the application. Generally, the tubing will extend from the cannula beyond their distal end by about 1 mm to about 1 cm (or any length therebetween), for example, about 1 to about 50 mm (or any length therebetween), or about 1 mm to about 25 mm (or any length therebetween, such as, but not limited to, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm or 25 mm), for example, 10 mm.

[0180] The tubing extending through the cannula may have a coating or surrounding material in one or more areas, for example to protect the tubing in contact with the infusion fluid. Thus, in certain embodiments, tubing (e.g., FEP (Teflon) tubing) protects the portion of the fused silica tubing that extends beyond the proximal end of the stainless steel cannula. The fused silica tubing may be used in conjunction with any suitable fitting, such as, but not limited to, a Luer style compression fitting. The syringe may be connected by any suitable means, and the syringe may be driven by a syringe pump (manual, electronic and / or computerized). It will be appreciated that the size of the syringe may be selected by the operator to deliver the appropriate amount of product. Thus, 1 mL, 2.5 mL, 5 mL, or even larger syringes may be used.

[0181] The graduated cannula can be made from a variety of physiologically acceptable materials, including, but not limited to, stainless steel (e.g., 316SS or 304SS), metals, metal alloys, polymers, organic fibers, inorganic fibers, and / or combinations thereof.

[0182] In some cases, the surface that contacts the infusion fluid (e.g., tubing or coating) may extend through the lumen of the cannula. The surface that contacts the infusion fluid may also use various materials, including, but not limited to, metals, metal alloys, polymers, organic fibers, inorganic fibers, and / or combinations thereof. In some embodiments, the surface that contacts the product is not stainless steel. In such embodiments, the outer cannula is still made of a material that is physiologically compatible with the target tissue, but may not be compatible with the biologically active agent or product formulation because it is not in contact with the product.

[0183] In some embodiments, the penetration of the infusion fluid is further increased by the use of a facilitating agent, which can further facilitate the delivery of the infusion fluid to a target tissue (e.g., a CNS target tissue). A non-limiting example of a facilitating agent is low molecular weight heparin (see, e.g., U.S. Pat. No. 7,922,999).

[0184] Suitable packaging for the pharmaceutical compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These articles of manufacture may be further sterilized and / or sealed.

[0185] Further provided herein is a method for treating a CNS disorder in a mammal, comprising administering to the mammal rAAV particles according to the methods described herein. Further provided is a method for treating Huntington's disease in a mammal, comprising administering to the mammal rAAV particles according to the methods described herein using the systems described herein.

[0186] The present invention also provides a kit for administering the rAAV particles described herein to a mammal according to the method of the present invention.The kit may include any of the rAAV particles or rAAV particle compositions of the present invention.For example, the kit may include an rAAV particle that includes an rAAV vector that encodes a heterologous nucleic acid that is expressed at least in the cerebral cortex and striatum of a mammal.In some embodiments, the kit further includes any of the devices or systems described above.

[0187] In some embodiments, the kit further includes instructions for CNS delivery (e.g., delivery to the striatum of a mammal) of the composition of rAAV particles. The kits described herein may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and inserts containing instructions for carrying out any of the methods described herein. Suitable packaging materials may also be included, and may be any packaging material known in the art, such as vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed. In some embodiments, the kits may further include instructions for the methods and methods described herein. and / or instructions for using any of the rAAV particles to treat a CNS disorder described herein. The kit may include a pharma- ceutically acceptable carrier suitable for injection into the CNS of an individual, as well as one or more of buffers, diluents, filters, needles, syringes, and package inserts containing instructions for performing an injection into the striatum of a mammal.

[0188] In some embodiments, the kits further contain one or more of the buffers and / or pharma- ceutically acceptable excipients described herein (e.g., as described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991)). In some embodiments, the kits include one or more pharma- ceutically acceptable excipients, carriers, solutions, and / or additional components described herein. The kits described herein can be packaged in single unit dosage forms or in multi-dose dosage forms. The contents of the kits are generally formulated as sterile and may be lyophilized or provided as a substantially isotonic solution. EXAMPLES

[0189] The present invention will be better understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are merely for illustrative purposes, and various modifications or changes in view thereof are expected to be suggested to those skilled in the art, and are encompassed within the spirit and intent of this application and the scope of the appended claims.

[0190] Example 1: Widespread GFP expression following AAV1 vector delivery in the striatum The ability of AAV1 to efficiently target both striatal and cortical structures in the rhesus monkey brain when delivered via convection-enhanced delivery (CED) was evaluated. An AAV vector containing a GFP cDNA under the control of the cytomegalovirus enhancer / chicken beta-actin (CBA) promoter was infused into the caudate and putamen of nine adult male rhesus monkeys using CED (see, e.g., Bankiewicz et al. (2000) Exp. Neurol. 164:2-14 and WO2010 / 088560).

[0191] method surgical delivery Nine adult male Rhesus macaques (Macaca mulatta; 8.9-11.9 kg) were enrolled in this study. All animals were infused with AAV vectors into both the caudate and putamen using MRI-guided CED (Richardson, RM et al. (2011) Neurosurgery (2011) Stereotact. Funct. Neurosurg. 69:154-163; Richardson, RM et al. (2011) Stereotact. Funct. Neurosurg. 89:141-151; Richardson, RM et al. (2011) Mol. Ther. 19:1048-1057). Immediately prior to surgery, animals were anesthetized with ketamine HCL (10 mg / kg), weighed, intubated, and maintained on 1-5% isoflurane. The head was placed in a stereotactic frame, and the animal was transported to an MRI (Siemens 3.0 T Trio MR unit) for T1-weighted planning scans. After scanning, the animal was transferred to the operating room, the head was prepared for the implantation procedure, and a ceramic, custom-designed fused silica reflux-resistant cannula with a 3 mm graduated tip was used for infusion. Temporary guide cannulas were implanted in both hemispheres (one for each hemisphere) using standard methods.

[0192] Animals were transfused into both the caudate and putamen with either AAV1-eGFP or AAV2-eGFP vectors obtained by two different production methods: triple transfection (TT) or producer cell line (PCL). Table 5 shows the vector concentrations and use. The amounts are listed. Animals were tested for the presence of anti-AAV1 and anti-AAV2 neutralizing antibodies as previously described, and were considered seronegative if they displayed an antibody titer of <1:32 (Bevan, AK et al. (2011) Mol. Ther. 19:1971-1980). Survival time in all animals was 1 month after AAV delivery.

[0193] Each animal received up to three microinjections per hemisphere to target the caudate and putamen (anterior and posterior commissure) regions as shown in Table 2. Prohance (2 mM gadoteridol) was added to the virus to visualize the distribution of the infusion during MRI. Using the convection-enhanced delivery (CED) method, approximately 30 μl of AAV was administered to the caudate and 60 μl to the putamen (i.e., 90 μL per hemisphere). The infusion rate was increased to a maximum of 5 μL / min.

[0194] [Table 3]

[0195] The first cohort of animals contained 1.7 × 10 11 A second cohort of animals received 1.7 × 10 11 vg AAV1-eGFP(PCL) (n = 2), or 1.3 × 10 11 vg AAV2-eGFP(PCL) (n=2). Continuous MRI was acquired to monitor the distribution of infusion within each target site and provide real-time feedback to the team. Immediately after the intraparenchymal administration procedure, the animals were transferred to the operating room, the guide cannula was removed, and the wound site was closed in anatomical layers. All experiments were performed in accordance with National Institutes of Health guidelines and protocols approved by the Animal Care and Ethics Committee. Immediately after the surgical procedure, the animals were transferred to the MRI room for the AAV administration procedure. Table 2 Comments regarding dosing procedures for each of the subjects indicated are provided below.

[0196] Administration Procedure - Treatment Group 2 (AAV1-eGFP TT) Subject #1. Approximately 60 μL of infusion was administered per hemisphere into each putamen via two orbits (30 μL / deposition). Coronal images from infusions into the anterior putamen showed the majority of gadolinium signal within each targeted structure. In the right hemisphere, there was minimal perivascular transport in the ventral putamen and distribution to the anterior commissure.

[0197] Subject #2. A T1 MRI was acquired after infusion of 0.127 mL into the putamen and 0.207 mL into the thalamus. The post-infusion T1 MRI showed widespread distribution of infusate within the right thalamus measuring approximately 1 cm anterior-posterior and 1 cm dorsoventral. Coronal T1 showed gadolinium distribution within the target area extending medially towards the internal capsule and superiorly towards the dorsal putamen. The majority of the infusate was contained within the rim of the putamen; however, transport through the perivascular space was also present within the white matter tracts of the internal capsule and the anterior commissure. Analysis revealed a 1:2 ratio of gadolinium infusion volume (Vi) to volume of distribution (Vd) in the thalamus and putamen.

[0198] In-life observations Detailed observations of animal health and neurological symptoms were performed daily for 5 days after dosing; subsequently, detailed observations were performed weekly until the end of the study. Observations and daily mortality checks were performed. Body weight assessments were performed before intracranial dosing, at the time of blood collection procedure, and at the time of necropsy. No significant differences in body weight were observed between treatment groups before surgery or at the time of necropsy (Figure 1). Whole blood, serum, and cerebrospinal fluid (CSF) were collected for hematology, serum chemistry, AAV1 and AAV2 capsid antibody assays, and eGFP mRNA level analysis.

[0199] Live Blood Collection and Processing Blood (approximately 5 mL) was collected prior to injection, approximately 72 hours post-injection, and at necropsy according to the blood sample collection schedule (see Table 3 below). Approximately 0.5-1.0 mL of whole blood was collected in EDTA tubes for hematological analysis. Approximately 2.0 mL of whole blood was collected in serum separator tubes (gel-containing, BD Microtainer) and processed to obtain serum for general chemistry analysis and AAV1 and AAV2 capsid antibody analysis.

[0200] [Table 4]

[0201] CSF collection and processing CSF was collected at two separate time points: prior to intracranial dosing and at necropsy. CSF collection was performed with animals placed in prone position and under anesthesia by cervical spinal tap. Prior to test article administration, 1-2 mL of CSF was collected, frozen on dry ice, and stored at ≦-60°C. At necropsy (prior to PBS perfusion), 2-4 mL of CSF was collected, filtered through a 0.8 micrometer syringe filter into a labeled collection tube, transferred in duplicate to Eppendorf tubes (1 mL aliquots for capsid antibody analysis and 2-4 mL aliquots for GFP analysis), immediately frozen on dry ice, and stored at ≦-60°C.

[0202] Necropsy and tissue collection All animals were euthanized approximately 30 days after the intracranial administration procedure. Each animal was euthanized using intravenous administration of sodium pentobarbital. After euthanasia and blood and CSF collection, the body was perfused transcardially with PBS (under RNase-free conditions) followed by PFA. The descending aorta was clamped to reduce fixation of peripheral tissues. This procedure was used to collect fixed brain tissue in addition to fresh peripheral tissue samples for GFP analysis by QPCR. The tissue collection table lists the tissues collected (Table 4). During PBS perfusion (before the start of 4% PFA perfusion), biopsy samples (0.5–1.0 cm) of select tissues (also listed in the tissue collection table) were collected. 3 ) were collected under RNase-free conditions using a disposable sterile scalpel (a new scalpel for each individual biopsy) into RNase-free tubes and stored frozen at ≦−60° C.

[0203] [Table 5]

[0204] Brain and Spinal Cord Processing The entire brain was carefully removed from the animal and photographed with a ruler for scale. Once the brain was removed from the skull, it was placed in a brain matrix and sliced ​​coronally into 6 mm blocks. The coronal blocks were stored in PFA and processed for histology. Relevant blocks containing the frontal cortex and midbrain regions were fragmented into free-floating 40 micrometer pieces. The entire spinal cord was carefully removed from the animal. Segments of the spinal cord were stored in PFA and processed for histology. Representative segments from the cervical, thoracic, and lumbar regions were fragmented into free-floating 40 micrometer pieces.

[0205] After perfusion with PBS-heparin followed by 4% buffered paraformaldehyde (PFA), brains from each animal were cut into 6 mm blocks (coronal plane) using disposable blades and a monkey brain matrix. Consecutive blocks (11-12 brain slabs per animal) were mounted horizontally on a whiteboard and photographed with the letters aligned sequentially. All brain blocks were then post-fixed for 24 hours in 4% buffered PFA. The quality of fixation of each block was visually inspected (no pink color was observed within the block). After post-fixation with PFA, each brain block was processed for free-floating sections by rinsing three times with PBS and immersion in 30% sucrose (cryopreservation) before cutting into 40 μm free-floating sections.

[0206] AAV vector production Prior to clinical evaluation, AAV vectors are typically produced via a standard triple transfection method (TT) in which HEK293 cells are transfected with two or three plasmids encoding the cis (vector genome) and trans (AAV rep and cap genes; adenoviral helper genes E2A, E4, and VA) elements required for vector packaging (Hauck et al., (2009) Mol. Ther. 17:144-152). Because the plasmid DNA input can be easily and quickly modified, this method allows for the evaluation of vectors based on diverse serotypes and the inclusion of various expression cassettes. Despite its flexibility and relatively rapid conversion time, the transfection method has challenges regarding scalability that limit the suitability of this method for large-scale rAAV vector production for clinical use.

[0207] At present, clinical grade rAAV is produced on a large scale via helper virus-free transient transfection methods, recombinant baculovirus or herpes simplex virus-based production systems, or packaging / producer cell lines (Ayuso et al., (2010) Curr. Gene Ther. 10:423-436). Adeno-associated virus producer cell lines (PCLs) are an effective method for large-scale production of clinical grade AAV vectors. In this system, a single plasmid containing three elements, namely, vector sequences, AAV rep and cap genes, and a gene for a selectable marker, is stably transfected into HeLaS3 cells. However, it is desirable to determine whether AAV vectors derived from producer cell lines are equivalent in potency to vectors produced by other methods, such as standard transient transfection methods.

[0208] For this study, AAV viral vectors were generated using two different production methods: triple transfection (TT) and producer cell line (PCL). Recombinant AAV vectors AAV1-GFP (TT) and AAV2-GFP (TT) were produced by triple transfection (using calcium phosphate) of human embryonic kidney carcinoma 293 cells (HEK-293) (see Xiao et al. (1998) Journal of Virology 72:2224-2232). Briefly, for production of AAV vectors by transient transfection, HEK293 cells were transfected using polyethylenimine (PEI) and a 1:1:1 ratio of three plasmids (ITR vector, AAV2rep / cap2 or AAV2rep / cap1, and pAd helper plasmid). The ITR vector plasmid encoded the cDNA for EGFP downstream of a cytomegalovirus enhancer / chicken beta actin-hybrid promoter (CBA). The pAd helper used was pHelper (Stratagene / Agilent Technologies, Santa Clara, Calif.).

[0209] The recombinant AAV vectors AAV1-GFP (PCL) and AAV2-GFP (PCL) were produced using an AAV producer cell process (Thorne et al., (200 9) Human Gene Therapy 20:707-714 and Martin et al., (2013) Human Gene Therapy Methods 24:253-269). Briefly, product-specific producer cell lines were generated by stably transfecting HeLa-S3 cells (ATCC CCL-2.2) with a plasmid containing the rep gene from serotype 2 and the capsid gene from either serotype 1 or 2, a promoter-heterologous nucleic acid sequence, a vector genome flanked by AAV2 inverted terminal repeats (ITRs), and a puromycin resistance gene. The vector genome harbored a cDNA for EGFP downstream of a cytomegalovirus enhancer / chicken beta actin-hybrid promoter, CBA. Transfected cells were grown in the presence of puromycin and stable integrants were isolated. The resulting cell lines were screened to select lead clones. Product-specific cell clones were then expanded in production bioreactors and infected with wild-type adenovirus as a helper to initiate AAV production. Virus was harvested 72 hours postinfection, and adenovirus was heat inactivated and removed by anion exchange methods.

[0210] Purification of AAV from both production platforms was performed as previously described (Qu, G. et al. (2007) J. Virol. Methods 140:183-192). Table 5 shows the titers obtained for all AAV1 and AAV2-GFP vectors. All vectors were incubated in 180 mM sodium chloride; 10 mM sodium phosphate (5 mM NaH 2 PO 4 2H 2 O + 5 mM Na 2 HPO 4 H 2 0); and 0.001% poloxamer 188 (Lutrol F68), pH 7.5 in water.

[0211] [Table 6]

[0212] immunohistochemistry Immunostaining with an antibody against GFP (1:500, AB3080; Chemicon) was performed on Zamboni-fixed 40-μm circular sections covering the entire frontal cortex and extending posteriorly towards the level of the striatum. The localization of GFP-immunopositive neurons was analyzed with reference to The Rhesus Monkey Brain in Stereotactic Coordinates (Paxinos, GHX and Toga, AW (2000) San Diego, CA: Academic Press) to identify specific areas of immunostaining in the cortex and striatum.

[0213] GFP staining with 3,3'-diaminobenzidine (DAB): Sections (three per 6 mm block, 2 mm apart) were washed three times in PBST for 5 min each, followed by 1% H 2 O 2 The cells were then treated with Sniper blocking solution (biocare.net / Sections were incubated in PBS containing 0.1% Tween-20 (available online at: product / background-sniper / ) for 30 minutes at room temperature, followed by overnight incubation with primary anti-GFP antibody (available online at: www.lifetechnologies.com / ) diluted 1:1000 in Da Vinci Green diluent (available online at: biocare.net / ). After rinsing three times for 5 minutes each with PBS containing 0.1% Tween-20 (PBST), sections were incubated in Mach 2HRP polymer (http: / / biocare.net / ) for 1 hour, followed by three washes and colorimetric development (DAB). Immunostained sections were counterstained with cresyl violet, mounted on slides, and sealed with Cytoseal® (available online at: www.thermoscientific.com / ).

[0214] Calculation of the coverage of GFP expression in non-human primate (NHP) brains: GFP staining from consecutive sections stained with matching IHC was projected onto the corresponding MRI-scanned brains (T1-weighted MR images in the coronal plane) of each individual monkey. Distribution / coverage of GFP expression was performed using OsiriX Imaging software version 3.1 (The OsiriX Foundation, Geneva, Switzerland).

[0215] Double immunofluorescence for vector affinity and efficiency of neuronal transduction: For double immunofluorescence staining of different cell markers (NeuN, S-100, Iba1) with GFP, a combination of primary antibodies was applied to the sections by overnight incubation at room temperature in PBST containing 20% ​​horse serum as a cocktail of primary antibodies. The primary antibodies used were: anti-GFP antibody (1:500, as above); anti-NeuN (1:500, available online at www.emdmillipore.com / ); anti-S-100 (1:300, available online at biocare.net / ), anti-Iba1 (1:500, available online at biocare.net / ); anti-Olig2 (1:50, available online at www.emdmillipore.com / ). After three washes with PBST, primary antibodies were visualized by incubation with the appropriate secondary fluorochrome-conjugated antibodies: goat anti-mouse DyLight549 and goat anti-rabbit DyLight488 (available online at www.biocare.net / ) for 2 hours in the dark. All secondary antibodies were diluted 1:1,000 in fluorescent antibody diluent (available online at biocare.net / ). In addition, sections were incubated in 0.1% Sudan Black solution (70% ethanol) to quench autofluorescence. After a final wash with PBS, sections were coverslipped with Vectashield Hard Set mounting medium for fluorescence (available online at www.vectorlabs.com / ). Control sections were treated without primary antibodies and no significant immunostaining was observed under these conditions.

[0216] Zeiss equipped with a CCD color video camera and image analysis system (Axiovision software, available online at www.zeiss.com / ) The presence of double-labeled cells (positive in both red and green channels) was determined using an Axioskop fluorescent microscope (available online at www.zeiss.com / ). Photomicrographs of double-labeled sections were obtained by merging images from two separate channels (red and green; colocalization appears yellow) without changing the section position or focus (20x objective). For double staining with GFP / NeuN, three sections from each monkey, spaced approximately 4 mm apart, were selected from the site of vector infusion. To evaluate the efficiency of neuronal transduction by AAV1-eGFP or AAV2-eGFP vectors in the targeted brain regions (caudate and putamen), five counting frames (700 μm × 550 μm) were randomly placed in the GFP+ region. The primary area of ​​transduction (PAT) was defined as a GFP-positive area ("cloud") covering more than 40% of the targeted structure. Similarly, to evaluate the efficiency of neuronal transduction outside the PAT (OPAT), Five counting frames (700 μm × 550 μm) from each section were selected with sufficient margins from the targeted structures (caudate and putamen) or from the GFP-positive "cloud" in the cortex (Figure 10C). To determine the ratio of GFP / NeuN positive cells, each counting frame was counted twice, once in the red channel for the number of NeuN+ cells and once in the combined red and green channels for the number of co-stained cells (GFP+ and NeuN+). At least 1,500 NeuN+ cells were counted for each of the three selected sections (five counting frames per section). Finally, the percentage of GFP+ / NeuN+ relative to the total number of NeuN+ was determined. All calculations for the striatum were done by adding the results from both hemispheres of each animal and combining the values ​​from the putamen and caudate nucleus, since the average transduction efficiency was identical in both structures of each animal.

[0217] Quantitative real-time PCR (TaqMan) GFP mRNA levels were measured by quantitative real-time PCR. Liver, heart, lung, kidney, and spleen samples were used for all RT-PCR analyses. Total RNA was extracted using the QIAGEN miRNeasy mini kit and then reverse transcribed and amplified using the High Performance cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative RT-PCR reactions were performed and analyzed on a QuantStudio12K Flex Real-Time PCR System (Applied Biosystems). Each sample was tested in duplicate and relative gene expression was determined using a standard curve.

[0218] Analysis of magnetic resonance imaging data Semiquantitative analysis (digital MRIs Vd / Vi): Volume of distribution (Vd) analysis was performed with OsiriX Imaging software version 3.1 (The OsiriX Foundation, Geneva, Switzerland). The infusion site, the cannula track and the cannula tip were identified on T1-weighted MR images in the coronal plane. Regions of interest (ROIs) were delineated to reveal the boundaries of the T1 gadolinium signal and the target sites (i.e. putamen and caudate nucleus). Three-dimensional volumetric reconstructions of the series of images and ROIs were analyzed to estimate the volume of distribution of the infusion (Vd) and its ratio to the volume of infusion (Vi).

[0219] Histological analysis of transgene expression: To assess transgene expression, brain sections were processed for immunohistochemical analysis (IHC). Four weeks after vector administration, animals were deeply anesthetized with sodium pentobarbital (25 mg / kg iv) and euthanized. Brains were removed and sectioned coronally into 6 mm blocks. Blocks were post-fixed in buffered paraformaldehyde (4%) for 24 hours, briefly washed in PBS, and prepared in 30% sucrose / PBS solution for cryopreservation. Formalin-fixed brain blocks were cut into 40 μm circular sections in a cryostat. Free-floating sections throughout the brain were collected serially and kept in antifreeze solution for further IHC analysis.

[0220] result Both AAV1-GFP and AAV2-GFP vectors resulted in abundant GFP expression from transduced neurons, as visualized by immunohistochemistry. After AAV1 infusion into the caudate and putamen by CED, extensive GFP immunostaining was detected in the substantia nigra (Fig. 2D) in addition to the caudate and putamen (Fig. 2C and D). In addition to the striatum, numerous cortical regions of the rhesus monkey brain were also transduced (Fig. 2A–D). Cortical GFP expression was most evident in frontal association cortical regions, premotor cortex, primary somatosensory cortical regions, and primary motor cortex, as well as extensive areas of the occipital cortex (Fig. 2A–D).

[0221] The majority of GFP-positive neurons in the cortex were morphologically identified as pyramidal neurons located in cortical layer IV with axonal projections to overlying layers. The density of GFP-positive neurons was It was particularly high in the frontal (Fig. 3A and B) and occipital cortices (Fig. 3C and D), where numerous neurons (Fig. 3B and D) were transduced in addition to astrocytes (Fig. 3A and C).

[0222] Example 2: Widespread GFP expression following AAV2 vector delivery in the striatum The ability of AAV2 to efficiently target both striatal and cortical structures in the rhesus monkey brain when delivered via convection-enhanced delivery (CED) was evaluated. An AAV vector containing GFP cDNA under the control of the cytomegalovirus enhancer / chicken beta-actin (CBA) promoter was infused into the caudate and putamen of eight adult rhesus monkeys using CED as described in Example 1 above.

[0223] Infusion of AAV2 into the striatum by CED resulted in GFP expression in the injected regions of the rhesus monkey brain (caudate and putamen) (Figure 4C), the substantia nigra (Figure 4D), and numerous cortical regions (Figure 4A-D). GFP expression in the striatum of AAV2-injected animals appeared to be slightly more restricted and localized when compared to the striatal coverage of the AAV1 vector. GFP expression within the NHP striatum was widespread but relatively contained within the gray matter boundaries of the targeted region, with no evidence of leakage or reflux of AAV2-GFP vector into adjacent non-targeted regions associated with significant infusion. Cortical GFP expression was evident in the same regions as seen with AAV1. Frontal association cortical regions, premotor cortex, primary somatosensory cortical regions, and primary motor cortex, as well as widespread areas of the occipital cortex, were well transduced (Figure 4A-D).

[0224] Example 3: Comparability of GFP expression following AAV1 and AAV2 vectors in the striatum generated by triple transfection or producer cell line processes. To date, the majority of preclinical studies have utilized AAV vectors produced by triple transfection, followed by purification using cesium chloride gradients or column chromatography. Therefore, to evaluate the impact of vector production on biodistribution in vivo, we compared two methods of vector production, triple transfection (TT) or producer cell line (PCL). AAV1 and AAV2 vectors produced by these two different manufacturing platforms were administered via CED and their distribution in the rhesus monkey brain was compared.

[0225] Infusion of AAV1-GFP vectors produced by triple transfection resulted in comparable GFP distribution and coverage when compared to AAV1-GFP vectors produced by the producer cell line process. GFP distribution was comparable for AAV1-GFP(TT) (Figure 5C and D) and AAV1-GFP(PCL) (Figure 5A and B) vectors 30 days after injection into the striatum of rhesus monkeys.

[0226] Similar results were seen with the AAV2-GFP vector: GFP distribution was similar and comparable in brains injected with AAV2-GFP(TT) (Fig. 6C and D) and AAV2-GFP(PCL) (Fig. 6A and B).

[0227] To measure infusion performance, AAV1-eGFP(TT); AAV2-eGFP(TT); AAV1-eGFP(PCL); and AAV2-eGFP(PCL) were infused into each striatum (60 μl into the putamen and 30 μl into the caudate) using 90 μl of each vector mixed with gadolinium contrast agent (2 mM Prohance; Bracco Diagnostics, Inc.). Magnetic resonance images (MRI) from each infusion confirmed that the positioning of each cannula was accurate and that the infusion covered the target area. All infusions were well contained within the target structures. Infusions generated from gadolinium signal on MR images. Three-dimensional reconstruction of the distribution showed that both configurations and infusion distributions were well consistent across all animals. In addition, the ratio of the volume of distribution (Vd) to the volume of infusion (Vi) (Vd / Vi) was calculated for each delivery, and the data were consistent across all infusions. Vd was approximately 3-fold greater than Vi (range 2.1-4.6) (Tables 6 and 7).

[0228] [Table 7]

[0229] [Table 8]

[0230] After bilateral injection of both AAV1-eGFP and AAV2-eGFP (prepared by both production methods, TT and PCL) into the striatum of NHPs, robust expression of eGFP was evident throughout both target structures (putamen and caudate nucleus) as well as regions of projection fields (globus pallidus external and internal segments - GPe and GPi, substantia nigra - SN, subthalamic nucleus - STN, cortical areas - neuronal layers IV and V) (Figure 7).

[0231] To evaluate the role of gadolinium (Gd) as a marker of vector distribution, we calculated the ratio of the area of ​​GFP expression (from tissue sections) to the area of ​​gadolinium signal in the corresponding MR scan. For monkeys infused with serotype AAV1, this ratio was 1. 21±0.10 for AAV1 versus 0.74±0.04 for AAV2 (FIG. 8). A ratio of 1.0 indicates perfect agreement between GFP expression and vector distribution as determined by MRI. This difference indicated that AAV1 vectors dispersed beyond the Gd signal and achieved greater spread than AAV2 in the primary area of ​​transduction.

[0232] To assess the distribution of the infused AAV vectors within the brain, representative free-floating brain sections from each animal (three per block; 40 μm thick) were stained with rabbit anti-GFP antibody (Millipore; catalog no. 3850, 1:500 dilution). Immunohistochemical evaluation revealed dark brown DAB signals within multiple regions projecting from the injected area (cortical regions) in addition to the injected target (right and left putamen and caudate nucleus).

[0233] By projecting the degree of GFP expression onto the MRI scans of each monkey's brain, the percentage coverage of cortical regions (brain regions relevant for HD) was calculated (see Table 6 for summary and Table 7 for further details). In all monkeys, on average, 24% of the striatum was transduced, which resulted in substantial GFP expression in the cortex (Figure 7). The degree of GFP expression in the cortex did not correlate with the AAV serotype used (AAV1 vs. AAV2) or vector production method (TT vs. PCL). A broader distribution of the infusion within the infusion site (striatum) appears to be the primary driving mechanism for the degree of transduction in the cortex. One NHP (subject no. 1; AAV1-eGFP[TT]) showed a particularly robust spread of GFP expression to cortical regions (layers IV and V) throughout the brain (both frontal and occipital - see Figure 7). Other animals showed that variability in cortical expression was associated with variation in both degree and local anatomical area within the caudate and putamen. Because the anterior and commissural regions of the striatum were targeted, GFP was detected more in the frontal and parietal cortical regions and less in the occipital cortex. Below we summarize the histological analysis of each animal (grouped by treatment group).

[0234] Treatment group 2 (ssAAV2 / 1-CBA-eGFP TT) Immunohistochemical assessment of eGFP expression in the whole brain revealed robust signals in targeted sites (both putamen and caudate nucleus) and projected structures (globus pallidus, substantia nigra, thalamus, subthalamic nucleus, and cortical regions).

[0235] Subject No. 1. The subject showed a particularly robust spread of GFP expression to cortical regions (layers 4 and 5) throughout the brain (both frontal and occipital). The morphology of the GFP-positive cells suggested transduction of both neurons and astrocytes, which was later confirmed by double immunofluorescence staining (see below). Calculation of the coverage of GFP expression indicated that 91% (see Table 7) of the entire cortex was transduced (this calculation was performed by projecting the extent of the GFP signal onto the MRI scans of the monkey brains analyzed).

[0236] Subject #2. Subject showed robust transduction in the globus pallidus and substantia nigra of both hemispheres in addition to the putamen and caudate nucleus. Projection of GFP expression to the cortex was less pronounced than in subject #1 and was observed mainly in the frontal regions of the cortex. GFP signal was also detected in the occipital cortex, but the density of GFP-positive cells was significantly lower. The coverage of GFP expression in the cortex was 50% (Table 7). Similar to subject #1, the GFP-positive cells had both neuronal and astrocytic morphology, which was confirmed by double immunofluorescence.

[0237] Subject #3. The subject showed strong GFP expression in all projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus, and cortex) in addition to the putamen and caudate nucleus. Although GFP signal was clearly detected in some areas of the cortex, GFP expression only accounted for 41% of its total cortical coverage (the lowest of all monkeys tested; see Table 7). Both neurons and astrocytes were transduced. A large portion of the right anterior corona radiata also showed GFP-positive signal. We showed that the IL-10 expression level increased with increasing IL-10 expression, which may be the result of vector efflux from the cannula primarily through the striatum.

[0238] Treatment group 3 (ssAAV2 / 2-CBA-eGFP TT) Subject #6. The subject showed strong GFP signal in both targeted structures (putamen and caudate nucleus). Dense scattered positive cells were detected in both of these regions. GFP expression was also widespread in frontal cortical regions, globus pallidus, substantia nigra, subthalamic nucleus, and parts of the thalamus. The coverage of GFP expression in the cortex accounted for 75% (Table 7). GFP-positive cells had mostly neuronal morphology, which was later confirmed by double immunofluorescence. Astrocyte-like GFP-positive cells were detected in the internal capsule as well as in a few cortical spots and in closely adjacent white matter areas with clearly visible tracks of the infusion cannula.

[0239] Subject #7. The subject showed GFP-positive signals in the right and left striatum (both putamen and caudate nucleus). The distribution was rather sparse when compared to other infused monkeys, and the pattern appeared more "spot-like" than uniform. As a result, the GFP signal in all projected brain structures appeared weaker as well. The coverage of GFP expression in the cortex accounted for 47% (Table 7). The GFP-positive cells had mostly neuronal morphology, which was later confirmed by double immunofluorescence. Astrocyte-like GFP-positive cells were detected in the internal capsule, as well as in a few cortical spots and in closely adjacent white matter areas, with a clearly visible track of the infusion cannula.

[0240] Treatment group 4 (ssAAV2 / 1-CBA-eGFP PCLs) Subject #4. The subject showed very robust GFP expression in the targeted structures, putamen and caudate nucleus. GFP-positive signals were also detected in the globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions. The coverage of GFP expression in the cortex accounted for 61% (Table 7). The anterior portion of the corona radiata also showed GFP-positive signals, which may be the result of vector efflux from the cannula, primarily through the striatum. The positive cells had both neuronal and astrocytic morphology, which was later confirmed by double immunofluorescence.

[0241] Subject #5. Subject showed robust GFP expression in the striatum (both putamen and caudate nucleus). GFP signal was also detected in projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions). Coverage of GFP expression in the cortex accounted for 68% (Table 7). The anterior portion of the corona radiata also showed GFP positive signal, which may be the result of vector efflux from the cannula primarily through the striatum. Positive cells had both neuronal and astrocytic morphology.

[0242] Treatment group 5 (ssAAV2 / 2-CBA-eGFP PCLs) Subject #9. Subject showed very robust GFP expression in the striatum (both putamen and caudate nucleus). GFP signal was also detected in projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions). The coverage of GFP expression in the cortex accounted for 73% (Table 7). GFP positive cells had mostly neuronal morphology, although astrocytic GFP positive cells were also detected within white matter tracts (internal capsule) and immediately adjacent to the cannula track.

[0243] Subject #8. Subject showed robust GFP expression in the striatum and projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions). Coverage of GFP expression in the cortex accounted for 50% (Table 7). GFP positive cells had mostly neuronal morphology, although astrocytic GFP+ cells were also detected within white matter tracts (internal capsule, corona radiata) and immediately adjacent to the cannula track.

[0244] Double immunofluorescence For both groups of NHPs transduced with AAV1-eGFP vectors (TT and PCL), the morphology of GFP-positive cells suggested transduction of both neurons and astrocytes (Figures 9A-9D). This was confirmed by double immunofluorescence staining with a combination of antibodies against GFP and NeuN (neuronal marker) or GFP and S-100 (astrocyte marker) (Figures 10A-10C). In contrast, AAV2-eGFP (both TT and PCL) was predominantly directed towards transduction of neurons (Figures 9E-9G and 10D). GFP-positive cells of the astrocyte lineage were detected in the internal capsule (Figure 9H) as well as in the cortical regions of the white matter, where the track of the infusion cannula was visible.

[0245] Based on double immunofluorescence, the efficiency of neuronal transduction in the striatal and cortical regions was calculated for all NHPs (in the coronal plane of the infusion site). Figure 11A summarizes the findings in the striatum. Table 8 below shows the individual calculations for each animal.

[0246] [Table 9]

[0247] Transduction of striatal neurons in the area of ​​primary transduction, as shown by MRI, ranged from 50% to 65%. The maximum efficiency of transduction was observed when AAV1-eGFP(TT) The lowest efficiency was observed in the transfused NHPs, with a mean of 64.2 ± 5.9%, and the lowest efficiency was observed in group AAV2-eGFP(TT), with a mean of 46.6 ± 11.7% (p<0.05; two-way ANOVA). This suggests that serotype AAV1 has about 18% higher efficiency than AAV2 in transducing neurons. AAV1-eGFP produced by PCLS demonstrated a weaker tendency to transduce more neurons (59.7 ± 8.1%) than AAV2-eGFP (50.1 ± 5.8%) (p>0.05; two-way ANOVA).

[0248] The above calculations were derived from the areas of strong GFP transduction as defined by MRI (primary area of ​​transduction - PAT). In addition, the striatal transduction efficiency of neurons in areas outside the PAT was calculated to check whether GFP-positive cells could also be detected outside the clear border of strong GFP signal ("outside the primary area of ​​transduction" - OPAT), suggesting that all tested vectors probably spread in the same manner. Figure 11B illustrates the scheme of how these areas were chosen (random selection of 5 counting frames). A dramatic difference in the estimation of transduction efficiency in OPAT between serotypes AAV1 and AAV2 was observed (Figure 11C), with AAV1 transducing many more neurons than AAV2 (8.1 ± 3.8% vs. 0.74 ± 0.25% for TT group, 7.2 ± 3.5% vs. 2.16 ± 1.8% for PCL group; p < 0.05, two-way ANOVA for both comparisons). Table 9 below shows the individual calculations for each animal.

[0249] [Table 10]

[0250] In addition, the efficiency of transduction in cortical regions projecting to the striatum was calculated. Because the degree of cortical coverage varied between animals, random cortical regions were counted in sections with adjacent GFP-positive striatum. Clear discrepancies were observed between animals (Table 8), with no apparent correlation to the serotype used. The mean transduction efficiency of neurons with AAV1 was 13.6±8.3% compared to 13.4±9.0% with AAV2 (p>0.97).

[0251] As mentioned above, AAV1-eGFP transduced more astrocytes (S-100 marker) than neurons. GFP-positive astrocytes were detected at the primary transduction site (striatum) as well as in cortical regions projecting to the striatum. Figure 10C shows an example of a GFP-transduced astrocyte. With the AAV2-eGFP vector, only scattered GFP+ astrocytes could be detected around the infusion cannula track. To determine whether the vector transduced other antigen-presenting cells in the brain, representative brain sections from all monkeys were stained with both antibodies against GFP and Iba-1, which is specific for microglia. This possibility was excluded, as no animal showed double-labeled cells (Figure 10E). Accordingly, staining for Olig-2, a marker for oligodendrocytes, showed very few cells positive for both GFP and Olig-2 in all monkeys tested. These sparse cells were mainly associated with the cannula. was detected near the track of the

[0252] Brain sections were stained with hematoxylin-eosin (H&E) to determine whether these vectors caused neuroinflammation. Sections were examined primarily for the presence of perivascular wrapping, i.e., accumulation of lymphocytes or plasma cells in dense masses around blood vessels. Varying degrees of such infiltration were detected in all monkeys, but no histological findings associated with other vectors / transgenes were observed. However, AAV1 was observed to cause a slightly more prominent macrophage and lymphocyte infiltration within the primary area of ​​transduction than in the case of AAV2 (Figures 12A and 12B). Vectors produced by triple transfection also appeared to cause more extensive perivascular wrapping than vectors generated by the producer cell line process. Of note, no infiltration was detected in the projection area of ​​transduction (cortical regions).

[0253] Example 4: Absence of detectable GFP expression in peripheral tissues outside the central nervous system (CNS) At necropsy, peripheral organ tissues including kidney, liver, lung, heart, and spleen were collected to assess whether expression of the AAV-GFP transgene administered by CED could be detected outside the CNS.

[0254] result No detectable levels of GFP were detected in any of the collected organs. Both the AAV-GFP(TT) (Figure 13A) and AAV-GFP(PCL) (Figure 13B) vectors showed no detectable peripheral GFP expression. As a positive control for this assay, mouse brain tissue injected with the AAV2 / 1-GFP(TT) vector was used.

[0255] conclusion Efficient delivery of therapeutic proteins to the brain remains a serious obstacle to achieving clinical efficacy while minimizing adverse effects. Advances in gene delivery have provided the opportunity to establish biologics production within the brain parenchyma. These advances have led to the initiation of multiple clinical trials in which AAV vectors have become the preferred vector system for treating neurological disorders. Although focal targeting of specific nuclei can be reliably achieved by stereotactic neurosurgical infusion, the extensive and intricate layout of the human cortex is not easily targeted by direct infusion of viral vectors. The difficulty of safely achieving widespread gene expression in the brain has hindered the development of potential treatments for neurological disorders that require cortical delivery.

[0256] As described herein, AAV vectors (e.g., AAV1 and AAV2) can provide widespread delivery throughout the primate striatum (caudate and putamen), as well as delivery to a significant number of cells within the cerebral cortex (including the frontal cortex, occipital cortex, and layer IV), thalamus, and hippocampus. In the studies discussed herein, GFP, a reporter protein with no known function in the cerebral cortex, was utilized. AAV1 and AAV2-GFP infused into the caudate and putamen using the CED delivery method caused high levels of GFP expression in both the caudate and putamen, as well as several cortical regions. GFP-positive neurons in the frontal cortex were located >20 mm from the site of AAV-GFP infusion, demonstrating axonal transport of GFP protein and AAV vector. Without wishing to be limited by theory, it is believed that since GFP remains in the cytoplasm and is not a secreted protein, the presence of GFP in the cortex indicates direct cell transduction of the AAV2 vector and active transport along a single axonal projection. Huntington's disease is an exemplary disease in which striatal delivery of AAV (e.g., striatal delivery of CED) may be useful. Huntington's disease Because it affects both striatal and cortical regions, a therapeutic strategy targeting both regions would be ideal.

[0257] The findings disclosed herein clearly demonstrate the feasibility of AAV vectors (e.g., AAV1 and AAV2) delivery to transduce neurons located at a considerable distance from the infusion site in the striatum.Therefore, intrastriatal administration of AAV vectors (e.g., AAV1 and AAV2) is ideal for use in the treatment of CNS disorders that require the delivery of therapeutic molecules to the striatum and cortex, such as, but not limited to, Huntington's disease.In addition, the AAV vectors produced by the triple transfection method and the producer cell line method show comparable transgene expression patterns and transduction levels, making the triple transfection and producer cell line AAV vector production method suitable for use in the present invention.

Claims

1. A method for delivering recombinant adeno-associated virus (rAAV) particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.

2. A method for delivering rAAV particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal, and the rAAV particles comprising an AAV serotype 1 (AAV1) capsid.

3. A method for delivering rAAV particles to the central nervous system of a mammal, comprising administering the rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal, and the rAAV particles comprising an AAV serotype 2 (AAV2) capsid.

4. The method according to any one of claims 1 to 3, wherein the mammal is a human.

5. The method of any one of claims 1 to 4, wherein the rAAV particles are administered to the putamen and caudate nucleus of the striatum.

6. The method of claim 5, wherein the rAAV particles are administered to the putamen and caudate nucleus of each hemisphere of the striatum.

7. The method of claim 5 or 6, wherein the rAAV particles are administered to one site in the caudate nucleus and two sites in the putamen.

8. The method of any one of claims 5 to 7, wherein the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:

1.

9. The method of any one of claims 1 to 8, wherein the heterologous nucleic acid is expressed in the frontal cortex, the occipital cortex, and / or layer IV of the mammal.

10. 10. The method of claim 9, wherein the heterologous nucleic acid is expressed in a frontal association cortical region, a premotor cortex, a primary somatosensory cortical region, a sensorimotor cortex, a parietal cortex, an occipital cortex, and / or a primary motor cortex.

11. The method of any one of claims 1 to 10, wherein the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex.

12. The method of any one of claims 1 to 11, wherein the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and / or hippocampus.

13. rAAV particles of claims 1 or 4-12, comprising an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid.

2. The method according to any one of the preceding claims.

14. 14. The method of claim 13, wherein the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrhlO.

15. The method of any one of claims 1 to 14, wherein the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.

16. The method of claim 15, wherein the heterologous nucleic acid is flanked by two AAV ITRs.

17. 17. The method of claim 15 or 16, wherein the AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV serotype ITR.

18. The method according to any one of claims 15 to 17, wherein the AAV ITR is an AAV2 ITR.

19. The method of claim 15 or 16, wherein the ITRs and capsid of the rAAV particle are derived from the same AAV serotype.

20. The method of any one of claims 3 to 12, wherein the ITRs and capsid are derived from AAV2.

21. The method of claim 15 or 16, wherein the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes.

22. The method of any one of claims 2 and 4 to 12, wherein the ITRs are derived from AAV2 and the capsid is derived from AAV1.

23. The method of any one of claims 1 to 22, wherein the heterologous nucleic acid is operably linked to a promoter.

24. 24. The method of claim 23, wherein the promoter expresses the heterologous nucleic acid in a cell of the CNS.

25. 25. The method of claim 23 or 24, wherein the promoter expresses the heterologous nucleic acid in cells of the brain.

26. The method according to any one of claims 23 to 25, wherein the promoter expresses the heterologous nucleic acid in neuronal and / or glial cells.

27. 27. The method of claim 26, wherein the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of cortical layer IV and / or neurons of cortical layer V.

28. The method according to any one of claims 23 to 27, wherein the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter.

29. The method of any one of claims 23 to 27, wherein the promoter is inducible.

30. The method of any one of claims 23 to 29, wherein the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal.

31. The method of any one of claims 1 to 30, wherein the rAAV vector is a self-complementary rAAV vector.

32. 32. The method of claim 31 , wherein the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, and the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length.

33. 33. The method of claim 32, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, the mutated AAV ITR comprising a deletion of the D region and comprising a terminal resolution sequence mutation.

34. The method of any one of claims 1 to 33, wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

35. 35. The method of claim 34, wherein the heterologous nucleic acid encodes a therapeutic polypeptide.

36. Therapeutic polypeptides include enzymes, neurotrophic factors, polypeptides that are deficient or mutated in individuals with CNS-related disorders, antioxidants, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MA), and the like. N2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosamine sulfohydrolase (NPS ... glucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartyl glucosaminidase, 36. The method of claim 35, wherein the enzyme is alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuramidase.

37. 36. The method of claim 35, wherein the heterologous nucleic acid encodes a therapeutic nucleic acid.

38. 38. The method of claim 37, wherein the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or DNAzyme.

39. The method of any one of claims 34 to 38, wherein the therapeutic polypeptide or nucleic acid is used to treat a disorder of the CNS.

40. 40. The method of claim 39, wherein the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or brain cancer.

41. Disorders include aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten type CNL4, Batten type CNL5, Batten type CNL6, Batten type CNL7, Batten type CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, beta-mannosidosis, and Mammary gland syndrome.

41. The method of claim 40, wherein the lysosomal storage disease is selected from the group consisting of Rothau-Lamy, Metachromatic Leukodystrophy, Morquio A, Morquio B, Mucolipidosis II / III Disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe Disease, Sandhoff Disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler Disease, Schindler-Kanzaki, Sialidosis, Sly Disease, Tay-Sachs Disease, and Wollman Disease.

42. The method of any one of claims 1 to 41, wherein the rAAV particles are in the form of a composition.

43. 43. The method of claim 42, wherein the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient.

44. 44. The method of any one of claims 1 to 43, wherein the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, wherein transfection of the nucleic acids into the host cell produces a host cell capable of producing rAAV particles.

45. 44. The method of any one of claims 1 to 43, wherein the rAAV particles are produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

46. The method of any one of claims 1 to 45, wherein the rAAV particles are delivered by stereotactic delivery.

47. The method of any one of claims 1 to 45, wherein the rAAV particles are delivered by convection-enhanced delivery.

48. The method of claim 47, wherein the rAAV particles are delivered using a CED delivery system.

49. 49. The method of claim 48, wherein the CED system includes a cannula and / or a pump.

50. 50. The method of claim 49, wherein the cannula is a reflux resistant cannula or a stepwise cannula. Method of posting.

51. 51. The method of claim 50, wherein the pump is a manual pump.

52. 51. The method of claim 50, wherein the pump is an osmotic pump.

53. 51. The method of claim 50, wherein the pump is an infusion pump.

54. A method for treating a CNS disorder in a mammal, comprising administering to said mammal an effective amount of rAAV particles by the method of any one of claims 1 to 53.

55. A method for treating Huntington's disease in a mammal, comprising administering an effective amount of rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.

56. The method of claim 55, wherein the rAAV particle comprises an AAV1 capsid or an AAV2 capsid.

57. A method for treating Parkinson's disease in a mammal, comprising administering an effective amount of rAAV particles to the striatum, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.

58. The method of claim 57, wherein the rAAV particle comprises an AAV2 capsid.

59. The method of any one of claims 55 to 58, wherein the mammal is a human.

60. The method of any one of claims 55 to 59, wherein the rAAV particles are administered to the putamen and caudate nucleus of the striatum.

61. 59. The method of claim 58, wherein the rAAV particles are administered to the putamen and caudate nucleus of each hemisphere of the striatum.

62. The method of claim 60 or 61, wherein the rAAV particles are administered to one site in the caudate nucleus and two sites in the putamen.

63. 63. The method of any one of claims 55-62, wherein the ratio of rAAV particles administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:

1.

64. 64. The method of any one of claims 55 to 63, wherein the heterologous nucleic acid is expressed in the frontal cortex, the occipital cortex, and / or layer IV of the mammal.

65. 63. The method of claim 62, wherein the heterologous nucleic acid is expressed in a frontal association cortical region, a premotor cortex, a primary somatosensory cortical region, a sensorimotor cortex, a parietal cortex, an occipital cortex, and / or a primary motor cortex.

66. The method of any one of claims 55 to 63, wherein the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex.

67. The heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and / or hippocampus; 67. The method according to any one of claims 55 to 66.

68. 66. The method of any one of claims 55 or 59-65, wherein the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid.

69. 69. The method of claim 68, wherein the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrhlO.

70. 70. The method of any one of claims 55 to 69, wherein the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.

71. 71. The method of claim 70, wherein the heterologous nucleic acid is flanked by two AAV ITRs.

72. The method of claim 70 or 71, wherein the AAV ITR is an ITR of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV serotype.

73. The method according to any one of claims 70 to 72, wherein the AAV ITR is an AAV2 ITR.

74. The method of claim 70 or 71, wherein the ITRs and capsid of the rAAV particle are derived from the same AAV serotype.

75. 75. The method of claim 74, wherein the ITRs and capsid are derived from AAV2.

76. The method of claim 70 or 71, wherein the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes.

77. 77. The method of claim 76, wherein the ITRs are derived from AAV2 and the capsid is derived from AAV1.

78. The method of any one of claims 55 to 77, wherein the heterologous nucleic acid is operably linked to a promoter.

79. 79. The method of claim 78, wherein the promoter expresses the heterologous nucleic acid in a cell of the CNS.

80. 80. The method of claim 78 or 79, wherein the promoter expresses the heterologous nucleic acid in cells of the brain.

81. The method of any one of claims 78 to 80, wherein the promoter expresses the heterologous nucleic acid in neuronal and / or glial cells.

82. 82. The method of claim 81, wherein the neuron is a medium spiny neuron of the caudate nucleus, a medium spiny neuron of the putamen, a neuron of layer IV of the cortex and / or a neuron of layer V of the cortex.

83. 83. The method of any one of claims 78 to 82, wherein the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter.

84. The method of any one of claims 78 to 82, wherein the promoter is inducible.

85. The method of any one of claims 78 to 84, wherein the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal.

86. The method of any one of claims 55 to 85, wherein the rAAV vector is a self-complementary rAAV vector.

87. 87. The method of claim 86, wherein the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length.

88. 88. The method of claim 87, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, the mutated AAV ITR comprising a deletion of the D region and comprising a terminal resolution sequence mutation.

89. The method of any one of claims 55 to 88, wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

90. 90. The method of any one of claims 55, 56 or 58-88, wherein the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid that inhibits expression of HTT or inhibits accumulation of HTT in cells of the mammalian CNS.

91. The method of claim 89 or 90, wherein the heterologous nucleic acid encodes an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or DNAzyme.

92. 89. The method of any one of claims 55, 56, or 58-88, wherein the heterologous nucleic acid encodes a miRNA that targets huntingtin.

93. 93. The method of claim 92, wherein the huntingtin comprises a mutation associated with Huntington's disease.

94. 89. The method of any one of claims 57 to 88, wherein the heterologous nucleic acid encodes a therapeutic polypeptide, said therapeutic polypeptide being glial cell-derived growth factor (GDNF), brain-derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), and / or amino acid decarboxylase (AADC).

95. The method of any one of claims 55 to 94, wherein the rAAV particles are in the form of a composition.

96. 96. The method of claim 95, wherein the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient.

97. 97. The method of any one of claims 55-96, wherein the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, wherein transfection of the nucleic acids into the host cell produces a host cell capable of producing rAAV particles.

98. 97. The method of any one of claims 55-96, wherein the rAAV particles are produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

99. The method of any one of claims 55 to 98, wherein the rAAV particles are delivered by stereotactic delivery.

100. 100. The method of any one of claims 55-99, wherein the rAAV particles are delivered by convection-enhanced delivery.

101. The method of claim 97, wherein the rAAV particles are delivered using a CED delivery system.

102. 102. The method of claim 101, wherein the CED system includes a cannula and / or a pump.

103. 103. The method of claim 102, wherein the cannula is a reflux resistant cannula or a graded cannula.

104. 103. The method of claim 102, wherein the pump is a manual pump.

105. 103. The method of claim 102, wherein the pump is an osmotic pump.

106. 103. The method of claim 102, wherein the pump is an infusion pump.

107. A system for expression of heterologous nucleic acids in the mammalian cerebral cortex and striatum, comprising: a) a composition comprising an rAAV particle, the rAAV particle comprising an rAAV vector encoding the heterologous nucleic acid; and b) A device for delivering the rAAV particles to the striatum. The system comprising:

108. The system of claim 107, wherein the rAAV particle comprises an AAV1 capsid or an AAV2 capsid.

109. The system of claim 107 or 108, wherein the mammal is a human.

110. The system of any one of claims 107 to 109, wherein the rAAV particles are administered to the putamen and caudate nucleus of the striatum.

111. The system of claim 110, wherein the rAAV particles are administered to the putamen and caudate nucleus of each hemisphere of the striatum.

112. The system of any one of claims 107 to 111, wherein the heterologous nucleic acid is expressed in the frontal cortex, the occipital cortex, and / or layer IV of the mammal.

113. The system of claim 112, wherein the heterologous nucleic acid is expressed in a frontal association cortical region, a premotor cortex, a primary somatosensory cortical region, a sensorimotor cortex, a parietal cortex, an occipital cortex, and / or a primary motor cortex.

114. The system of any one of claims 107 to 113, wherein the rAAV particles undergo retrograde or anterograde transport in the cerebral cortex.

115. The system of any one of claims 107 to 114, wherein the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and / or hippocampus.

116. 116. The system of any one of claims 107 or 109-115, wherein the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid.

117. The system of claim 116, wherein the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrhlO.

118. The system of any one of claims 107 to 117, wherein the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.

119. The system of claim 118, wherein the heterologous nucleic acid is flanked by two AAV ITRs.

120. The system of claim 118 or 119, wherein the AAV ITR is an ITR of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype.

121. The system of any one of claims 118 to 120, wherein the AAV ITR is an AAV2 ITR.

122. The system of claim 118 or 119, wherein the ITRs and capsid of the rAAV particle are derived from the same AAV serotype.

123. The system of claim 122, wherein the ITRs and capsid are derived from AAV2.

124. The system of claim 118 or 119, wherein the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes.

125. The system of claim 124, wherein the ITRs are derived from AAV2 and the capsid is derived from AAV1.

126. The system of any one of claims 107 to 125, wherein the heterologous nucleic acid is operably linked to a promoter.

127. The system of claim 126, wherein the promoter expresses the heterologous nucleic acid in cells of the CNS.

128. The system of claim 126 or 127, wherein the promoter expresses the heterologous nucleic acid in cells of the brain.

129. The system of any one of claims 126 to 128, wherein the promoter expresses the heterologous nucleic acid in a neuronal or glial cell.

130. 130. The system of claim 129, wherein the neurons are medium spiny neurons of the caudate nucleus, medium spiny neurons of the putamen, neurons of layer IV of the cortex and / or neurons of layer V of the cortex.

131. The system of any one of claims 126 to 130, wherein the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter or a GUSB promoter.

132. The system of any one of claims 126 to 130, wherein the promoter is inducible.

133. The system of any one of claims 126 to 132, wherein the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal.

134. The system of any one of claims 107 to 133, wherein the rAAV vector is a self-complementary rAAV vector.

135. The system of claim 134, wherein the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, and the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length.

136. The system of claim 135, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, the mutated AAV ITR comprising a deletion of the D region and comprising a terminal resolution sequence mutation.

137. The system of any one of claims 107 to 136, wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

138. The system of claim 137, wherein the heterologous nucleic acid encodes a therapeutic polypeptide.

139. Therapeutic polypeptides include enzymes, neurotrophic factors, polypeptides that are deficient or mutated in individuals with CNS-related disorders, antioxidants, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-synuclein, alpha-glucosidase, beta-galactosidase-1 (GLB1), alpha-galactosidase-1 (GLB1), alpha-galactosidase-1 (GALC ... α-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, 139. The system of claim 138, wherein the enzyme is acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuramidase.

140. The system of claim 138, wherein the heterologous nucleic acid encodes a therapeutic nucleic acid.

141. The system of claim 140, wherein the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or DNAzyme.

142. The system of any one of claims 137 to 141, wherein the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.

143. 143. The system of claim 142, wherein the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or brain cancer.

144. Disorders include aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten type CNL4, Batten type CNL5, Batten type CNL6, Batten type CNL7, Batten type CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, beta-mannosidosis, Marot's disease, and cystinosis. The system of claim 143, wherein the lysosomal storage disease is selected from the group consisting of Lamy, Metachromatic Leukodystrophy, Morquio A, Morquio B, Mucolipidosis II / III disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe disease, Sandhoff disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler disease, Schindler-Kanzaki, Sialidosis, Sly disease, Tay-Sachs disease, and Wollman disease.

145. The system of any one of claims 107 to 144, wherein the rAAV particles are in the form of a composition.

146. The system of claim 145, wherein the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient.

147. The system of any one of claims 107 to 146, wherein the rAAV particles are produced by triple transfection of a host cell with nucleic acid encoding an rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, wherein transfection of the nucleic acids into the host cell generates a host cell capable of producing rAAV particles.

148. The system of any one of claims 107 to 146, wherein the rAAV particles are produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

149. The system of any one of claims 107 to 148, wherein the rAAV particles are delivered by stereotactic delivery.

150. The system of any one of claims 107 to 149, wherein the rAAV particles are delivered by convection-enhanced delivery.

151. The system of claim 150, wherein the rAAV particles are delivered using a CED delivery system.

152. The system of claim 151, wherein the CED system includes a cannula and / or a pump.

153. 153. The system of claim 152, wherein the cannula is a reflux resistant cannula or a graded cannula.

154. 153. The system of claim 152, wherein the pump is a manual pump.

155. The system of claim 152, wherein the pump is an osmotic pump.

156. The system of claim 152, wherein the pump is an infusion pump.

157. 107. A kit for use in the method of any one of claims 1 to 106, comprising rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid to be expressed in at least the cerebral cortex and striatum of a mammal.

158. The kit of claim 157, wherein the rAAV particles comprise an AAV serotype 1 (AAV1) capsid.

159. The kit of claim 158, wherein the rAAV particles comprise an AAV serotype 2 (AAV2) capsid.

160. A kit for treating Huntington's disease in a mammal, comprising a composition comprising an effective amount of rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal.

161. The kit of claim 160, wherein the rAAV particles comprise an AAV serotype 1 (AAV1) capsid or an AAV serotype 2 (AAV2) capsid.

162. A method for treating Parkinson's disease in a mammal, comprising administering to said mammal a composition comprising an effective amount of rAAV particles.

13. A kit for administering a rAAV particle to a mammal, the kit comprising: an rAAV vector encoding a heterologous nucleic acid to be expressed in at least the cerebral cortex and striatum of the mammal.

163. The kit of claim 161, wherein the rAAV particles comprise an AAV serotype 1 (AAV1) capsid or an AAV serotype 2 (AAV2) capsid.

164. The kit of any one of claims 157 to 163, further comprising a device for delivering rAAV particles to the striatum.

165. The kit of any one of claims 157 to 164, wherein the rAAV particles are in the form of a composition.

166. 166. The kit of claim 165, wherein the composition comprises a buffer and / or a pharma- ceutically acceptable excipient.

167. The kit of any one of claims 157 to 166, further comprising instructions for delivering the composition of rAAV particles to the striatum.

168. rAAV particles for use in any one of the methods according to claims 1 to 106.

169. A recombinant adeno-associated virus (rAAV) particle for use in delivering an rAAV particle to the central nervous system of a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid to be expressed in at least the cerebral cortex and striatum of the mammal.

170. 1. A recombinant adeno-associated virus (rAAV) particle for use in delivering rAAV particles to the central nervous system of a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid to be expressed in at least the cerebral cortex and striatum of the mammal, and further comprising an AAV serotype 1 (AAV1) capsid.

171. 1. A recombinant adeno-associated virus (rAAV) particle for use in delivering rAAV particles to the central nervous system of a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid expressed in at least the cerebral cortex and striatum of the mammal, and further comprising an AAV serotype 2 (AAV2) capsid.

172. 1. An rAAV particle for use in treating Huntington's disease in a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.

173. 1. An rAAV particle for use in treating Parkinson's disease in a mammal, the rAAV particle comprising an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.

174. The rAAV of claim 173, wherein the rAAV particle comprises an AAV2 capsid.

175. The rAAV particle of any one of claims 169 to 174, wherein the mammal is a human.

176. The rAAV particle of any one of claims 169 to 175, for administration to the putamen and caudate nucleus of the striatum.

177. The rAAV particle described in claim 176, for administration to the putamen and caudate nucleus of each hemisphere of the striatum.

178. The rAAV particle of any one of claims 169-177, wherein the heterologous nucleic acid is expressed in the frontal cortex, occipital cortex, and / or layer IV of a mammal.

179. The rAAV particle of claim 178, wherein the heterologous nucleic acid is expressed in a frontal association cortex region, a premotor cortex, a primary somatosensory cortex region, a sensorimotor cortex, a parietal cortex, an occipital cortex, and / or a primary motor cortex.

180. The rAAV particle of any one of claims 169 to 179, which undergoes retrograde or anterograde transport in the cerebral cortex.

181. The rAAV particle of any one of claims 169-180, wherein the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra, and / or hippocampus.

182. 182. The rAAV particle of any one of claims 169, 170, or 175-181, wherein the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid.

183. The rAAV particle of claim 182, wherein the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrhlO.

184. The rAAV particle of any one of claims 169 to 183, wherein the rAAV vector comprises a heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.

185. The rAAV particle of claim 184, wherein the heterologous nucleic acid is flanked by two AAV ITRs.

186. The rAAV particle of claim 184 or 185, wherein the AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR.

187. The rAAV particle of any one of claims 184 to 186, wherein the AAV ITR is an AAV2 ITR.

188. The rAAV particle of claim 184 or 185, wherein the ITRs and the capsid of the rAAV particle are derived from the same AAV serotype.

189. The rAAV particle of claim 188, wherein the ITRs and capsid are derived from AAV2.

190. The rAAV particle of claim 184 or 185, wherein the ITRs and the capsid of the rAAV viral particle are derived from different AAV serotypes.

191. The rAAV particle of claim 190, wherein the ITRs are derived from AAV2 and the capsid is derived from AAV1.

192. The rAAV particle of any one of claims 169 to 171, wherein the heterologous nucleic acid is operably linked to a promoter.

193. The rAAV particle of claim 192, wherein the promoter expresses a heterologous nucleic acid in a cell of the CNS.

194. The rAAV particle of claim 192 or 193, wherein the promoter expresses a heterologous nucleic acid in cells of the brain.

195. The rAAV particle of any one of claims 191 to 194, wherein the promoter expresses a heterologous nucleic acid in a neuronal or glial cell.

196. The rAAV particle of claim 195, wherein the neuron is a medium spiny neuron of the caudate nucleus, a medium spiny neuron of the putamen, a neuron in layer IV of the cortex and / or a neuron in layer V of the cortex.

197. The rAAV particle of any one of claims 192 to 196, wherein the promoter is a CBA promoter, a minimal CBA promoter, a CMV promoter, or a GUSB promoter.

198. The rAAV particle of any one of claims 192 to 196, wherein the promoter is inducible.

199. The rAAV particle of any one of claims 192 to 198, wherein the rAAV vector comprises one or more of an enhancer, a splice donor / splice acceptor pair, a matrix attachment site, or a polyadenylation signal.

200. The rAAV particle of any one of claims 169 to 199, wherein the rAAV vector is a self-complementary rAAV vector.

201. The rAAV particle of claim 200, wherein the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence is capable of forming intrastrand base pairs with the second nucleic acid sequence over most or all of its length.

202. The rAAV particle of claim 200, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, the mutated AAV ITR comprising a deletion of the D region and comprising a mutation in the terminal resolution sequence.

203. The rAAV particle of any one of claims 169-202, wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

204. The rAAV particle of claim 203, wherein the heterologous nucleic acid encodes a therapeutic polypeptide.

205. Therapeutic polypeptides include enzymes, neurotrophic factors, polypeptides that are deficient or mutated in individuals with CNS-related disorders, antioxidants, anti-apoptotic factors, Anti-angiogenic and anti-inflammatory factors, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine -1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick type C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, alpha-glucosaminidase 205. The rAAV particle of claim 204, wherein the rAAV protein is selected from the group consisting of N-acetylglucosamine-6-sulfatase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, and alpha-neuramidase.

206. The rAAV particle of claim 203, wherein the heterologous nucleic acid encodes a therapeutic nucleic acid.

207. The rAAV particle of claim 206, wherein the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or DNAzyme.

208. The rAAV particle of any one of claims 203 to 207, wherein the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.

209. The rAAV particle of claim 208, wherein the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or brain cancer.

210. Disorders include aspartylglucosaminuria, Fabry, Batten disease of infancy (CNL1), classical late infantile Batten disease (CNL2), juvenile Batten disease (CNL3), Batten type CNL4, Batten type CNL5, Batten type CNL6, Batten type CNL7, Batten type CNL8, cystinosis, Farber, fucosidosis, galactosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, beta-mannosidosis, and Maroteaux disease.

210. The rAAV particle of claim 209, wherein the lysosomal storage disease is selected from the group consisting of: Ramie, Metachromatic Leukodystrophy, Morquio A, Morquio B, Mucolipidosis II / III Disease, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe Disease, Sandhoff Disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler Disease, Schindler-Kanzaki, Sialidosis, Sly Disease, Tay-Sachs Disease, and Wollman Disease.

211. The rAAV particle of any one of claims 169 to 210, in the form of a composition.

212. The composition of claim 211, wherein the composition is a pharmaceutical composition comprising a pharma- ceutical acceptable excipient. rAAV particles.

213. The rAAV particle of any one of claims 169 to 212, wherein the rAAV particle is produced by triple transfection of a host cell with nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions, wherein transfection of the nucleic acids into the host cell produces a host cell capable of producing the rAAV particle.

214. The rAAV particle of any one of claims 169 to 212, produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

215. The rAAV particle of any one of claims 169 to 214, which is delivered by stereotactic delivery.

216. The rAAV particle of any one of claims 169 to 214, delivered by convection-enhanced delivery.

217. The rAAV particle described in claim 216, which is delivered using a CED delivery system.

218. The rAAV particle of claim 217, wherein the CED system comprises a cannula and / or a pump.

219. The rAAV particle of claim 218, wherein the cannula is a reflux resistant cannula or a stepwise cannula.

220. The rAAV particle of claim 218, wherein the pump is a manual pump.

221. The rAAV particle of claim 218, wherein the pump is an osmotic pump.

222. The rAAV particle of claim 218, wherein the pump is an infusion pump.

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