NZ735290B2

NZ735290B2 - Enhanced delivery of viral particles to the striatum and cortex

Info

Publication number: NZ735290B2
Application number: NZ735290A
Authority: NZ
Inventors: Lamya Shihabuddin; Lisa M Stanek
Original assignee: Genzyme Corporation
Filing date: 2016-02-09
Publication date: 2024-01-30

Abstract

Provided herein are novel methods for delivering recombinant adeno-associated viral (rAAV) particles to the central nervous system of a mammal (e.g., a human). In aspects, the methods involve administering rAAV particles containing a heterologous nucleic acid to the striatum and causing expression of the heterologous nucleic acid in at least the cerebral cortex and the striatum of the mammal.

Description

ENHANCED DELIVERY OF VIRAL LES TO THE STRIATUM AND CORTEX CROSS—REFERENCE TO RELATED APPLICATIONS This application claims priority to US. Provisional Application No. 62/114,544, filed on February 10, 2015, and US. Provisional Application No. 62/220,997, filed on September 19, 2015, the content of each of which is hereby incorporated by reference in its entirety for all purposes.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 159792012740SEQLIST.txt, date recorded: February 4, 2016, size: 1 KB).

FIELD OF THE INVENTION The present ion s to the delivery of AAV gene therapy vectors to the brain, e. g., the striatum and/or cortex.

SUMMARY OF THE INVENTION Adeno—associated virus based vectors have become the preferred vector system for neurologic gene therapy, with an excellent safety record ished in multiple al trials (Kaplitt et al., (2007) Lancet 97—2105; Eberling et al., (2008) Neurology 70:1980—1983; Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27—35). ive treatment of neurologic disorders has been ed by problems associated with the delivery of AAV vectors to affected cell populations. This delivery issue has been especially problematic for disorders involving the cerebral cortex. Simple injections do not distribute AAV vectors effectively, g on diffusion, which is effective only within a 1— to 3—mm radius. An alternative method, convection—enhanced delivery (CED) (Nguyen et al., (2003) J. Neurosurg. 98:5 84—590), has been used clinically in gene therapy (AAV2— hAADC) for Parkinson's disease (Fiandaca et al., (2008) Exp. Neurol. 209:51—57). The underlying principle of CED involves pumping infusate into brain parenchyma under sufficient pressure to overcome the hydrostatic pressure of interstitial fluid, y forcing the infused particles into close contact with the dense perivasculature of the brain. Pulsation of these vessels acts as a pump, distributing the particles over large ces throughout the parenchyma (Hadaczek et al., (2006) Hum. Gene Ther. 17:291—302). To increase the safety and efficacy of CED a re?ux—resistant cannula (Krauze et al., (2009) Methods Enzymol. 465:349—362) can be employed along with monitored delivery with real—time MRI.

Monitored delivery allows for the quantification and control of aberrant events, such as a re?ux and leakage of infusate into ventricles ing et al., (2008) Neurology 70:1980—1983; Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27—35; Saito et al., (2011) Journal ofNeurosurgery Pediatrics 7:522—526). However, there is still a need for improved procedures to achieve widespread expression of AAV vectors in the cortex and/or striatum.

The invention provides a method for ring a recombinant adeno—as sociated viral (rAAV) particle to the central nervous system of a mammal comprising stering the rAAV particle to the striatum, n the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and um of the mammal. In some s, the invention provides a method for delivering a rAAV particle to the l nervous system of a mammal comprising administering the rAAV particle to the um, wherein the rAAV particle comprises an rAAV vector encoding a heterologous nucleic acid that is sed in at least the cerebral cortex and striatum of the mammal and wherein the rAAV particle ses an AAV serotype 1 (AAVl) capsid. In some aspects, the invention provides a method for delivering a rAAV particle to the central nervous system of a mammal comprising administering the rAAV particle to the striatum, wherein the rAAV particle comprises an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and um of the mammal and wherein the rAAV particle comprises an AAV serotype 2 (AAV2) capsid. In some embodiments, the mammal is a human.

In some embodiments, the rAAV particle is administered to at least the putamen and the caudate nucleus of the striatum. In some embodiments, the rAAV particle is stered to at least the putamen and the caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particle is 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 e 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 at least in the prefrontal association cortical areas, the premotor cortex, the primary somatosensory cortical areas, sensory motor cortex, parietal , occipital cortex, and/or primary motor cortex. In some embodiments, the rAAV le undergoes retrograde or anterograde transport in the cerebral cortex. In some embodiments, the logous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and/or ampus.

In some embodiments, the rAAV particle is administered to the caudate nucleus and the putamen at a rate of greater than 1 uL/min to about 5 uL/min.

In some embodiments of the above aspects and embodiments, the rAAV particle comprises an AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAVl/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAVZ/HBon serotype capsid. In some embodiments, the AAV serotype is AAVl, AAVZ, AAVS, AAV6, AAV7, AAVS, , R, AAV9, AAVlO, or . In some embodiments, the rAAV vector comprises the logous nucleic acid ?anked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is ?anked by two AAV ITRs. In some embodiments, the AAV ITRs are AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAV12, 71A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid of the rAAV particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid are derived from AAVZ. In other embodiments, the ITR and the capsid of the rAAV viral les are derived from different AAV serotypes. In some embodiments, the ITR is derived from AAV2 and the capsid is derived from AAVl.

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 brain cell. 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 s, a medium spiny neuron of the putamen, a neuron of the cortex layer IV and/or a neuron of the cortex layer V. In some embodiments, the glial cell is an astrocyte. In some embodiments, the promoter is a CBA promoter, a minimum CBA promoter, a CMV er 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 . In some embodiments, the rAAV vector is a self—complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding the heterologous nucleic acid and a second nucleic acid sequence encoding a ment of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a d AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.

In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an , a neurotrophic factor, a polypeptide that is deficient or mutated in an individual with a CNS —related disorder, an antioxidant, an anti—apoptotic factor, an anti—angiogenic , and an anti—in?ammatory factor, alpha—synuclein, acid beta—glucosidase (GBA), beta—galactosidase—l (GLB l), ate 2—sulfatase (IDS), osylceramidase (GALC), a mannosidase, D— mannosidase (MANZB l), beta—mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N— acetylglucosamine—l—phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann—Pick C protein (NPCl), acid l,4—glucosidase (GAA), hexosaminidase beta subunit, HEXB, N—sulfoglucosamine sulfohydrolase ), N—alpha— acetylglucosaminidase (NAGLU), heparin acetyl—CoA, alpha—glucosaminidase N— acetyltransferase (MPS3C), N—acetylglucosamine—6—sulfatase (GNS), alpha—N— galactosaminidase (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 logous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme. In some embodiments, the therapeutic polypeptide or the eutic nucleic acid is used to treat a er of the CNS.

In some embodiments of the above aspects and embodiments, the disorder of the CNS is a lysosomal storage disease (LSD), gton's disease, epilepsy, Parkinson's disease, Alzheimer's e, stroke, corticobasal degeneration (CBD), corticogasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or cancer of the brain. In some embodiments, the er is a lysosomal storage disease ed from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNLl), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNL5, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, dosis, osidosialidosis Gaucher e type , 1, r e type 2, Gaucher disease type 3, GMl gangliosidosis, Hunter disease, Krabbe disease, 0t mannosidosis disease, [3 mannosidosis disease, MaroteauX—Lamy, metachromatic leukodystrophy disease, Morquio A, Morquio B, mucolipidosisII/III disease, Niemann—Pick A disease, n—Pick B disease, Niemann—Pick C disease, Pompe disease, Sandhoff disease, Sanfillipo A disease, Sanfillipo B disease, Sanfillipo C disease, Sanfillipo D disease, Schindler disease, Schindler—Kanzaki, sialidosis, Sly disease, Tay—Sachs e, and Wolman disease.

In some embodiments of the above aspects and embodiments, the rAAV particle is in a composition. In further ments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

In some embodiments of the above aspects and embodiments, the rAAV particle was produced by triple transfection of a c acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV particles. In other embodiments, the rAAV particle was produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

In some embodiments of the above aspects and ments, the rAAV particle is delivered by stereotactic ry. In some embodiments, the rAAV particle is delivered by convection enhanced delivery. In some embodiments, the rAAV particle is delivered using a CED delivery system. In some embodiments, the CED system ses a cannula. In some embodiments, the cannula is a resistant cannula or a stepped cannula. In some embodiments, the CED system comprises 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.

In some aspects, the ion provides a method for delivering rAAV particles to the central nervous system of a mammal comprising administering a composition comprising the rAAV particles to the striatum by CED, n the composition is stered to the striatum at a rate of greater than 1 uL/min to about 5 uL/min. In some aspects, the invention provides a method for delivering rAAV particles to the central nervous system of a mammal comprising administering a composition comprising the rAAV particles to the striatum by CED, wherein the composition comprises rAAV particles and poloxamer. In some ments, the poloxamer is poloxamer 188. In some embodiments, the concentration of poloxamer in the composition is ranges from about 0.0001% to about 0.01%. In some embodiments, the concentration of poloxamer in the composition is about . In some embodiments, the composition further comprises sodium de, wherein the concentration of sodium chloride in the composition ranges from about 100 mM to about 250 mM. In some embodiments, the concentration of sodium chloride in the composition is about 180 mM. In some ments, the composition further comprises sodium phosphate, wherein the concentration of sodium ate in the ition ranges from 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, wherein 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 the putamen at a rate of greater than 1 uL/min to about 5 uL/min. In some embodiments, the amount of the composition delivered to the putamen is about twice the volume delivered to the caudate s. In some embodiments, about 20 uL to about 50 uL of the composition is administered to the caudate nucleus of each here and about 40 uL to about 100 uL of the composition is administered to the putamen of each hemisphere. In some embodiments, about 30 uL of the composition is stered to the caudate nucleus of each hemisphere and about 60 uL of the ition is administered to the putamen of each hemisphere.

In some embodiments, the invention provides a method of ng a disorder of the CNS in a mammal comprising administering an effective amount of a rAAV particle to the mammal by the methods described above.

In some aspects, the invention es a method of treating Huntington’s e in a mammal comprising administering an effective amount of a rAAV particle to the striatum, wherein the rAAV particle comprises 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 rAAV particle comprises an AAVl capsid or an AAV2 capsid. In other aspects, the invention provides a method of treating Parkinson’s disease in a mammal comprising administering an effective amount of a rAAV le to the striatum, wherein the rAAV particle ses a rAAV vector encoding a heterologous c acid that is expressed in at least the cerebral cortex and striatum of the mammal. In some embodiments, the rAAV particle comprises an AAV2 capsid. In some embodiments, the mammal is a human.

In some embodiments, the rAAV particle is administered to at least the putamen and the caudate nucleus of the striatum. In some embodiments, the rAAV particle is administered to at least the putamen and the caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particle is administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV particles stered 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 at least in the prefrontal association cortical areas, the or cortex, the primary sensory cortical areas, y motor cortex, parietal cortex, occipital cortex, and/or primary motor cortex. In some embodiments, the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex. In some embodiments, the heterologous nucleic acid is further expressed in the thalamus, subthalamic s, globus pallidus, substantia nigra and/or ampus.

In some embodiments of the above aspects and embodiments, the rAAV particle comprises an AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, R, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAVl/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAVZ/HBon serotype capsid. In some embodiments, the AAV serotype is AAVl, AAVZ, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, or AAVrth. In some embodiments, the rAAV vector comprises the heterologous c acid ?anked by one or more AAV inverted terminal repeat (ITR) sequences. In some ments, the heterologous nucleic acid is ?anked by two AAV ITRs. In some embodiments, the AAV ITRs are AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, 71A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid of the rAAV particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid are derived from AAVZ. In other embodiments, the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes. In some ments, the ITR is derived from AAV2 and the capsid is derived from AAVl.

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 c acid in a brain cell. In some embodiments, the er 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 cortex layer IV and/or a neuron of the cortex layer V. In some embodiments, the glial cell is an astrocyte. In some embodiments, the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible. In further ments, the rAAV vector comprises one or more of an enhancer, a splice donor/splice or 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 the heterologous c acid and a second c acid sequence encoding a ment of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first c 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.

In some embodiments of the above aspects and ments, the logous nucleic acid s a therapeutic ptide or eutic 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 the 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 logous nucleic acid encodes a miRNA that targets huntingtin. In some embodiments, the gtin comprises a on associated with Huntington’s disease.

In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for ng Huntington’s disease. In some ments, 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 the 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 a miRNA that targets huntingtin. In some embodiments, the huntingtin comprises a mutation associated with Huntington’s disease.

In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for treating Parkinson’s disease. In some embodiments, the therapeutic polypeptide is glial—derived growth factor (GDNF), brain—derived growth factor (BDNF), tyrosine hydroxlase (TH), GTP—cyclohydrolase ), and/or amino acid decarboxylase (AADC).

In some embodiments of the above aspects and embodiments, the rAAV le is in a composition. In further embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

In some embodiments of the above aspects and embodiments, the rAAV particle was produced by triple transfection of a nucleic acid encoding the rAAV , a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV particles. In other embodiments, the rAAV particle was produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV , a nucleic acid ng AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

In some embodiments of the above aspects and embodiments, the rAAV particle is delivered by stereotactic delivery. In some embodiments, the rAAV particle is delivered by convection enhanced delivery. In some embodiments, the rAAV particle is delivered using a CED ry system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a re?ux—resistant a or a stepped cannula. In some ments, the CED system comprises 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.

In some aspects, the invention provides a system for expression of a logous nucleic acid in the cerebral cortex and striatum of a mammal, comprising a) a composition comprising rAAV particles, wherein the rAAV particles comprise a rAAV vector encoding the logous nucleic acid; and b) a device for delivery of the rAAV particles to the striatum. In some embodiments, the rAAV particle comprises an AAVl capsid or an AAV2 capsid. In some embodiments, the mammal is a human.

In some embodiments of the system of the invention, the rAAV le is administered to the putamen and the caudate nucleus of the um. In some embodiments, the rAAV particle is administered to at least one site in the caudate nucleus and two sites in the putamen. In some embodiments, the ratio of rAAV les 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 , occipital cortex, and/or layer IV of the mammal. In some embodiments, the heterologous nucleic acid is sed at least in the prefrontal association cortical areas, the premotor cortex, the primary somatosensory cortical areas, sensory motor cortex, parietal , occipital cortex, and/or primary motor cortex. In some embodiments, the rAAV particle undergoes 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 of the system of the invention, the rAAV particle comprises an AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAVl/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAVZ/HBon pe capsid. In some embodiments, the AAV serotype is AAVl, AAVZ, AAVS, AAV6, AAV7, AAVS, AAVrhS, R, AAV9, AAVlO, or . In some embodiments, the rAAV vector comprises the logous nucleic acid ?anked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is ?anked by two AAV ITRs. In some ments, the AAV ITRs are AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV pe ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid of the rAAV particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid are d from AAVZ. In other embodiments, the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes. In some embodiments, the ITR is derived from AAV2 and the capsid is derived from AAVl.

In some embodiments of the system of the invention, 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 brain cell. 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 e nucleus, a medium spiny neuron of the putamen, a neuron of the cortex layer IV and/or a neuron of the cortex layer V. In some embodiments, the glial cell is an astrocyte. In some embodiments, the promoter is a CBA promoter, a m CBA promoter, a CMV promoter or a GUSB promoter. In other ments, 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 ment 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 the 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 along most or all of its . In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a d AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.

In some embodiments of the system of the invention, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a eutic polypeptide. In some embodiments, the therapeutic polypeptide is an enzyme, a neurotrophic factor, a polypeptide that is deficient or mutated in an individual with a CNS—related disorder, an antioxidant, an anti—apoptotic factor, an ngiogenic factor, and an anti—in?ammatory , alpha—synuclein, acid beta—glucosidase (GBA), beta—galactosidase—l (GLB l), iduronate 2—sulfatase (IDS), galactosylceramidase (GALC), a idase, alpha—D—mannosidase (MANZB 1), beta— mannosidase (MANBA), pseudoarylsulfatase A (ARSA), ylglucosamine—l— phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann—Pick C protein (NPCl), acid alpha—l,4—glucosidase (GAA), hexosaminidase beta subunit, HEXB, N— lucosamine sulfohydrolase (MPS3A), a—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 , Aspartylglucosaminidase, Alpha—galactosidase A, Palmitoyl protein thioesterase, Tripeptidyl peptidase, Lysosomal transmembrane protein, Cysteine transporter, Acid ceramidase, Acid L—fucosidase, cathepsin A, alpha—L—iduronidase, Arylsulfatase B, Arylsulfatase A, N—acetylgalactosamine—6—sulfate, Acid beta—galactosidase, or alpha—neuramidase. In other ments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a me or a e. In some embodiments, the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.

In some embodiments of the system of the invention, the disorder of the CNS is a lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal ration (CBD), corticogasal ganglionic degeneration (CBGD), temporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or cancer of the brain. In some embodiments, the disorder is a mal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNLl), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten e (CNL3), Batten form CNL4, Batten form CNL5, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis Gaucher disease type , 1, Gaucher disease type 2, r disease type 3, GMl gangliosidosis, Hunter disease, Krabbe disease, 0t mannosidosis disease, [3 mannosidosis disease, MaroteauX—Lamy, romatic leukodystrophy disease, Morquio A, Morquio B, pidosisII/III e, Niemann—Pick A disease, Niemann—Pick B disease, Niemann—Pick C disease, Pompe disease, Sandhoff disease, Sanfillipo A disease, Sanfillipo B disease, Sanfillipo C disease, Sanfillipo D disease, Schindler disease, Schindler—Kanzaki, sialidosis, Sly disease, Tay—Sachs disease, and Wolman disease.

In some embodiments, the rAAV of the invention comprises a logous nucleic acid encoding a therapeutic polypeptide or eutic nucleic acid for treating Huntington’s disease. In some embodiments, the therapeutic polypeptide or the therapeutic nucleic acid inhibits the expression of HTT or ts the accumulation of HTT in cells of the CNS of the 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 me or a DNAzyme. In some embodiments, the heterologous nucleic acid encodes a miRNA that targets huntingtin. In some embodiments, the gtin comprises a mutation associated with Huntington’s disease.

In some embodiments, the rAAV particle of the invention comprises a heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for treating Parkinson’s disease. In some embodiments, the therapeutic polypeptide is glial— derived growth factor (GDNF), brain—derived growth factor (BDNF), tyrosine hydroxlase (TH), GTP—cyclohydrolase (GTPCH), and/or amino acid decarboxylase (AADC).

In some ments of the system of the invention, the rAAV particle is in a composition. In further embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

In some embodiments of the system of the ion, the rAAV le was produced by triple transfection of a nucleic acid encoding the rAAV , a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell e of producing rAAV les. In other embodiments, the rAAV particle was produced by a producer cell line comprising one or more of c acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

In some embodiments of the system of the invention, the rAAV particle is delivered by stereotactic ry. In some embodiments, the rAAV particle is delivered by convection enhanced delivery. In some embodiments, the rAAV particle is red using a CED delivery system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a —resistant cannula or a stepped cannula. In some embodiments, the CED system comprises a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some ments, the pump is an infusion pump.

In some aspects, the invention provides a kit for use in any of the methods described above where the kit comprising rAAV particles, wherein the rAAV particles comprise a 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 rAAV les comprise an AAV serotype 1 (AAVl) capsid. In some embodiments, the rAAV particles se an AAV serotype 2 (AAVZ) capsid.

In some aspects, the 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 that is sed in at least the cerebral cortex and striatum of the mammal. In some aspects, the 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 se a 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 rAAV particles of the kits comprise an AAV serotype 1 (AAVl) capsid or an AAV pe 2 (AAVZ) capsid. In some embodiments, the kit further comprising a device for delivery of the rAAV particles to the striatum. In some embodiments, the rAAV particles of the kit are in a composition. In some ments, the composition comprises a buffer and/or a ceutically able excipient. In further embodiments, the kit comprises instructions for delivery of the composition of rAAV particles to the striatum.

In some aspects, the invention provides a rAAV particle for use in any of the methods described above. In some aspects, the ion provides a rAAV particle for use in delivering a recombinant associated viral (rAAV) particle to the l nervous system of a mammal, n the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal. In some aspects, the invention provides a rAAV particle for use in delivering a recombinant adeno—associated viral (rAAV) particle to the central nervous system of a mammal, wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal, and wherein the rAAV particle further ses an AAV serotype 1 (AAVl) capsid. In some aspects, the invention provide a rAAV particle for use in delivering a recombinant adeno—associated viral (rAAV) particle to the central nervous system of a mammal, wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal, and n the rAAV particle further ses an AAV serotype 1 (AAVZ) capsid.

In some aspects, the invention provides a rAAV le for use in treating Huntington’s disease in a mammal, wherein the rAAV le comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal. In some aspects, the invention provides a rAAV particle for use in treating Parkinson’s disease in a mammal, wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the . In some embodiments, the rAAV particle comprises an AAV2 capsid. In some embodiments, the mammal is a human.

In some embodiments, the rAAV particle of the invention is administered to at least the n and the caudate s of the striatum. In some embodiments, the rAAV particle is administered to at least the putamen and the caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particle is 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 les stered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is sed in at least the frontal cortex, occipital cortex, and/or layer IV of the mammal. In some embodiments, the heterologous nucleic acid is sed at least in the prefrontal association cortical areas, the premotor cortex, the primary somatosensory cortical areas, sensory motor cortex, parietal cortex, occipital cortex, and/or primary motor cortex. In some embodiments, the rAAV particle undergoes 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 of the above aspects and embodiments, the rAAV particle of the invention comprises an AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, 71A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAVl/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAVZ/HBon serotype . In some embodiments, the AAV serotype is AAVl, AAVZ, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, or AAVrth. In some embodiments, the rAAV vector comprises the logous nucleic acid ?anked by one or more AAV inverted terminal repeat (ITR) sequences. In some ments, the heterologous nucleic acid is ?anked by two AAV ITRs. In some embodiments, the AAV ITRs are AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, R, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid of the rAAV particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid are derived from AAVZ. In other embodiments, the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes. In some embodiments, the ITR is d from AAV2 and the capsid is derived from AAVl.

In some embodiments of the above aspects and ments, the heterologous nucleic acid is operably linked to a promoter. In some embodiments, the promoter expresses the logous nucleic acid in a cell of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in a brain cell. In some ments, 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 cortex layer IV and/or a neuron of the cortex layer V. In some embodiments, the glial cell is an astrocyte. In some embodiments, the promoter is a CBA promoter, a minimum CBA promoter, a CMV er 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 the logous nucleic acid and a second c acid sequence encoding a complement of the logous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second c 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.

In some ments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some ments, 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 an individual with a CNS —related disorder, an antioxidant, an anti—apoptotic factor, an ngiogenic factor, and an anti—in?ammatory factor, alpha—synuclein, acid beta—glucosidase (GBA), alactosidase—l (GLB l), iduronate 2—sulfatase (IDS), osylceramidase (GALC), a mannosidase, alpha—D— idase (MAN2B l), annosidase (MANBA), pseudoarylsulfatase A (ARSA), N— acetylglucosamine—l—phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann—Pick C n (NPCl), acid alpha—l,4—glucosidase (GAA), hexosaminidase beta t, HEXB, N—sulfoglucosamine sulfohydrolase (MPS3A), N—alpha— acetylglucosaminidase (NAGLU), heparin acetyl—CoA, alpha—glucosaminidase N— acetyltransferase ), N—acetylglucosamine—6—sulfatase (GNS), alpha—N— acetylgalactosaminidase (NAGA), beta—glucuronidase , hexosaminidase alpha t (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), Aspartylglucosaminidase, Alpha—galactosidase A, Palmitoyl n thioesterase, Tripeptidyl peptidase, Lysosomal embrane 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, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme. In some embodiments, the therapeutic ptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.

In some embodiments of the above aspects and embodiments, the er of the CNS is a lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticogasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or cancer of the brain. In some embodiments, the disorder is a lysosomal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNLl), c Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNL5, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis Gaucher e type , 1, Gaucher disease type 2, Gaucher disease type 3, GMl gangliosidosis, Hunter disease, Krabbe disease, 0t mannosidosis disease, [3 mannosidosis disease, MaroteauX—Lamy, romatic leukodystrophy disease, Morquio A, Morquio B, mucolipidosisII/III disease, Niemann—Pick A disease, Niemann—Pick B disease, Niemann—Pick C disease, Pompe disease, Sandhoff disease, Sanfillipo A disease, Sanfillipo B disease, Sanfillipo C e, Sanfillipo D disease, Schindler disease, Schindler-Kanzaki, osis, Sly disease, Tay-Sachs disease, and Wolman disease.

In some embodiments of the above aspects and embodiments, the rAAV particle is in a composition. In r embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

In some embodiments of the above aspects and embodiments, the rAAV particle was produced by triple ection of a nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV particles. In other embodiments, the rAAV le was produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, a nucleic acid ng AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

In some embodiments of the above s and embodiments, the rAAV particle is delivered by stereotactic delivery. In some embodiments, the rAAV particle is delivered by convection enhanced delivery. In some embodiments, the rAAV particle is delivered using a CED ry system. In some embodiments, the CED system ses a cannula. In some embodiments, the cannula is a -resistant cannula or a stepped cannula. In some embodiments, the CED system comprises a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an c pump. In some embodiments, the pump is an infusion pump. [0048a] In one aspect, there is provided use of a recombinant adeno-associated viral (rAAV) particle in the manufacture of a medicament for treating a disorder of the central nervous system (CNS) in a subject, n the rAAV particle is to be administered to the striatum by convection enhanced delivery (CED), wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the occipital cortex and/or layer IV of the cerebral cortex and striatum of the subject, wherein the rAAV particle comprises an AAV 2 capsid, and wherein the rAAV particle is stered to at least one site in the caudate nucleus and two sites in the putamen in each hemisphere of the striatum.

[PAGE 19a TO FOLLOW] 60_1 (GHMatters) P43229NZ00 [0048b] In another aspect, there is provided a method of treating a disorder of the central nervous system (CNS) in a non-human primate sing administering a recombinant adeno-associated viral (rAAV) le, wherein the rAAV particle is administered to the um by convection enhanced delivery (CED), wherein the rAAV particle ses a rAAV vector ng a heterologous nucleic acid that is expressed in at least the occipital cortex and/or layer IV of the cerebral cortex and striatum of the subject, wherein the rAAV particle comprises an AAV 2 capsid, and wherein the rAAV particle is administered to at least one site in the caudate nucleus and two sites in the n in each hemisphere of the striatum.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. r, it is to be understood that, if any prior art publication is referred to herein, such nce does not constitute an admission that the publication forms a part of the common general knowledge in the art, in New Zealand or any other country.

BRIEF DESCRIPTION OF THE DRAWINGS shows Rhesus monkey body s, taken immediately prior to surgery (black) and at the time of necropsy (gray), in animals administered AAV1 and AAV2 vectors made by triple transfection (TT) and producer cell line (PCL) processes.

FIGS. 2A-2D show representative brain sections stained for GFP 30 days after infusion of AAV1-GFP (TT) into Rhesus monkey caudate and putamen. Sections in FIGS.

[PAGE 20 TO FOLLOW] -19a- 20230660_1 (GHMatters) P43229NZ00 2A-2D extend through the brain in the rostral to caudal direction. Sections from three representative animals are displayed in each panel.

FIGS. 3A-3D show representative brain ns demonstrating al expression of GFP in the frontal cortex (FIGS. 3A & 3B) and occipital cortex (FIGS. 3C & 3D) in both astrocytes (FIGS. 3A & 3C) and cortical neurons (FIGS. 3B & 3D) after on of AAV1-GFP (TT) into Rhesus monkey caudate and putamen.

FIGS. 4A-4D show representative brain sections d for GFP 30 days after infusion of AAV2-GFP (TT) into Rhesus monkey caudate and putamen. Sections in FIGS. 4A-4D extend through the brain in the l to caudal direction. Sections from three representative animals are displayed in each panel.

FIGS. 5A-5D show representative brain sections stained for GFP 30 days after infusion of AAV1-GFP made by producer cell lines (PCL) (FIGS. 5A & 5B) or triple transfection (TT) (FIGS. 5C & 5D) processes into Rhesus monkey caudate and putamen.

FIGS. 6A-6D show entative brain sections stained for GFP 30 days after infusion of AAV2-GFP made by producer cell line (PCL) (FIGS. 6A & 6B) or triple transfection (TT) (FIGS. 6C & 6D) ses into Rhesus monkey caudate and putamen. shows the distribution of GFP in non-human primate (NHP) brains infused with AAV1-eGFP and AAV2-eGFP. AAV1-eGFP and AAV2-eGFP vectors were infused bilaterally into the striatum of 9 Rhesus macaques. Four weeks after the surgery, brains were processed for immunohistochemistry (IHC) against GFP. Columns show representative GFP-stained brain sections from 4 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 distribution of GFP sion throughout the entire brain from frontal cortex, striatum (infusion sites), midbrain, to occipital parts of the cortex. All groups showed robust GFP signal in the sites of injection (putamen and caudate nucleus) as well as extensive transport to cortical regions and substantia nigra. Based on the IHC staining, the coverage of GFP expression in both target structure (striatum) and cortical regions were calculated for each monkey and are summarized in Table 7. 19823731_2 (GHMatters) P43229NZ00 shows the ratios of primary areas of transduction (PAT) to vector distribution (Vd). Primary areas of GFP expression in the striatum were delineated on scans from the GFP—stained sections and their values divided by values obtained from matching MRI scans with Gadolinium signal. Ratios > 10 te that the extent of GFP expression exceeds the boundaries of Gadolinium signal after infusion. The results from monkeys infused with AAV vectors showed that AAVl s better in the brain parenchyma than AAV2 (1.21 i 0.1 vs. 0.74 i 0.04; p < 0.007 with 2—tailed unpaired t—test).

FIGS. 9A-9H show GFP expression in the NHP brain transduced with AAVl— eGFP and AAV2—eGFP. : High magnification (40X) of the target structure caudate nucleus transduced with AAVl—eGFP (TT) of subject number 1. Dark—brown GFP+ neurons stained by DAB are visible t densely stained network of positive neuronal fibers. Such a robust signal was ed in all monkeys injected with AAVl—eGFP vector produced by both TT and PCL methods. : Fragment of prefrontal cortex of subject number 1 ( demonstrating massive transport of vector GFP from the sites of injection (striatum) to cortical regions. Based on morphology of GFP+ cells, both neurons and astrocytes were detected in the cortex. : Higher magnification (40X) of the frame indicated in showing numerous cortical neurons expressing GFP. : High (40X) magnification of the cortex from subject number lshowing GFP+ cells of astrocytic morphology. : High magnification (40X) of the target structure putamen transduced with AAV2—eGFP (TT) of t number 6. Dark—brown DAB signal show expression of GFP in neurons and their densely stained network of fibers. : Fragment of ntal cortex of subject number 6 ( demonstrating massive transport of vector AAV2—eGFP from the striatum (injection site) to al regions. The vast majority of GFP—positive cells had neuronal morphology (magnification 2.5X). : Higher ication (40X) of the frame indicated in showing numerous cortical neurons sing GFP. : Higher magnification (20X) of internal capsule of t number 6 showing GFP+ cells with astrocytic morphology.

FIGS. 10A-10E show the cellular m of GFP and AAV2—eGFP injected into the monkey brain. Monkey brain sections were processed for double immuno?uorescence staining against GFP and various cellular markers to determine cellular tropism of the injected vectors. A: Section from e nucleus (target structure) from subject number 1 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and neuronal marker NeuN (red channel for DyLightTM 549 dye; middle column). Merged pictures (magnification 20X; right column) from both channels show numerous neurons expressing GFP, verifying neuronal tropism of AAVl— eGFP. B: The same staining was performed for a section from prefrontal cortex of t number 1 showing neuronal transduction in a distal brain structure receiving neuronal projections from the striatum and is evidence of retrograde transport of AAVl— eGFP. C: Section from caudate nucleus (target structure) from subject number 1 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and ytic marker S—lOO (red channel for DyLightTM 549 dye; middle column). Merged pictures (magnification 20X; right column) from both channels show numerous astrocytes expressing GFP, verifying that AAVl—eGFP also transduces astrocytes. D: Section from caudate nucleus (target structure) from subject number 6 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and neuronal marker NeuN (red channel for DyLightTM 549 dye; middle column). Merged pictures (magnification 20X; right column) from both channels show us neurons expressing GFP, verifying neuronal tropism of AAV2—eGFP. E: Section from caudate nucleus (target structure) from subject number 3 d with antibodies against GFP (green l for DyLightTM 488 dye; left column) and microglia marker Iba—l (red channel for DyLightTM 549 dye; middle column). The lack of co—staining of both markers in merged picture (magnification 20X; right column) indicates that AAVl does not transduce lia, and this was also the case for AAV2 (data not shown).

FIGS. C show the ency of neuronal transduction in the striatum of NHP injected with AAVl—eGFP and AAV2—eGFP. Double immuno?uorescence staining against GFP and neuronal marker NeuN of monkey brain sections was performed to ate the efficiency of neuronal transduction within the um (target ure) and cortical regions. For the striatum, the efficiency of uction was calculated in the primary area of GFP transduction (PAT) where signal was robust with densely distributed GFP+ neurons (A). Neurons were also detected in regions e the primary areas of GFP transduction (OPAT; C). Scheme for the technique of counting GFP+ neurons in PAT (inner g) and OPAT (outer shading) is shown in B. Data from dual counts for each monkey and brain structure are shown in Table 8 (PAT) and Table 9 (OPAT).

FIGS. 12A & 12B show vector—related ogical findings. Independent evaluation of hematoxylin and eosin (H&E) staining of coronal sections from areas of primary uction (PAT) revealed normal gliosis related to cannula insertion in all experimental groups. H&E staining also revealed perivascular cellular infiltrates in all animals regardless of the vector used. The incidence and severity of perivascular cuffs was increased in groups injected with AAVl, especially when the vector was prepared by the TT method. A: H&E—stained section from subject number 3 shows numerous perivascular cuffs in the left putamen transduced with AAVl—eGFP (TT). One blood vessel is magnified (5X) in the right bottom corner. B: H&E—stained section from subject number 5 shows only a few localized scular cuffs in the left caudate nucleus transduced with AAVl—eGFP (PCL). A few blood vessels are magnified (5X) in the left bottom comer.

FIGS. 13A & 13B show quantitative PCT (QPCR) analysis of eGFP mRNA expression in liver, spleen, heart, kidney, and lung s 1 month ing injection of AAVl—eGFP into Rhesus monkey caudate and putamen. (A) AAVl and AAV2— eGFP vectors made by a triple transfection (TT) process. (B) AAVl and AAV2— eGFP vectors made by a producer cell line (PCL) process.

DETAILED DESCRIPTION As discussed in detail herein, the inventors have discovered that AAV vectors (e.g., AAVl and AAV2 vectors) efficiently target both striatal and cortical structures in the Rhesus monkey brain when delivered to the striatum (e.g., by convection ed delivery, CED). These studies also evaluated two ent manufacturing platforms, and these s demonstrate that AAV generated by triple transfection and er cell lines target both striatal and al structures in the Rhesus monkey brain. Intrastriatal delivery of rAAV particles (e.g., AAVl and AAV2 vectors) produced using both platforms was able to transduce neurons located a considerable distance from the infusion site (e.g., al structures), as well as neurons in the striatum. Accordingly, the present invention provides methods for delivering a recombinant associated viral (rAAV) particle containing a rAAV vector encoding a heterologous nucleic acid to the central s system of a mammal by administering the rAAV particle to the striatum where the heterologous nucleic acid is sed in at least the cerebral cortex and striatum of the mammal.

The invention also provides methods for treating a CNS disorder (e.g., Huntington’s disease) in a mammal by administering to the striatum a rAAV particle encoding a heterologous nucleic acid that is sed in at least the cerebral cortex and striatum, as well as systems and kits for expression of a heterologous nucleic acid in the cerebral cortex and striatum of a mammal using a rAAV particle described herein. The s in the invention may also e a delivery device (e.g., a CED ) for delivery of the rAAV particle to the striatum of a mammal, and likewise, the systems and kits of the invention may further include a device for delivery of the rAAV particle to the um of a mammal.

I. General Techniques The techniques and procedures bed or referenced herein are generally well understood and commonly ed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in lar Cloning: A Laboratory Manual ook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring , N.Y., 2012); Current Protocols in Molecular Biology (F.M.

Ausubel, et al. eds., 2003); the series Methods in Enzymology mic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and GR. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture ofAnimal Cells: A Manual c Technique and Specialized Applications (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J .E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue e (J .P. Mather and PE. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B.

Griffiths, and D.G. Newell, eds., J. Wiley and Sons, 1993—8); Handbook ofExperimental Immunology (D.M. Weir and CC. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and MP. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (CA. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988—1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., d ic Publishers, 1995); and Cancer: ples and Practice of Oncology (V.T.

DeVita et al., eds., J .B. Lippincott Company, 2011). 11. Definitions A "vector," as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.

The term ucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or ibonucleotides. Thus, this term includes, but is not limited to, single—, double— or multi—stranded DNA or RNA, genomic DNA, cDNA, DNA—RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non—natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate . Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P—NHZ) or a mixed oramidate— phosphodiester oligomer. In addition, a double—stranded polynucleotide can be obtained from the single ed polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under riate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate .

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non—natural amino acid residues, and include, but are not limited to, es, oligopeptides, dimers, trimers, and ers of amino acid residues. Both full—length proteins and fragments thereof are encompassed by the definition.

The terms also include post—expression modifications of the polypeptide, for example, glycosylation, ation, ation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These cations may be deliberate, as through site—directed nesis, or may be accidental, such as through mutations of hosts which produce the ns or errors due to PCR amplification.

A "recombinant viral " refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is ?anked by at least one inverted terminal repeat sequences (ITRs). In some embodiments, the inant nucleic acid is ?anked by two ITRs.

A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more logous sequences (i.e., nucleic acid sequence not of AAV origin) that are ?anked by at least one or two AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been ed with a le helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (Le. AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a d used for cloning or transfection), then the rAAV vector may be referred to as a "pro—vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle; for example, an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a "recombinant adeno—associated viral particle (rAAV particle)".

"Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a logous nucleotide ce with t to the vector. A heterologous nucleic acid may refer to a nucleic acid derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.

The term "heterologous c acid" refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and ally, translated and/or expressed under appropriate conditions. In some aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.

"Chicken B—actin (CBA) promoter" refers to a polynucleotide ce derived from a chicken B—actin gene (e.g., Gallus gallus beta actin, represented by GenB ank Entrez Gene ID 396526). As used herein, "chicken B—actin promoter" may refer to a promoter containing a galovirus (CMV) early er element, the er and first exon and intron of the chicken B—actin gene, and the splice acceptor of the rabbit beta—globin gene, such as the ces described in Miyazaki, J. et al. (1989) Gene 79(2):269—77. As used herein, the term "CAG promoter" may be used interchangeably. As used , the term "CMV early enhancer/chicken beta actin (CAG) promoter" may be used interchangeably.

The terms "genome particles (gp),:9 en genome equivalents," or "genome copies" as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome les in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for e, in Clark et al. (1999) Hum. Gene Ther., 10:1031— 1039; jk et al. (2002) M01. Then, 6:272—278.

The term "vector genome (vg)" as used herein may refer to one or more polynucleotides comprising a set of the 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, a 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 through recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence ?anking a promoter, a sequence of st (e.g., a heterologous nucleic acid), and a enylation sequence. A te vector genome may include a complete set of the polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector may be measured in terms of vg/mL. Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

The terms "infection unit (iu),39 c"infectious particle," or "replication unit," as used in reference to a viral titer, refer to the number of infectious and ation—competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in hlin er al. (1988) J. Virol., 62: 1963—1973.

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

An "inverted terminal repeat" or "ITR" sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in te orientation.

An "AAV ed terminal repeat (ITR)" sequence, a term well—understood in the art, is an approximately 145—nucleotide sequence that is present at both termini of the native single—stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and n the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of self—complementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand airing to occur within this portion of the ITR.

A "terminal resolution ce" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant al resolution sequence is refractory to cleavage by AAV rep proteins.

"AAV helper ons" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents.

A "helper virus" for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A helper virus provides "helper functions" which allow for the replication of AAV. A number of such helper viruses have been identified, including iruses, herpesviruses and, poxviruses such as vaccinia and baculovirus. The iruses encompass a number of different subgroups, although irus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non—human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex s (HSV), Epstein— Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). es of adenovirus helper functions for the replication of AAV include ElA functions, ElB functions, E2A functions, VA functions and E4orf6 functions. Baculoviruses ble from depositories include Autographa californica nuclear polyhedrosis virus.

A preparation of rAAV is said to be "substantially free" of helper virus if the ratio of infectious AAV particles to ious helper virus particles is at least about 102:1; at least about 104:1, at least about 106:1; or at least about 108:1 or more. In some embodiments, preparations are also free of equivalent amounts of helper virus proteins (i. 6., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form). Viral and/or cellular protein ination can generally be observed as the presence of sie staining bands on SDS gels (e.g., the appearance of bands other than those ponding to the AAV capsid proteins VPl, VP2 and VP3).

"AAV helper ons" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents. A "helper virus" for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A number of such helper s have been identified, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although irus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non—human ian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, e, for example, herpes simplex viruses (HSV), Epstein— Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).

"Percent (%) sequence identity" with t to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate ce that are identical with the amino acid residues or tides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the ce identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular y (Ausubel er al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST—2, ALIGN or Megalign (DNASTAR) software. An example of an alignment m is ALIGN Plus (Scientific and Educational Software, Pennsylvania).

Those d in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes , the % amino acid ce identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the on X/Y, where X is the number of amino acid es scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence ty of A to B will not equal the % amino acid sequence identity of B to A. For purposes , the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z, where W is the number of nucleotides scored as identical matches by the sequence ent program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be iated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid ce D, the % nucleic acid sequence identity of C to D will not equal the % c acid sequence identity of D to C.

An "isolated" molecule (e.g., nucleic acid or protein) or cell means it has been identified and separated and/or recovered from a component of its natural nment.

An "effective amount" is an amount sufficient to effect beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of clinical endpoints, and the like). An effective amount can be administered in one or more strations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay pment of a disease.

As used , the term "convection enhanced delivery (CED)" may refer to delivery of a eutic agent to the CNS by infusion at a rate in which hydrostatic pressure leads to convective distribution. In some embodiments, the infusion is done at a rate greater than 0.5 uL/min. However, any suitable ?ow rate can be used such that the ranial pressure is maintained at suitable levels so as not to injure the brain tissue.

CED may be accomplished, for example, by using a suitable catheter or cannula (e.g., a step—design re?ux—free cannula) through positioning the tip of the cannula at least in close proximity to the target CNS tissue (for example, the tip is inserted into the CNS tissue).

After the cannula is positioned, it is connected to a pump which delivers the therapeutic agent through the cannula tip to the target CNS tissue. A pressure gradient from the tip of the cannula may be ined during infusion. In some embodiments, infusion may be monitored by a tracing agent detectable by an imaging method such as intraoperative MRI (iMRI) or another real—time MRI que and/or delivered by standard stereotaxic injection equipment and techniques (e.g., the ClearPoint® system from MRI Interventions, s, TN).

As used herein, the term "poloxamer" may refer to a block copolymer made of a chain of polyoxypropylene ?anked by two chains of polyoxyethylene. Trade names under which poloxamers may be sold include without limitation PLURONIC® (BASF), KOLLIPHOR® (BASF), LUTROL® (BASF), and SYNPERONIC® (Croda International).

An idual" or "subject" is a . Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and ), primates (e.g., humans and non—human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For es of this invention, beneficial or desired al results include, but are not limited to, alleviation of symptoms, diminishment of extent of e, stabilized (e.g., not worsening) state of disease, preventing spread (e.g., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean ging survival as compared to expected survival if not receiving treatment.

As used herein, the term "prophylactic treatment" refers to treatment, wherein an individual is known or suspected to have or be at risk for having a disorder but has displayed no symptoms or minimal symptoms of the disorder. An individual undergoing prophylactic treatment may be treated prior to onset of symptoms.

"Huntington’s disease (HD)" refers to the progressive brain disorder typically caused by mutations in the HTT gene (aka huntingtin, HD or IT]5). It may be characterized by symptoms including abnormal movements (termed chorea), gradual loss of motor function, emotional or psychiatric ses, and progressively ed cognition.

Although most symptoms appear in the 30s and 40s, juvenile forms of the disease have also been observed. For further description of HD, see OMIM Entry No. 143100, which is hereby incorporated by reference in its entirety.

"Huntingtin (HTT)" may refer either to the gene or to a polypeptide product thereof associated with most cases of Huntington’s e. The normal function of gtin is not fully understood. However, ons in the huntingtin gene are known to cause HD. These mutations are typically inherited in an autosomal dominant fashion and involve expansion of trinucleotide CAG repeats in the HTT gene, leading to a polyglutamine (polyQ) tract in the Htt protein.

As used herein, a "therapeutic" agent (e.g., a therapeutic polypeptide, nucleic acid, transgene, or the like) is one that provides a beneficial or desired clinical result, such as the exemplary clinical results described above. As such, a therapeutic agent may be used in a treatment as described above.

Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X." As used herein, the singular form of the articles en :9 en a an," and "the" includes plural references unless indicated otherwise.

It is understood that s and embodiments of the ion described herein include "comprising,:9 6‘consisting," and/or "consisting ially of" aspects and embodiments.

III. Methods for delivering rAAV particles In some aspects, the invention provides methods for delivering a recombinant adeno—associated viral (rAAV) particle to the central nervous system of a mammal comprising administering the rAAV particle to the um, n the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal. In further s, the invention provides methods for delivering a rAAV particle to the central nervous system of a mammal comprising administering the rAAV particle to the striatum, wherein the rAAV particle comprises an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal and wherein the rAAV particle comprises an AAV serotype 1 (AAVl) capsid. In yet further aspects, the invention provides methods for delivering a rAAV le to the central nervous system of a mammal sing administering the rAAV particle to the striatum, wherein the rAAV particle comprises an rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal and n the rAAV particle ses an AAV serotype 2 (AAVZ) capsid. In still further aspects, the invention provides methods for treating Huntington’s e in a mammal comprising administering a rAAV particle to the striatum, wherein the rAAV particle comprises a 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 mammal is a human.

Certain aspects of the present disclosure relate to administration of a rAAV particle to one or more regions of the central nervous system (CNS). In some embodiments, the rAAV le is administered to the striatum. The striatum is known as a region of the brain that receives inputs from the cerebral cortex (the term "cortex" may be used interchangeably herein) and sends s to the basal ganglia (the striatum is also referred to as the striate nucleus and the neostriatum). As described above, the striatum controls both motor movements and nal control/motivation and has been implicated in many neurological diseases, such as Huntington’s disease. Several cell types of interest are located in the um, including t limitation spiny projection neurons (also known as medium spiny neurons), GABAergic intemeurons, and cholinergic intemeurons. Medium spiny neurons make up most of the striatal neurons. These neurons are GABAergic and express dopamine receptors. Each hemisphere of the brain contains a striatum.

Important substructures of the striatum include the caudate nucleus and the putamen. In some ments, the rAAV le is administered to the e s (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 implicated in l of functions such as directed movements, spatial working memory, memory, goal—directed actions, emotion, sleep, language, and learning. Each hemisphere of the brain contains a caudate nucleus.

In some embodiments, the rAAV particle is administered to the putamen. Along with the caudate nucleus, the putamen is known as a structure of the dorsal um. The putamen comprises part of the lenticular nucleus and connects the cerebral cortex with the substantia nigra and the globus pallidus. Highly integrated with many other structures of the brain, the putamen has been implicated in control of functions such as learning, motor learning, motor performance, motor tasks, and limb movements. Each hemisphere of the brain ns a n. rAAV particles may be administered to one or more sites of the striatum. In some embodiments, the rAAV particle is administered to the putamen and the caudate nucleus of the striatum. In some embodiments, the rAAV particle is administered to the n and the caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV particle is administered to at least one site in the caudate s and two sites in the putamen.

In some embodiments, the rAAV particle is stered to one hemisphere of the brain. In some embodiments, the rAAV particle is administered to both hemispheres of the brain. For example, in some embodiments, the rAAV particle is administered to the putamen and the 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 ning rAAV particles is administered to 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 e nucleus of the left hemisphere, the caudate nucleus of the right hemisphere, the putamen of the left hemisphere and the putamen of the right here.

In some ments, the composition ning rAAV particles is administered to more than one location simultaneously or sequentially. In some embodiments, multiple ions of the composition containing rAAV particles are no more than about any of one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours or 24 hours apart. In some embodiments, multiple injections of the composition containing rAAV les are more than about 24 hours apart.

Generally, from about luL to about lmL of a composition of the invention can be red (e.g., from about 100 uL to about 500 uL of a 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 twice the volume delivered to the caudate nucleus. In other embodiments, the amount of the composition delivered to the putamen is about any of IX, l.25X, l.5X. l.75X, 2X, 2.25X, 2.5X. 2.75X, 3X, 3.5X, 4X, 4.5X, 5X or 10X (or any ratio therebetween) the volume delivered to the e nucleus. For example, 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 (e.g., about 30 uL of the composition is administered to the caudate nucleus of each hemisphere and about 60 uL of the composition is stered to the putamen of each hemisphere). In some embodiments, about 20 uL to about 50 uL of the ition (or any amount therebetween) is administered to the caudate nucleus of each hemisphere, and about 40 uL to about 100 uL of the composition (or any amount therebetween) is administered to the putamen of each hemisphere. In some embodiments, the volume of the composition administered to the caudate s of each hemisphere is less than about any of the following volumes (in uL): 50, 45 , 40, 35 , 30, or 25. In some embodiments, the volume of the composition administered to the e nucleus of each hemisphere is greater than about any of the following volumes (in uL): 20, 25 , 30, 35, 40, or 45. That is, the volume of the composition administered to the caudate nucleus of each hemisphere can be any of a range of volumes having an upper limit of 50, 45, 40, 35, 30, or 25 and an independently ed lower limit of 20, 25, 30, 35, 40, or 45 wherein the lower limit is less than the upper limit. In some embodiments, the volume of the composition administered to the putamen of each hemisphere is less than about any of the following volumes (in uL): 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45. In some embodiments, the volume of the composition administered to the n of each hemisphere is greater than about any of the following volumes (in uL): 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, or 95. That is, the volume of the composition administered to the putamen of each hemisphere can be any of a range of s having an upper limit of 100, 95 , 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45 and an independently ed lower limit of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, wherein the lower limit is less than the upper limit.

In some ments, the composition is administered to the um at a rate of greater than 1 uL/min to about 5 uL/min. In some embodiments, the composition is administered to the caudate nucleus and the putamen at a rate of greater than 1 uL/min to about 5 uL/min. In some embodiments, the composition is administered to the striatum (the caudate nucleus and/or the putamen) at a rate of greater than about any of l uL/min, 2 uL/min, 3 uL/min, 4 , 5 uL/min, 6 uL/min, 7 uL/min, 8 uL/min, 9 uL/min, or 10 uL/min. In some embodiments, the composition is administered to the striatum (the caudate nucleus and/or the putamen) at a rate of any of about 1 uL/min to about 10 uL/min, about 1 uL/min to about 9 uL/min, about 1 uL/min to about 8 uL/min, about 1 uL/min to about 7 uL/min, about 1 uL/min to about 6 uL/min, about 1 uL/min to about 5 uL/min, about 1 uL/min to about 4 uL/min, about 1 uL/min to about 3 uL/min, about 1 uL/min to about 2 uL/min, about 2 uL/min to about 10 uL/min, about 2 uL/min to about 9 uL/min, about 2 uL/min to about 8 uL/min, about 2 uL/min to about 7 uL/min, about 2 uL/min to about 6 uL/min, about 2 uL/min to about 5 uL/min, about 2 uL/min to about 4 uL/min, about 2 uL/min to about 3 uL/min, about 3 uL/min to about 10 uL/min, about 3 uL/min to about 9 uL/min, about 3 uL/min to about 8 , about 3 uL/min to about 7 uL/min, about 3 uL/min to about 6 uL/min, about 3 uL/min to about 5 uL/min, about 3 uL/min to about 4 uL/min, about 4 uL/min to about 10 uL/min, about 4 uL/min to about 9 uL/min, about 4 uL/min to about 8 uL/min, about 4 uL/min to about 7 uL/min, about 4 uL/min to about 6 uL/min, about 4 uL/min to about 5 uL/min, about 5 uL/min to about 10 uL/min, about 5 uL/min to about 9 uL/min, about 5 uL/min to about 8 uL/min, about 5 uL/min to about 7 uL/min, about 5 uL/min to about 6 uL/min, about 6 uL/min to about 10 uL/min, about 6 uL/min to about 9 uL/min, about 6 uL/min to about 8 uL/min, about 6 uL/min to about 7 uL/min, about 7 uL/min to about 10 uL/min, about 7 uL/min to about 9 uL/min, about 7 uL/min to about 8 uL/min, about 8 uL/min to about 10 uL/min, about 8 uL/min to about 9 uL/min, or about 9 uL/min to about 10 uL/min. In some embodiments, the composition is administered in incrementai increases in flow rate during delivery (5.6., "stepping"), In some embodiments, administration of the rAAV particle is performed once. In other embodiments, stration of the rAAV particle is performed more than once. One of skill in the art may determine how many times to perform administration of the rAAV particle based in part on, e.g., the disorder being treated and/or the t response to treatment.

In some embodiments, the s comprise administration to CNS an ive amount of recombinant Viral particles to the striatum, wherein the rAAV le comprises a rAAV vector encoding a heterologous c acid that is expressed in at least the al cortex and striatum, In some embodiments, the Viral titer of the rAAV particles is at least about any of 5 x 1012, 6 x 1012, 7 x 1012, 8 x 1012, 9 x 1012, 10 x 1012, 11 x 1012, 15 x 1012, 20 x 1012, 25 x 1012, 30 x 1012, or 50 x 1012 genome copies/mL. In some embodiments, the Viral titer of the rAAV particles is about any of 5 x 1012 to 6 x 1012, 6 x 1012 to 7 x 1012, 7 x 1012 to 8 x1012,8 x 1012 to 9 x 1012, 9 x 1012 to 10 x 1012,10 x 1012 to 11 x 1012, 11 x 1012 to 15 x 1012, 15 x 1012 to 20 x 1012, 20 x 1012 to 25 x 1012, 25 x 1012 to 30 x 1012, 30 x 1012 to 50 x 1012 or 50 x 1012 to 100 x 1012 genome /mL. In some embodiments, the Viral titer of the rAAV particles is about any of 5 x 1012 to 10 x 1012, 10 x 1012 to 25 x 1012, or 25 x 1012 to 50 x lOldenome copies/mL. In some embodiments, the Viral titer of the rAAV particles is at least about any of5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 10 x 109, 11 x 109, 15 x 109, x 109, 25 x 109, 30 x 109, or 50 x 109 transducing units /mL. In some embodiments, the Viral titer of the rAAV particles is about any of 5 x 109 to 6 x 109, 6 x 109 to 7 x 109, 7 x 109 to 8 x109,8 x 109 to 9 x 109, 9 x 109 to 10 x109,10 x 109 to 11 x 109, 11 x 109 to 15 x109, x 109 to 20 x 109, 20 x 109 to 25 x 109, 25 x 109 to 30 x 109, 30 ><109 to 50 x 109 or 50 x 109 to 100 x 109 transducing units /mL. In some ments, the viral titer of the rAAV particles is about any of 5 x 109 to 10 x 109, 10 x 109 to 15 x 109, 15 x 109 to 25 x 109,01' 25 x 109 to 50 x 109 transducing units /mL. In some embodiments, the viral titer of the rAAV les is at least any of about 5 x 1010, 6 x 1010, 7 x 1010, 8 x 1010, 9 x 1010, 10 x 1010, ll x 1010, 15 x 1010, 20 x 1010, 25 x 1010, 30 x 1010, 40 x 1010, or 50 x lo10 infectious units/mL.

In some embodiments, the viral titer of the rAAV particles is at least any of about 5 x 1010 to 6 x 1010, 6 x 1010 to 7 x 1010, 7 x 1010 to 8 x1010,8 x 1010 to 9 x 1010, 9 x 1010 to 10 x 1010, x 1010 to ll x 1010, ll x 1010 to 15 x 1010,15 x 1010 to 20 x 1010, 20 x 1010 to 25 x1010, x 1010 to 30 x 1010, 30 x 1010 to 40 x 1010, 40 x 1010 to 50 x 1010, or 50 x 1010 to 100 x 1010 infectious units/mL. In some embodiments, the viral titer of the rAAV particles is at least any of about 5 x 1010 to 10 x 1010, 10 x 1010 to 15 x 1010, 15 x 1010 to 25 x 1010,01‘ 25 x 1010 to 50 x lo10 infectious units/mL.

In some embodiments, the methods comprise administration to CNS an effective amount of recombinant viral particles to the um, wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum. In some embodiments, the dose of viral les administered to the individual is at least about any of l x 108 to about l x 1013 genome copies/kg of body weight.

In some embodiments, the dose of viral particles stered to the dual is about l x 108 to l x 1013 genome copies/kg of body weight.

In some embodiments, the methods se administration to CNS an effective amount of recombinant viral particles to the striatum, wherein the rAAV particle comprises a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and um. In some embodiments, the total amount of viral particles administered to the individual is at least about l x 109 to about l x 1014 genome copies. In some embodiments, the total amount of viral particles administered to the individual is about l x 109 to about l x 1014 genome copies.

Compositions of the invention (e.g., rAAV particles) can be used either alone or in combination with one or more additional therapeutic agents for treating any or all of the disorders described herein. The interval between sequential stration can be in terms of at least (or, alternatively, less than) minutes, hours, or days.

IV. Expression Constructs In some aspects, the invention provides methods 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 a rAAV vector. The rAAV vector may encode a heterologous nucleic acid, (e.g., a heterologous nucleic acid expressed in at least the cerebral cortex and striatum). rAAV vectors are described in greater detail infra.

In some embodiments, the rAAV vector encodes a heterologous nucleic acid. In some embodiments, a heterologous nucleic acid may encode a therapeutic polypeptide or therapeutic nucleic acid. A therapeutic polypeptide or eutic nucleic acid may be used, for example, to ameliorate a m, prevent or delay progression, and/or provide a treatment of 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.

The heterologous c acid may be expressed in one or more regions of interest within the CNS. For example, in some ments, the heterologous nucleic acid is expressed in at least the cerebral cortex and striatum. The heterologous nucleic acid may be e of sion ubiquitously throughout the CNS, or it may be expressed in a subset of CNS cells.

In some ments, the logous nucleic acid is expressed in the frontal cortex, occipital cortex, and/or layer IV of the mammal. The cerebral cortex is known as the outer layer of the mammalian brain important for language, consciousness, memory, attention, and awareness. The cerebral cortex is subdivided into a number of different components and regions due to its extensive anatomy and x functions. It may be divided into left and right hemispheres. In on, it contains four gross lobes: frontal, parietal, temporal, and occipital. Frontal cortex may refer to the frontal lobe of the cortex and is known to provide a wide range of neurological functions related to non—task—based memory, social interactions, decision making, and other complex cognitive ons.

Occipital cortex may refer to the occipital lobe of the cortex and is known to be ed in visual processing. Parietal cortex may refer to the parietal lobe of the cortex and is known to be involved in language processing, proprioception, and sensory inputs related to touch.

Temporal cortex may refer to the temporal lobe of the cortex and is known to be involved in ge, memory, and emotional association.

In addition, three general types of areas of the cortex are described: sensory, motor, and association. These may be divided into 5 functional subdivisions: primary motor cortex (involved in muscle control), premotor cortex (higher order motor areas that command primary motor areas), association areas (e.g., parietal—temporal—occipital or prefrontal; these areas are involved in ng, memory, attention, and other higher cognitive tasks and assume the majority of the human cortex), higher order areas ry processing), and primary sensory areas (e.g., auditory, visual, and somatosensory). In some embodiments, the heterologous nucleic acid is expressed in the prefrontal association cortical areas, the premotor cortex, the primary somatosensory cortical areas, sensory motor cortex, parietal cortex, occipital cortex, and/or primary motor cortex.

In addition, the cerebral cortex may be divided into different cortical layers (moving from superficial to deep), each containing a characteristic pattern of al connectivities and cell types. These layers may be d into ranular layers s I—III), internal granular (IV), and infragranular (V and VI). ranular layers typically project to other cortical layers, whereas infragranular layers receive input from supragranular layers and send output to structures outside the cortex (e.g., motor, sensory, and thalamic regions). Layer V contains pyramidal neurons with axons that connect to subcortical structures like the basal a. Layer V neurons in the primary motor cortex also form the cortico spinal tract that is critical for voluntary motor control. Layer IV receives inputs from the thalamus and connects to the rest of the column, thereby ing critical functions related to integration of the thalamus and cortex. teristic cells of layer IV include stellate cells (e.g., spiny stellate cells) and pyramidal neurons.

In some embodiments, the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex. Retrograde transport refers to the enon by which cargo (e.g., rAAV particles) is moved from a neuronal s (e.g., an axon) to the cell body. Anterograde transport refers to movement from the cell body to the cell ne (e.g., a synapse). Retrograde transport of AAV particles is thought to occur via receptor— ed internalization at the axon terminal, followed by microtubule—mediated transport to the nucleus (see, e.g., Kaspar er al., (2002) M01. Ther. 5:50—56; Boulis er al., (2003) Neurobiol. Dis. 14:535-541; Kaspar er al., (2003) Science 301:839—842). It is known that the striatum ns projections from other brain regions, such as regions of the cortex. Both anterograde and rade transport may allow rAAV particles to be distributed throughout the brain, such as between the cortex and thalamus (see, e.g., Kells, A.P. er al. (2009) Proc.

Natl. Acad. Sci. 106:2407—2411). Therefore, t wishing to be bound to theory, it is thought that injection of AAV particles into one brain region (e.g., the striatum, caudate nucleus, and/or putamen) may allow the AAV particles to be delivered to other areas of the brain (e.g., the cortex) h retrograde transport.

In some embodiments, the heterologous nucleic acid is r expressed in the thalamus, substantia nigra and/or hippocampus. As described above, mechanisms such as anterograde and/or retrograde transport may allow rAAV particles injected into the cerebral cortex and/or striatum to be distributed to other s of the brain, particularly those that connect to the cortex. The thalamus is between the cortex and in, sends signals (e.g., sensory and motor) to the cortex from subcortical areas, and plays a role in alertness and sleep. The thalamus also connects to the hippocampus, part of the limbic system and a critical mediator of long—term memory consolidation. Part of the basal ganglia, the ntia nigra contains many dopaminergic neurons and is important for movement and reward. CNS disorders like Parkinson’s disease are associated with loss of dopaminergic neurons in the substantia nigra. It further es dopamine to the striatum that is critical for proper striatal function.

In some aspects, the invention provides rAAV vectors for use in methods of preventing or treating one or more gene defects (e.g., heritable gene defects, somatic gene tions, and the like) in a mammal, such as for example, a gene defect that results in a polypeptide ency or polypeptide excess in a subject, or for treating or reducing the severity or extent of ency in a subject manifesting a CNS—associated er linked to a deficiency in such polypeptides in cells and s. In some embodiments, methods involve administration of a rAAV vector that encodes one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, NAs, antisense nucleotides, etc. in a pharmaceutically—acceptable carrier to the subject in an amount and for a period of time sufficient to treat the CNS—associated disorder in the subject having or suspected of having such a disorder.

A rAAV vector may comprise as a transgene, a nucleic acid encoding a protein or functional RNA that modulates or treats a CNS—associated disorder. The following is a non— limiting list of genes associated with CNS—associated ers: neuronal apoptosis inhibitory n (NAIP), nerve growth factor (NGF), glial—derived growth factor (GDNF), brain— derived growth factor (BDNF), ciliary rophic factor (CNTF), tyrosine hydroxlase (TM, GTP—cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase (SODl) and amino acid decarboxylase (AADC). For example, a useful ene in the treatment of Parkinson's disease encodes TH, which is a rate limiting enzyme in the synthesis of dopamine. A transgene encoding GTPCII, which generates the T11 cofactor tetrahydrobiopterin, may also be used in the treatment of Parkinson's e. A transgene ng GDNF or BDNF, or AADC, which facilitates conversion of L—Dopa to DA, may also be used for the treatment of Parkinson's disease. For the treatment of ALS, a useful transgene may encode: GDNF, BDNF or CNTF. Also for the treatment of ALS, a useful transgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibits the expression of SODl. For the treatment of ischemia a useful transgene may encode NAIP or NGF. A transgene ng Beta—glucuronidase (GUS) may be useful for the treatment of certain lysosomal storage diseases (e.g., Mucopolysacharidosis type VII (MPS VID). A transgene ng a prodrug activation gene, e.g., HSV—Thymidine kinase which converts ganciclovir to a toxic nucleotide which disrupts DNA synthesis and leads to cell death, may be useful for treating certain cancers, e. g., when administered in ation with the prodrug. A transgene encoding an endogenous opioid, such a B—endorphin may be useful for treating pain. Other examples of enes that may be used in the rAAV vectors of the invention will be apparent to the skilled artisan (See, e. g., Costantini LC, et al., Gene Therapy (2000) 7, 93—109).

In some embodiments, the heterologous nucleic acid may encode a therapeutic nucleic acid. In some embodiments, a therapeutic nucleic acid may include without limitation an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme. As such, a therapeutic c acid may encode an RNA that when ribed from the nucleic acids of the vector can treat a disorder of the invention (e.g., a disorder of the CNS) by ering with translation or transcription of an al or excess protein associated with a disorder of the invention. For example, the nucleic acids of the invention may encode for an RNA which treats a disorder by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins. Therapeutic RNA sequences e RNAi, small inhibitory RNA (siRNA), micro RNA (miRNA), and/or ribozymes (such as hammerhead and hairpin ribozymes) that can treat disorders by highly ic elimination or reduction of mRNA ng the abnormal and/or excess proteins.

In some embodiments, the heterologous nucleic acid may encode a therapeutic polypeptide. A therapeutic polypeptide may, e.g., supply a polypeptide and/or enzymatic activity that is absent or present at a reduced level in a cell or organism. Alternatively, a therapeutic polypeptide may supply a polypeptide and/or enzymatic activity that indirectly counteracts an imbalance in a cell or organism. For e, a eutic polypeptide for a disorder d to buildup of a metabolite caused by a deficiency in a metabolic enzyme or activity may supply a missing metabolic enzyme or activity, or it may supply an alternate metabolic enzyme or activity that leads to reduction of the metabolite. A therapeutic polypeptide may also be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain—of—function mutation, or whose activity is otherwise misregulated) by acting, e. g., as a dominant—negative polypeptide.

In some ments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS. Without wishing to be bound to theory, it is thought that a therapeutic polypeptide or therapeutic nucleic acid may be used to reduce or eliminate the expression and/or activity of a ptide whose gain—of—function has been associated with a disorder, or to enhance the expression and/or activity of a ptide to complement a deficiency that has been associated with a disorder (e.g., a mutation in a gene whose expression shows similar or d activity). Non—limiting examples of CNS disorders of the invention that may be treated by a eutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes that may be targeted or supplied are provided in parenthesis for each disorder) include stroke (e.g., caspase-3, Beclin], Ask], PAR], HIFIa, PUMA, and/or any of the genes described in Fukuda, A.M. and Badaut, J. (2013) Genes ) 4:435—456), Huntington’s disease (mutant HTT), epilepsy (e.g., SCNIA, NMDAR, ADK, and/or any of the genes described in , D. (2010) Epilepsia 51:1659—1668), Parkinson’s disease (alpha— synuclein), Lou Gehrig’s disease (also known as amyotrophic l sclerosis; SODJ), mer’s disease (tau, amyloid precursor protein), corticobasal degeneration or CBD (tau), corticogasal ganglionic degeneration or CBGD (tau), temporal dementia or FTD (tau), progressive supranuclear palsy or PSP (tau), multiple system y or MSA (alpha— synuclein), cancer of the brain (e. g., a mutant or overexpressed oncogene ated in brain cancer), and mal storage diseases (LSD). Disorders of the invention may include those that involve large areas 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 may be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention e traumatic brain injury, enzymatic dysfunction disorders, psychiatric disorders (including post—traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementias, autism, and depression). Enzymatic dysfunction disorders include t limitation leukodystrophies (including Canavan’s disease) and any of the lysosomal storage diseases bed below.

In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat a lysosomal storage disease. As is commonly known in the art, lysosomal storage disease are rare, inherited metabolic disorders characterized by defects in lysosomal function. Such disorders are often caused by a deficiency in an enzyme required for proper mucopolysaccharide, glycoprotein, and/or lipid metabolism, leading to a pathological accumulation of lysosomally stored cellular materials. Non—limiting examples of lysosomal storage diseases of the invention that may be treated by a eutic polypeptide or therapeutic nucleic acid of the invention lary genes that may be targeted or supplied are provided in parenthesis for each disorder) include Gaucher disease type 2 or type 3 (acid beta—glucosidase, GBA), GMl gangliosidosis (beta—galactosidase—l, GLBI), Hunter disease (iduronate 2—sulfatase, IDS), Krabbe e (galactosylceramidase, GALC), a mannosidosis disease (a mannosidase, such as alpha—D—mannosidase, MANZB] ), B idosis e (beta—mannosidase, MANBA), metachromatic leukodystrophy disease (pseudoarylsulfatase A, ARSA), mucolipidosisII/III disease (N—acetylglucosamine—l—phosphotransferase, GNPTAB), n—Pick A disease (acid sphingomyelinase, ASM), Niemann—Pick C disease (Niemann— Pick C protein, NPC] ), Pompe e (acid alpha—l,4—glucosidase, GAA), Sandhoff disease (hexosaminidase beta subunit, HEXB), Sanfillipo A e (N—sulfoglucosamine sulfohydrolase, MPS3A), Sanfillipo B disease (N—alpha—acetylglucosaminidase, NAGLU), lipo C disease (heparin acetyl—CoA:alpha—glucosaminidase N—acetyltransferase, , Sanfillipo D disease (N—acetylglucosamine—6—sulfatase, GNS), ler disease (alpha—N—acetylgalactosaminidase, NAGA), Sly disease (beta—glucuronidase, GUSB), Tay— Sachs disease (hexosaminidase alpha subunit, HEXA), and Wolman e (lysosomal acid lipase, LIPA).

Additional lysosomal e diseases, as well as the defective enzyme associated with each e, are listed in Table 1 below. In some embodiments, a disease listed in the table below is treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention that complements or otherwise compensates for the corresponding enzymatic defect.

Table 1. Lysosomal storage disorders and associated defective enzymes.

Lysosomal e disease Defective enzyme Aspartylglusoaminuria Aspartylglucosaminidase Fabry Alpha—galactosidase A Infantile Batten Disease (CNLl) Palmitoyl protein terase c Late Infantile Batten Disease Tripeptidyl peptidase (CNL2) Juvenile Batten Disease (CNL3) Lysosomal transmembrane protein Batten, other forms (CNL4—CNL8) multiple gene products Cystinosis Cysteine transporter Farber Acid ceramidase Fucosidosis Acid alpha—L—fucosidase Galactosidosialidosis tive n/cathep sin A Gaucher types 1, 2, and 3 Acid beta—glucosidase GMl gangliosidosis Acid beta—galactosidase Hunter Iduronate—2—sulfatase Hurler—Scheie Alpha—L—iduronidase Krabbe Galactocerebrosidase alpha—mannosidosis Acid mannosidase beta—mannosidosis Acid beta—mannosidase MaroteauX—Lamy Arylsulfatase B Metachromatic leukodystrophy Arylsulfatase A Morquio A N—acetylgalactosamine—6—sulfate Morquio B Acid beta—galactosidase Mucolipidosis lI/III N—acetylglucosamine— l—phosphotransferase n—Pick A, B Acid sphingomyelinase n—Pick C NPC—l Pompe acid alpha—glucosidase Sandhoff beta—hexosaminidase B Sanfilippo A Heparan N—sulfatase Sanfilippo B alpha—N—acetylglucosaminidase Sanfilippo C Acetyl—CoA:alpha—glucoasaminide N— acetyltransferase Sanfilippo D N—acetylglucosamine—6—sulfate Schindler disease alpha—N—acetylgalactosaminidase Schindler—Kanzaki alpha—N—acetylgalactosaminidase Sialidosis alpha—neuramidase Cl) _ beta—glucuronidase Tay—S achs exosaminidase A Wolman Acid lipase As such, in some embodiments, the therapeutic polypeptide is caspase—3, Beclinl, Askl, PARl, , PUMA,SCNlA, NMDAR, ADK, alpha—synuclein, SODl, acid beta— glucosidase (GBA), beta—galactosidase—l (GLB l), iduronate 2—sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha—D—mannosidase (MANZB 1), beta— mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N—acetylglucosamine—l— phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann—Pick C protein (NPCl), acid alpha—l,4—glucosidase (GAA), minidase beta subunit, HEXB, N— sulfoglucosamine sulfohydrolase (MPS3A), N—alpha—acetylglucosaminidase (NAGLU), heparin acetyl—CoA, alpha—glucosaminidase N—acetyltransferase (MPS 3C), N— glucosamine—6—sulfatase (GNS), N—acetylgalactosaminidase (NAGA), beta— glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), or lysosomal acid lipase (LIPA). The therapeutic polypeptide may increase or decrease the function of the target polypeptide in the subject (e.g., it may supply the g function in a lysosomal e disease, or reduce the level of alpha—synuclein in MSA, such as by blocking its function or dysfunction). In some embodiments, the therapeutic nucleic acid is caspase—3, Beclinl, Askl, PARl, HIFlOt, CNlA, NMDAR, ADK, alpha—synuclein, SODl, acid beta—glucosidase (GBA), alactosidase—l (GLB l), iduronate 2—sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha—D—mannosidase (MANZB l), beta—mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N—acetylglucosamine—l— phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann—Pick C n (NPCl), acid alpha—l,4—glucosidase (GAA), hexosaminidase beta subunit, HEXB, N— lucosamine sulfohydrolase (MPS3A), N—alpha—acetylglucosaminidase (NAGLU), heparin acetyl—CoA, alpha—glucosaminidase yltransferase (MPS 3C), N— acetylglucosamine—6—sulfatase (GNS), alpha—N—acetylgalactosaminidase (NAGA), beta— glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), or lysosomal acid lipase . The therapeutic nucleic acid may increase or decrease the on of the target polypeptide in the subject (e.g., it may supply the missing function in a lysosomal e disease, or reduce the level of synuclein in MSA, such as by RNAi).

An exemplary disease for which AAV expression in the cortex and um may be useful is Huntington’s disease (HD). Huntington’s disease is caused by a CAG repeat expansion mutation that encodes an elongated polyglutamine (polyQ) repeat in the mutant huntingtin protein (mHTT). HD is a particularly attractive target for DNA— and RNA—based therapies as it is an autosomal dominant disease resulting from mutation on a single allele.

AAV vectors provide an ideal delivery system for nucleic acid eutics and allow for long lasting and continuous expression of these huntingtin lowering molecules in the brain.

To achieve maximal clinical efficacy in HD, delivery to both the striatum and cortex will likely be required. Postmortem analysis of HD patient brains revealed extensive medium spiny neuronal loss in the striatum, in addition to loss of pyramidal neurons in the cerebral cortex and hippocampus. It was recently shown using conditional enic mouse models of HD that genetically reducing mHTT expression in neuronal populations in the striatum and cortex provides significantly more efficacy than reducing mHTT in either site alone (Wang et al., (2014) Nature medicine 20:536—541). er, this evidence suggests that delivery of gene therapy agents to both al and cortical regions may be ideal for maximal therapeutic efficacy.

The use of gene therapy vectors to deliver biologics to critical brain regions implicated in Huntington’s disease pathogenesis has been a challenge, due in large part to the physical constraints of effectively delivering a vector specifically to the striatum and the cerebral cortex. Although multiple direct infusions can be effective in small animal brains, as the architecture and volume of brain tissue increases in primates, it becomes more difficult to achieve widespread striatal and cortical delivery through single site infusion. Therefore, the inventors’ discovery that striatal administration can achieve read rAAV distribution, including the cortex and striatum, has utility in treating Huntington’s disease.

Accordingly, certain s of the invention relate to methods for treating gton’s disease in a mammal comprising administering a rAAV particle to the striatum, wherein the rAAV particle ses a rAAV vector encoding a logous nucleic acid that is expressed in at least the cerebral cortex and um of the mammal. HD is characterized by ssive symptoms related to overall movement and motor control, cognition, and mental health. While the precise nature and extent of symptoms vary between individuals, symptoms generally progress over time. In most cases, symptoms begin to appear between 30 and 40 years of age with subtle disruptions in motor skills, cognition, and personality. Over time, these progress into jerky, uncontrollable movements and loss of muscle control, dementia, and atric illnesses such as depression, sion, anxiety, and obsessive—compulsive behaviors. Death typically occurs 10—15 years after the onset of symptoms. Less than 10% of HD cases involve a juvenile—onset form of the disease, characterized by a faster disease progression. It is thought that approximately 1 in 10,000 Americans has HD.

Most cases of HD are associated with a trinucleotide CAG repeat expansion in the HTT gene. The number of CAG repeats in the HTT gene is strongly correlated with the manifestation of HD. For example, individuals with 35 or fewer repeats typically do not develop HD, but individuals with between 27 and 35 repeats have a greater risk of having offspring with HD. Individuals with between 36 and 40—42 repeats have an incomplete penetrance of HD, whereas individuals with more than 40—42 repeats show complete penetrance. Cases of juvenile—onset HD may be associated with CAG repeat sizes of 60 or more .

The polyQ—expanded Htt protein resulting from this CAG repeat ion is ated with cellular aggregates or ion bodies, perturbations to protein homeostasis, and transcriptional dysregulation. While these toxic ypes may be ated with several parts of the body, they are most typically ated with neuronal cell death. HD patients often display cortical thinning and a striking, progressive loss of striatal neurons.

The um appears to be the most vulnerable region of the brain to HD (particularly the striatal medium spiny neurons), with early effects seen in the n and caudate nucleus.

Cell death in the striatal spiny neurons, increased numbers of astrocytes, and activation of microglia are observed in the brains of HD patients. HD may also affect certain regions of the hippocampus, cerebral cortex, thalamus, alamus, and cerebellum.

Animal models of HD may be used to test potential therapeutic gies, such as the compositions and methods of the present disclosure. Mouse models for HD are known in the art. These include mouse models with fragments of mutant HTT such as the R6/l and Nl7l—82Q HD mice r er al., (2005) Proc. Natl. Acad. Sci. USA 102:5820—5825, uez—Lebron et al., (2005) M01. Ther. 12:618—633, Machida et al., (2006) Biochem.

Biophys. Res. Commun. 343: 190—197). Another e of a mouse HD model described herein is the YACl28 mouse model. This model bears a yeast artificial chromosome (YAC) expressing a mutant human HTT gene with 128 CAG repeats, and YACl28 mice exhibit significant 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. M01.

Genet. 2l:22l9—2232).

Other animal models for HD may also be used. For example, transgenic rat (von Horsten, S. et al. (2003) Hum. M01. Genet. —24) and rhesus monkey (Yang, S.H. et al. (2008) Nature 453:921—4) models have been described. Non—genetic models are also known.

These most often involve the use of excitotoxic compounds (such as quinolinic acid or kainic acid) or mitochondrial toxins (such as 3—nitropropionic acid and malonic acid) to induce striatal neuron cell death in rodents or non—human primates (for more description and references, see Ramaswamy, S. et al. (2007) ILAR J. —73).

In some s, the invention es methods for ameliorating a symptom of HD, comprising administration of a rAAV particle sing a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum to the striatum. In some embodiments, the symptoms of HD include, but are not d to, chorea, rigidity, uncontrollable body movements, loss of muscle control, lack of coordination, restlessness, slowed eye movements, abnormal posturing, instability, ataxic gait, abnormal facial expression, speech problems, difficulties chewing and/or swallowing, disturbance of sleep, seizures, dementia, cognitive deficits (e. g., diminished abilities related to planning, abstract thought, flexibility, rule acquisition, interpersonal sensitivity, self—control, attention, learning, and memory), depression, anxiety, changes in ality, aggression, sive behavior, obsessive—compulsive behavior, hypersexuality, sis, apathy, irritability, suicidal thoughts, weight loss, muscle y, heart failure, reduced glucose tolerance, testicular atrophy, and osteoporosis.

In some s, the invention provides methods to prevent or delay progression of HD. Autosomal dominant HD is a genetic disease that can be genotyped. For example, the number of CAG repeats in HTT may be determined by sed repeat sizing. This type of diagnosis may be performed at any stage of life through directly testing juveniles or adults (e. g., along with presentation of al symptoms), prenatal screening or prenatal exclusion testing (e. g., by chorionic villus sampling or amniocentesis), or preimplantation screening of embryos. Additionally, HD may be diagnosed by brain imaging, g for shrinkage of the caudate nuclei and/or putamen and/or enlarged ventricles. These symptoms, ed with a family history of HD and/or clinical symptoms, may indicate HD.

Means for determining amelioration of the symptoms of HD are known in the art.

For example, the Unified Huntington’s Disease Rating Scale (UHDRS) may be used to assess motor function, cognitive function, behavioral abnormalities, and functional capacity (see, e. g., Huntington Study Group (1996) Movement Disorders 11:136—42). This rating scale was developed to provide a uniform, comprehensive test for multiple facets of the e pathology, incorporating elements from tests such as the HD Activities and Daily Living Scale, Marsden and Quinn’s chorea severity scale, the Physical Disability and Independence scales, the HD motor rating scale ), the HD functional ty scale (HDFCS), and the quantitated neurological exam (QNE). Other test useful for ining amelioration of HD symptoms may include Without limitation 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 the Symbol Digit Modalities Test.

In some aspects of the invention, the methods are used for the treatment of humans with HD. As described above, HD is inherited in an mal dominant manner and caused by CAG repeat expansion in the HTT gene. rAAV particles may include, e. g., a heterologous nucleic acid encoding a therapeutic polypeptide or nucleic acid that targets HTT. Juvenile— onset HD is most often ted from the paternal side. Huntington disease—like phenotypes have also been correlated with other genetic loci, such as HDL], PRNP, HDL2, HDL3, and HDL4. It is thought that other genetic loci may modify the manifestation of HD symptoms, including mutations in the GRINZA, GRINZB, MSX], GRIKZ, and APOE genes.

In some embodiments, delivery of recombinant viral particles is by injection of viral les to the striatum. Intrastriatal stration delivers recombinant viral particles to an area of the brain, the striatum (including the n and caudate nucleus), that is highly affected by HD. In addition, and Without Wishing to be bound to theory, it is thought that recombinant viral particles (e.g., rAAV particles) injected into the striatum may be also dispersed (e. g., through retrograde transport) to other areas of the brain, including t limitation projection areas (e.g., the cerebral ). In some ments, the recombinant viral particles are delivered by convection enhanced delivery (e.g., tion enhanced delivery to the striatum).

In some embodiments, the transgene (e.g., 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, a simian virus 40 (SV40) promoter and a CK6 promoter, a transthyretin er (TTR), a TK promoter, a tetracycline responsive promoter (TRE), an HBV promoter, an hAAT promoter, a LSP promoter, chimeric liver—specific promoters (LSPs), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer/chicken beta—actin/Rabbit B—globin promoter (CAG promoter; Niwa et 611., Gene, 1991, : 193—9) and the elongation factor l—alpha promoter (EFl—alpha) promoter (Kim et al., Gene, 1990, 91(2):217—23 and Guo et al., Gene Ther., 1996, 3(9):802—10). In some embodiments, the promoter comprises a human B— glucuronidase er or a cytomegalovirus enhancer linked to a chicken B—actin (CBA) promoter. The er can be a constitutive, inducible or repressible promoter. In some embodiments, the invention provides a recombinant vector comprising nucleic acid encoding a logous nucleic acid of the present disclosure operably linked to a CBA promoter. In some embodiments, the er is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. es of constitutive ers include, Without limitation, 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., t 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 EFla promoter rogen].

Inducible ers allow regulation of gene sion and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific logical state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and ble systems are ble from a variety of commercial sources, including, Without limitation, 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 regulated by exogenously supplied promoters include the zinc—inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)—inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ne insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346— 3351 (1996)), the tetracycline—repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 7—5551 ), the ycline—inducible system (Gossen et al., Science, 268:1766— 1769 (1995), see also 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)). Still other types of ble promoters which may be useful in this context are those which are ted by a specific logical state, e. g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter, or fragment thereof, for the ene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue— specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue—specific gene expression capabilities. In some cases, the tissue—specific regulatory sequences bind 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 —specific regulatory sequences include, but are not limited to the following tissue specific promoters: neuronal such as neuron—specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol, —15 (1993)), 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 nuclei (NeuN), glial fibrillary acidic protein , adenomatous polyposis coli (APC), and d calcium—binding adapter le 1 (Iba—l). Other appropriate tissue specific promoters will be apparent to the skilled artisan. In some ments, the promoter is a chicken Beta—actin promoter.

In some embodiments, the promoter expresses the heterologous nucleic acid in a cell of the CNS. As such, in some embodiments, a therapeutic ptide or a therapeutic nucleic acid of the ion may be used to treat a disorder of the CNS. In some embodiments, the promoter expresses the heterologous nucleic acid in a brain cell. A brain cell may refer to any brain cell known in the art, including Without limitation a neuron (such as a sensory neuron, motor neuron, interneuron, dopaminergic neuron, medium spiny neuron, cholinergic neuron, GABAergic neuron, pyramidal neuron, etc), a glial cell (such as microglia, macroglia, astrocytes, oligodendrocytes, ependymal cells, radial glia, etc), a brain parenchyma cell, microglial cell, ependymal cell, and/or a Purkinje cell. In some embodiments, the promoter expresses the heterologous nucleic acid in a neuron and/or glial cell. In some embodiments, the neuron is a medium spiny neuron of the caudate s, a medium spiny neuron of the putamen, a neuron of the cortex layer IV and/or a neuron of the cortex layer V.

Various ers that express transcripts (e.g., a heterologous ene) in CNS cells, brain cells, neurons, and glial cells are known in the art and described . Such promoters can comprise control sequences normally associated with the selected gene or heterologous control sequences. Often. useful heterologous control sequences include those derived from sequences encoding mammalian or viral genes. Examples e, without limitation, the SVé’il) early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late prrnnoter (Ad half), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the (ills/EV ate early promoter region (CM VIE), a rous sarcoma Virus (RSV) promoter, synthetic promoters, hybrid promoters. and the like. In addition, sequences d from nonviral genes, such as the murine metallotliionein gene, may also be used. Such promoter sequences are commercially available from, egg, Stratagene (San Diego, CA). CNS—specific promoters and inducible promoters may be used.

Examples of Cl‘sS specific promoters include t limitation those ed from CNS- specific genes such as myelin basic protein (MBP), glial ?brillary acid protein , and neuron specific enolase (NSE). Examples of inducible promoters include DNA responsive elements for ecdysonei tetracycline, metallothionein, and hypoxia, inter alia.

The present invention plates the use of a recombinant viral genome for introduction of one or more nucleic acid sequences encoding for a logous nucleic acid or packaging into an AAV viral particle. The recombinant viral genome may include any element to establish the expression of a heterologous transgene, for example, a promoter, a heterologous c acid, an ITR, a ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal, and/or origin of replication. In some ments, 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 administration of an effective amount of rAAV particles sing 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., the 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 neurons are transduced. In some ments, 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 uced.

Methods to fy neurons transduced by recombinant viral particles expressing miRNA are known in the art; for example, immunohistochemistry, RNA ion (e.g., qPCR, Northern blotting, q, in situ hybridization, and the like) or the use of a co—expressed marker such as enhanced green ?uorescent protein can be used to detect expression.

In some aspects, the invention es viral particles comprising a recombinant self—complementing genome (e.g., a self—complementary rAAV vector). AAV viral les with self—complementing vector genomes and methods of use of self—complementing AAV genomes are described in US Patent Nos. 6,596,535; 717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang 2., et al., (2003) Gene Ther :2105—2111, each of Which are incorporated herein by reference in its entirety. A rAAV comprising a self—complementing genome Will quickly form a double stranded DNA le by virtue of its partially complementing sequences (e.g., complementing coding and non—coding strands of a heterologous nucleic acid). In some embodiments, the vector comprises first nucleic acid sequence encoding the heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the nucleic acid, Where the first nucleic acid sequence can form intrastrand base pairs with the second c acid sequence along most or all of its length.

In some embodiments, the first heterologous nucleic acid sequence and a 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’— CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG — 3’ (SEQ ID NO: 1).

The mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence. As a result, on replicating an AAV viral genome, the rep proteins will not cleave the viral genome at the mutated ITR and as such, a recombinant viral genome comprising the following in 5' to 3' order will be packaged in a viral capsid: an AAV ITR, the first heterologous polynucleotide ce including regulatory sequences, the mutated AAV ITR, the second heterologous cleotide in reverse orientation to the first heterologous polynucleotide and a third AAV ITR.

V. Viral particles and methods of producing viral particles rAAV viral particles The invention provides methods and systems for administering rAAV particles. In some ments, the rAAV le comprises a rAAV vector. In some embodiments, the viral particle is a inant AAV particle comprising a nucleic acid comprising a heterologous nucleic acid ?anked by one or two AAV inverted terminal repeats . The nucleic acid is encapsidated in the AAV particle. The AAV particle also comprises capsid proteins. In some embodiments, the nucleic acid comprises the coding sequence(s) of interest (e. g., a heterologous nucleic acid) ively linked components in the direction of transcription, control sequences including transcription initiation and termination sequences, thereby forming an expression cassette. The expression cassette is ?anked on the 5' and 3' end by at least one functional AAV ITR sequence. By "functional AAV ITR ce" it is meant that the ITR sequence functions as intended for the , replication and packaging of the AAV virion. See Davidson et al., PNAS, 2000, 97(7)3428—32; Passini et al., J. , 2003, 77(12):7034—40; and Pechan et 611., Gene Ther., 2009, 16:10—16, all of which are incorporated herein in their entirety by reference. For practicing some aspects of the invention, the inant vectors comprise at least all of the sequences of AAV essential for encapsidation and the al ures for infection by the rAAV. AAV ITRs for use in the vectors of the invention need not have a wild—type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793—801), and may be altered by the insertion, deletion or substitution of nucleotides or the AAV ITRs may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are tly known, 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. Use of any AAV serotype is considered Within the scope of the present invention. In some embodiments, a rAAV vector is a vector derived from an AAV serotype, including Without tion, AAV ITRs are AAVl, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAV10, AAVrth, AAVl l, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV or the like. In some embodiments, the c acid in the AAV ITRs are AAVl, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAV10, AAVrth, AAVl l, AAV12, 71A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.

In certain embodiments, the c acid in the AAV ses an AAV2 ITR.

In some embodiments, a vector may include a r nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green ?uorescent protein. In some embodiments, the stuffer nucleic acid may be located between the promoter and the nucleic acid encoding the RNAi.

In r embodiments, the rAAV particles comprise an AAVl capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 , an AAVS capsid, an AAV6 capsid (e.g., a Wild— type AAV6 capsid, or a variant AAV6 capsid such as ShHlO, as described in U.S. PG Pub. 2012/0164106), an AAV7 capsid, an AAVS capsid, an AAVrhS capsid, an AAVrhSR capsid, an AAV9 capsid (e.g., a Wild—type AAV9 capsid, or a modified AAV9 capsid as described in U.S. PG Pub. 2013/0323226), an AAVlO , an AAVrth capsid, an AAVll capsid, an AAVl2 capsid, a tyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R47 lA capsid, an AAVAAV2/2—7m8 capsid, an AAV DJ capsid (e.g., an AAV—DJ/S , an AAV—DJ/9 capsid, or any other of the capsids described in U.S. PG Pub. 2012/0066783), an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAVl/AAV2 chimeric capsid, a bovine AAV , a mouse AAV capsid, a rAAV2/HBon capsid, or an AAV capsid bed in U.S. Pat. No. 8,283,151 or International Publication No. WO/2003/042397. In some embodiments, a mutant capsid protein maintains the ability to form an AAV capsid. In some embodiments, the rAAV particle comprises AAVS tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci U S A 105(22):7827—7832. In further embodiments, the rAAV le comprises capsid proteins of an AAV serotype from Clades A—F (Gao, et al., J. Virol. 2004, 78(12):6381). In some embodiments, the rAAV particle comprises an AAVl capsid protein or mutant thereof. In other embodiments, the rAAV particle comprises an AAV2 capsid protein or mutant thereof. In some embodiments, the AAV serotype is AAVl, AAV2, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, or AAVrth. In some embodiments, the rAAV le comprises an AAV serotype 1 (AAVl) capsid. In some embodiments, the rAAV particle comprises an AAV serotype 2 (AAV2) capsid.

Different AAV serotypes are used to optimize transduction of particular target cells or to target specific cell types within a particular target tissue (e.g., a CNS tissue). A rAAV particle can se viral proteins and viral nucleic acids derived from the same pe or different serotypes (e.g., a mixed serotype). For e, in some embodiments a rAAV particle can comprise AAVl capsid proteins and at least one AAV2 ITR or it can comprise AAV2 capsid proteins and at least one AAVl ITR. Any combination of AAV serotypes for tion of a rAAV particle is provided herein as if each combination had been expressly stated herein. In some embodiments, the invention provides rAAV particles comprising an AAVl capsid and a rAAV vector of the present disclosure (e.g., an expression cassette sing a heterologous c acid), ?anked by at least one AAV2 ITR. In some embodiments, the invention provides rAAV particles comprising an AAV2 capsid. In some embodiments, the ITR and the capsid are derived from AAV2. In some embodiments, the ITR is derived from AAV2 and the capsid is derived from AAVl.

Production ofAAVparticles us methods are known in the art for production of rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus—AAV hybrids, herpesvirus—AAV hybrids (Conway, JE et al., (1997) J.

Virology 7l(l l):8780—8789) and baculovirus—AAV hybrids. rAAV production cultures for the production of rAAV virus particles all require; 1) suitable host cells, including, for example, human—derived cell lines such as HeLa, A549, or 293 cells, or —derived cell lines such as SF—9, in the case of baculovirus tion systems; 2) suitable helper virus function, provided by ype or mutant irus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a nucleic acid (such as a therapeutic nucleic acid) ?anked by at least one AAV ITR sequences ; and 5) suitable media and media components to support rAAV production. In some embodiments, the AAV rep and cap gene products may be from any AAV pe. In l, but not obligatory, the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome as long as the rep gene products may function to replicated and package the rAAV genome. Suitable media known in the art may be used for the production of rAAV s. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf—900 II SFM media 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 tion of recombinant AAV vectors. In some embodiments, the AAV helper functions are provided by adenovirus or HSV. In some embodiments, the AAV helper functions are ed by baculovirus and the host cell is an insect cell (e. g., Spodopterafrugiperda (Sf9) .

In some embodiments, rAAV les may be produced by a triple ection method, such as the exemplary triple transfection method provided infra. Brie?y, a plasmid containing a rep gene and a capsid gene, along with a helper adenoviral plasmid, may be transfected (e. g., using the calcium phosphate method) into a cell line (e. g., HEK—293 cells), and virus may be collected and optionally purified. As such, in some ments, the rAAV particle was produced by triple transfection of a nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a c acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV particles.

In some embodiments, rAAV particles may be produced by a producer cell line method, such as the exemplary producer cell line method provided infra (see also (referenced in Martin et al., (2013) Human Gene Therapy Methods 24:253—269). Brie?y, a cell line (e.g., a HeLa cell line) may be stably transfected with a d containing a rep gene, a capsid gene, and a promoter—heterologous nucleic acid sequence. Cell lines may be screened to select a lead clone for rAAV production, which may then be expanded to a production bioreactor and infected with an adenovirus (e.g., a wild—type adenovirus) as helper to initiate rAAV production. Virus may subsequently be harvested, adenovirus may be vated (e. g., by heat) and/or removed, and the rAAV particles may be purified. As such, in some embodiments, the rAAV le was produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.

In some aspects, a method is provided for producing any rAAV particle as disclosed herein comprising (a) culturing a host cell under a condition that rAAV les are produced, wherein the host cell comprises (i) one or more AAV package genes, wherein each said AAV packaging gene encodes an AAV replication and/or encapsidation protein; (ii) a rAAV pro—vector comprising a nucleic acid encoding a heterologous nucleic acid as described herein ?anked by at least one AAV ITR, and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell. In some embodiments, said at least one AAV ITR is selected from the group consisting of AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.

In some embodiments, said encapsidation protein is selected from the group consisting of AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, AAVrhSR, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAVl/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAVZ/HBon serotype capsid proteins or mutants thereof. In some ments, the encapsidation protein is an AAVS capsid protein including AAVS capsid ns having tyrosine capsid mutations. In some embodiments, the encapsidation protein is an AAVS capsid protein including AAVS capsid proteins having ne capsid mutations and the ITR is an AAV2 ITR. In r embodiments, the rAAV particle comprises capsid proteins of an AAV serotype from Clades A—F. In some embodiments, the rAAV particles se an AAVl capsid and a recombinant genome sing AAV2 ITRs, a mutant AAV2 ITR and c acid encoding a therapeutic transgene/nucleic acid. In some ments, the AAV ITRs are AAV ITRs are AAVl, AAVZ, AAV3, AAV4, AAVS, AAV6, AAV7, AAVS, AAVrhS, R, AAV9, AAVlO, AAVrth, AAVl l, AAVlZ, AAV2R47lA, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In certain embodiments, the AAV ITRs are AAV2 ITRs. le rAAV tion culture media of the present invention may be supplemented with serum or serum—derived recombinant proteins at a level of 0.5%—20% (v/v or w/v). Alternatively, as is known in the art, rAAV vectors may be produced in serum—free conditions which may also be referred to as media with no animal—derived products. One of ordinary skill in the art may appreciate that commercial or custom media designed to support production of rAAV vectors may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in tion cultures. rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) le to the particular host cell being utilized. As is known in the art, rAAV production cultures include attachment— dependent cultures which can be cultured in suitable attachment—dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed—bed or ?uidized— bed bioreactors. rAAV vector tion cultures may also e sion—adapted host cells such as HeLa, 293, and SF—9 cells which can be cultured in a variety of ways including, for example, spinner ?asks, stirred tank bioreactors, and disposable systems such as the Wave bag system. rAAV vector particles of the invention may be harvested from rAAV production cultures by lysis of the host cells of the tion e or by harvest of the spent media from the tion culture, provided the cells are cultured under conditions known in the art to cause release of rAAV les into the media from intact cells, as described more fully in US. Patent No. 6,566,118). Suitable methods of 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.

In a further embodiment, the rAAV particles are purified. The term "purified" as used herein includes a preparation of rAAV particles devoid of at least some of the other components that may also be present where the rAAV particles naturally occur or are initially prepared from. Thus, for e, isolated rAAV les may be ed using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase—resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity, or it can be measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, including production culture contaminants or in—process contaminants, including helper virus, media components, and the like.

In some embodiments, the rAAV production e harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is ied by tion through a series of depth filters including, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade AlHC Millipore Millistak+ HC Pod Filter, and a 0.2 um Filter Opticap XLlO Millipore s SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any ose acetate filter of 0.2 um or greater pore size known in the In some embodiments, the rAAV production culture harvest is further treated with ase® to digest any high molecular weight DNA present in the production culture. In some embodiments, the Benzonase® digestion is performed under standard conditions known in the art including, for example, 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. rAAV particles may be isolated or ed using one or more of the following purification steps: equilibrium centrifugation; flow—through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat vation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method comprises all the steps in the order as bed below. Methods to purify rAAV particles are found, for example, in Xiao er al., (1998) Journal of Virology 72:2224—2232; US Patent Numbers 6,989,264 and 8,137,948; and In some embodiments, the rAAV particle is in a pharmaceutical composition. The pharmaceutical compositions may be suitable for any mode of administration described . A pharmaceutical ition of a recombinant viral le comprising a nucleic acid ng a therapeutic transgene/nucleic acid can be introduced to the CNS (e.g., the striatum and/or cerebral cortex).

In some embodiments, the rAAV particle is in a pharmaceutical composition comprising a pharmaceutically acceptable excipient. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients e but are not limited to stabilizing agents, wetting and emulsifying , salts for varying osmolarity, encapsulating agents, pH buffering substances, and buffers. Such excipients include any pharmaceutical agent suitable for direct delivery to the eye which may 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, ol and ethanol. Pharmaceutically acceptable salts can be included therein, for e, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as es, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON’S CEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991).

In some embodiments, the stered composition includes rAAV particles and poloxamer. The term "poloxamer" may encompass many compounds e different lengths for the polyoxypropylene and polyoxyethylene chains may be used in combination.

For example, a mer may have the chemical formula of HO(C2H4O)H(C3H6O)m(C2H4O)nH, where n (i.e., the polyoxyethylene chain length) has a value from about 60 to about 150, and m (i.e., the polyoxypropylene chain length) has a value from about 25 to about 60.

In some embodiments, the mer is poloxamer 188 (e.g., CAS No. 9003—11—6).

Poloxamers may be described by a numbering system that designates their imate molecular weight and percentage of polyoxyethylene content. These values often refer to an average value in a poloxamer composition, rather than an absolute value of each poloxamer le in the composition. Under this ology, the first two digits are multiplied by 100 to give the approximate molecular weight of the polyoxypropylene block, and the third digit is multiplied by 10 to give the percentage by weight of the polyoxyethylene block. For example, poloxamer 188 may refer to a poloxamer with n having a value of about 80 and with m having a value of about 27 as in the formula depicted above. Poloxamer 188 may have an average molecular weight of from about 7680 to about 9510 g/mol.

Poloxamers sold under a trade name such as PLURONIC® may be named under a different methodology. A letter may be used to indicate the al state (e.g., F for solid, P for paste, or L for liquid). A 2 or 3 digit number may be used to indicate the chemical properties. The first one or two digits are multiplied by 300 to give the approximate molecular weight of the polyoxypropylene block, and the third digit is multiplied by 10 to give the percentage by weight of the yethylene block. For example, IC® or LUTROL® F68 may refer to a solid poloxamer with n having a value of about 80 and with m having a value of about 27 as in the formula depicted above. Therefore, in some embodiments, the poloxamer 188 may be PLURONIC® F68 or LUTROL® F68.

In some embodiments, the concentration of poloxamer in the composition ranges from about % 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 r than about any of the ing percentages: 0.0001, 0.0005, 0.001, or 0.005. That is, the concentration of poloxamer in the composition can be any of a range of percentages having 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 , wherein the lower limit is less than the upper limit. In certain ments, the concentration of poloxamer in the composition is about 0.001%.

In some embodiments, the composition further ses sodium chloride. In some embodiments, the concentration of sodium chloride in the composition ranges from about 100 mM to about 250 mM. In some embodiments, the concentration of sodium de in the composition is less than about any of the following concentrations (in mM): 250, 225 , 200, 175, 150, or 125. In some ments, 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 can 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 or 225 wherein , 200, , the lower limit is less than the upper limit. In certain embodiments, the concentration of sodium chloride in the composition is about 180 mM.

In some embodiments, the composition further comprises sodium phosphate.

Sodium phosphate may refer to any single species of sodium phosphate (e.g., sic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, and so forth), or it may refer to sodium phosphate , a mixture of monobasic and dibasic sodium phosphate solutions. Recipes for sodium ate buffers across a range of pH may be found in a variety of rd molecular biology protocols, such as the Promega Protocols & Applications Guide, "Buffers for Biochemical Reactions," Appendix B part C.

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 ate in the composition is less than about any of the following concentrations (in mM): 20, 15, or 10. In some embodiments, the tration of sodium phosphate in the ition 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 can 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, wherein the lower limit is less than the upper limit. In certain embodiments, the concentration of sodium phosphate in the composition is about 10 mM.

In some embodiments, the pH of sodium phosphate in the composition is about 7.0 to about 8.0. For e, in some embodiments, the pH of sodium ate 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 sodium phosphate in the composition is about 7.5. Any of the pH values for sodium phosphate bed herein may be combined with any of the concentration values for 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.

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

Such pharmaceutically acceptable rs can be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable or tic origin, such as peanut oil, soybean oil, l oil, and the like. Saline solutions and aqueous se, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may further comprise additional ingredients, for example preservatives, buffers, ty agents, antioxidants and stabilizers, nonionic wetting or clarifying , viscosity—increasing agents, and the like. The pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms. The compositions are generally formulated as sterile and substantially isotonic solution.

VI. Systems for delivery of rAAV particles Also ed are systems for expression of a heterologous nucleic acid in the al cortex and striatum of a mammal, comprising (a) a composition comprising rAAV particles, wherein the rAAV particles comprise a rAAV vector encoding the heterologous nucleic acid; and (b) a device for delivery of the rAAV particles to the striatum. The s and devices of the invention may be used to deliver any of the rAAV particles bed herein to the CNS (e. g., the striatum) of a mammal. As described above, a rAAV particle delivered to the striatum may be used to introduce a rAAV vector ng a heterologous nucleic acid for expression in the al cortex and striatum.

In some embodiments, the rAAV le is delivered by convection enhanced delivery (CED). CED is based on pumping an infusate (e.g., a composition containing a rAAV particle) into the CNS under re in which the hydrostatic pressure of the interstitial ?uid is overcome. This brings the infusate into contact with the CNS sculature, which is utilized like a pump to distribute the infusate through convection and enhance the extent of its delivery (see, e. g., Hadaczek et al., (2006) Hum. Gene Ther. 17:291—302; Bankiewicz et al., (2000) Exp. Neurol. 164:2—14; Sanftner, LM et al., (2005) Exp. Neurol. l94(2):476—483; Forsayeth, JR et al., (2006) M01. Ther. l4(4):57l—577; US.

Pat. No. 6,953,575; US. Pat. App. Pub. No. 2002/0141980; US. Pat. App. Pub. No. 2007/0259031; WO 99/61066; and As described herein, an advantage of nsing CED is the enl'ianced distribution of the infusate throughout the brain. CED may result in improved delivery at the site of injection within the brain (eg. the striatum, eaudate nucleus, and/or putamen). In addition, delivery to other regions of the brain (e. g., the al cortex, frontal cortex, prefrontal association cortical areas, premotor cortex, primary somatosensory cortical areas, and/or primary motor cortex) may be achieved h CED. Without wishing to be bound to theory, it is also thought that inant viral les (e.g., rAAV particles) injected into the striatum may be also dispersed (e.g., through retrograde ort) to other areas of the brain, including without limitation projection areas (e.g., the cortex).

In some embodiments, the rAAV particle is delivered using a CED delivery system.

AAV particles may be red by CED (see, e.g., WO 99/61066). As bed herein, CED may be accomplished using any of the s described herein. Devices for CED (e.g., for delivery of a composition ing rAAV les) are known in the art and generally employ a pump (e.g., an osmotic and/or infusion pump, as described below) and an injection device (e.g., a catheter, cannula, etc). Optionally, an imaging technique may be used to guide the injection device and/or monitor delivery of the infusate (e.g., a composition including rAAV particles). The injection device may be inserted into the CNS tissue in the subject. One of skill in the art is able to determined suitable coordinates for positioning the injection device in the target CNS tissue. In some embodiments, positioning is accomplished through an anatomical map obtained for e by CT and/or MRI imaging of the t’s brain to guide the injection device to the target CNS tissue. In some embodiments, iMRI and/or ime imaging of the delivery may be performed. In some embodiments, the device is used to administer rAAV particles to a mammal by the methods of the invention.

In some ments, intraoperative magnetic resonance imaging (iMRI) and/or real—time imaging of the delivery may be performed. In some embodiments, the device is used to administer rAAV particles to a mammal by the methods of the invention. iMRI is known in the art as a technique for MRI—based g of a patient during surgery, which helps confirm a successful surgical procedure (e.g., to deliver rAAV particles to the CNS) and reduces the risk of damaging other parts of the tissue (for further descriptions, see, e.g., Fiandaca er al., (2009) Neuroimage 47 Suppl. 2:T27—35). In some embodiments, a tracing agent (e.g., an MRI contrast enhancing agent) may be co—delivered with the infusate (e.g., a composition including rAAV les) to provide for real—time monitoring of tissue distribution of infusate. See for example Fiandaca er al., (2009) Neuroimage 47 Suppl. 2:T27—35; US. PG Pub 2007/0259031; and US. Patent No. 7,922,999. Use of a tracing agent may inform the cessation of delivery. Other tracing and imaging means known in the art may also be used to follow infusate distribution.

In some embodiments, rAAV particles may be administered by standard stereotaxic injection using devices and methods known in the art for delivery of rAAV particles.

Generally, these methods may use an injection device, a ng system for translating a region of the tissue targeted for delivery into a series of nates (e.g., parameters along the latero—lateral, dorso—ventral, and rostro—caudal axes), and a device for stereotaxic localization ing to the planned nates (a stereotactic device, optionally including the probe and a structure for fixing the head in place 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, s, TN).

In some embodiments, the device for convection enhanced ry comprises 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., on Corp., ALZA Inc. in Palo Alto, CA). Pump systems may be table. Exemplary pump systems may be found, e.g., in US. Patent Nos. 7,351,239; 7,34l,577; 6,042,579; 5,735,8l5; and 147. In some embodiments, the pump is a manual pump. Exemplary s for CED, including reflux~ resistant and d cannulae, may be found in WC 991461066 and W0 2006f04209t), which are hereby incorporated by reference in its entirety.

In some embodiments, the device for convection enhanced delivery comprises a reflux—resistant cannula (e.g., a re?ux—free step design cannula). Further descriptions and exemplary re?ux—resistant cannulae may be found, for example, in Krauze er al., (2009) Methods Enzymol. 465:349—362; U.S. PG Pub 2006/0135945; US. PG Pub 2007/0088295; and PC'F/‘UStlS/?a’lt)l l . 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 comprises a re?ux—resistant cannula joined with a pump that produces enough pressure to cause the infusate to flow through the cannula to the target tissue at eontrolled rates, Any le flow rate ean be used such that the intraeranial re is maintained at suitable levels so as not to injure the brain tissue. in some embodiments, the cannula is a stepped cannula. As described. 82. in WO 2006/042090, a stepped cannula has a number of steps (egc, four in of WO 2006/042090). The steps nearest the distal end of the cannula are those that enter the target tissue first, and, accordingly, the number of steps entering the target tissue (6.3;, the striatum) will depend on the depth of penetration needed to reach that target in the subject, With respect to ry to the brain, the operator can readily determine the appropriate depth of penetration, talting into account both the size of the subject being treated and the location within the brain that is being targeted.

The cannula may be connected to a pump through a system of tubing. Tubing extends through the lumen of cannula. and the intusate may he delivered through this tubing. in embodiments containing the tubing, the tubing may be flush with the distal end of the cannula. Altematively, the tubing s from the distal end of the cannula, hi such embodiments, the amount which the tubing extends may vary depending on the application. lly, the tubing will extend from about 1 min to about 1 cm, from the cannula. (or any length therebetween‘), eg, from about 1 to about 50 nun (or any length therebetween), or from about l nun to about 25 nun (or any length therebetween, including, but not limited to, 1 mm, '2 mm, 3 mm, 4 mm, 5 nun, 6 mm, 7 mm, 8 mm, 9 mm, ll) mm, ll mm, l2 mni, l3 mm, 14 mm, 15 mm, in mm, 17 mm, 18 mm, l9 mm, 20 mm, 21 mm, 2‘2 mm, 23 mm, 24 mm or 25 min), such as ll) min beyond the distal end thereof.

The tubing ing through the cannula may have a. coating or surrounding material in one or more s, for example to t the tubing in t with the infusate.

Thus, in certain ments, tubing (tag, FEP (Teflon) tubing) protects the portion of the fused silica tubing extending beyond the proximal end of the stainless steel cannula. The ltisetl silica. tubing maybe connected to the syringe by any suitable means, including, but not limited to, a Luer compression fitting, and the e is driven by a syringe pump (manual, electronic and/or computerized). it will apparent that the syringe size can be selected by the operator to r the appropriate amount of productts). Thus, l, mL, 2.5 mL, 5 oils, or even larger syringes maybe used. d cannulae may be made out of the variety of materials that are logically acceptable, including without limitation stainless steel (6.5;, M633 or 30433), metal, metal alloys, polymers, organic fibers, inorganic fibers and/or combinations thereof.

Optionally, an infusate-contact surface (eg, tubing or coating) may extend through the lumen of the a A variety of materials may also be used for the o infusate—contact surface, including but not limited to metals, metal alloys, polymers, organic fibers, inorganic libers and/or combinations thereof. ln some embodiments, the product~contact surface is not stainless steel. in such embodiments, the outer cannula may still be made of a material physiologically compatible with the target tissue, but there since there is no product contact it need not be ible with the ically active agent or product formulation.

In some embodiments, penetration of the infusate is further augmented by the use of a facilitating agent. A facilitating agent is capable of further facilitating the delivery of infusate to target tissue (65)., CNS target tissue). A ruin—limiting example of a facilitating agent is low molecular weight heparin (see, 6.3., US. l>atent No. 7,922,999).

Suitable packaging for pharmaceutical compositions described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, ?exible packaging (e.g., sealed Mylar or plastic bags), and the like. These es of manufacture may further be ized and/or sealed. r provided herein are methods for treating a disorder of the CNS in a mammal comprising administering a rAAV particle to the mammal ing to the methods described herein. Yet further provided are methods for treating Huntington’s e in a mammal comprising administering a rAAV particle to the mammal according to the methods described herein using a system as described .

The present invention also provides kits for administering a rAAV particle described herein to a mammal ing to the methods of the invention. The kits may comprise any of the rAAV particles or rAAV particle compositions of the invention. For example, the kits may include rAAV particles with a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of a mammal. In some embodiments, the kits further comprise any of the devices or systems described above.

In some embodiments, the kits further include 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 e other als desirable from a commercial and user oint, ing other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described . Suitable packaging materials may also be included and may be any packaging materials known in the art, including, for e, vials (such as sealed vials), vessels, ampules, bottles, jars, ?exible 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 comprise instructions for treating a disorder of the CNS described herein using any of the methods and/or rAAV particles described herein. The kits may e a ceutically acceptable carrier suitable for injection into the CNS of an individual, and one or more of: a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for performing injections into the striatum of a .

In some embodiments, the kits further contain one or more of the buffers and/or pharmaceutically 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 pharmaceutically acceptable excipients, carriers, ons, and/or onal ingredients described herein. The kits described herein can be packaged in single unit dosages or in multidosage forms. The contents of the kits are generally formulated as sterile and can be lyophilized or provided as a substantially isotonic solution.

EXAMPLES The invention will be more fully understood by reference to the following examples.

They should not, however, be construed as limiting the scope of the ion. It is understood that the examples and ments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Widespread GFP Expression after Intrastriatal AAVl Vector Delivery The ability of AAVl to efficiently target both striatal and al structures in the Rhesus monkey brain when delivered via convection—enhanced ry (CED) was evaluated. AAV vectors containing GFP cDNA under the control of cytomegalovirus enhancer/chicken beta—actin (CBA) promoter were d into the e and putamen of 9 adult male Rhesus monkeys using CED (see, e.g., Bankiewicz er al., (2000) Exp. Neurol. 164:2-14 and Methods Surgical Delivery Nine adult male Rhesus macaques (Macaca mulatta; 8.9 — 11.9 kg) were ed in this study. All animals received an infusion of AAV vector rally into caudate nucleus and putamen by means of MRI—guided CED (Richardson, R.M. et al. (2011) Neurosurgery 69:154—163; Richardson, R.M. et al. (2011) Stereotact. Funct. urg. 89:141—151; Richardson, R.M. et al. (2011) M01. 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 mounted onto a stereotaXic frame, and the animal transported to the MRI (Siemens 3.0 T Trio MR unit) for a Tl—weighted planning scan. After scanning, animals were transferred to the operating room and the head prepared for an implantation procedure, and a ceramic custom—designed fused silica reflux—resistant cannula with a 3—mm stepped tip was used for the infusion. Temporary guide cannula were implanted bilaterally (one per hemisphere) using standard methods.

Animals received bilateral infusions into caudate nucleus and n of either AAVl—eGFP or AAV2—eGFP vectors obtained with 2 different production methods: Triple Transfection (TT) or er Cell Line (PCL). Vector concentrations and doses are described in Table 5. Animals were tested for the ce of anti—AAVl and AV2 neutralizing antibodies as previously described and were considered seronegative as they presented antibody titers of < 1:32 , A.K. et al. (2011) M01. Ther. 19:1971—1980).

Survival time was 1 month after AAV delivery for all the animals.

Each animal received up to three microinjections per hemisphere to target the caudate and the putamen ommissural and post—commissural) regions, as shown in Table 2. To visualize infusate distribution during MRI, Prohance (2 mM ridol) was added to the virus. Approximately 30 ul of AAV was stered into the caudate and 60 ul into the putamen using the convection enhanced ry (CED) method (i.e., 90 uL per hemisphere).

The infusion rate was ramped up to a maximum of 5 uL/min.

Table 2. Parenchymal dose volumes per site.

Vector Dose per . Number Target Production Hemisphere Hemisphere ofDosing Structure Sites 3o+3o Pummen AAw-eGFP 3o+3o W10" 1 (m - Caudam Vector Dose per Number Dose t Target Production Hemisphere Hemisphere ofDosing Volume Number Structure Method (vg) Sites (.uL) Right 30 + 30 Putamen Left 30 + 30 Right 30 Caudate bCD11" 30 Right 30 + 30 Left 30 + 30 Right 30 Caudate rCD11" 30 Right 30 + 30 Putamen Left 60 Right 30 Caudate GFP rCD11" 15 + 15 1.71;;1011 (TT) Right 60 Putamen Left 30 + 30 Right 30 Caudate rCD11" 30 Right 60 Putamen Left 60 1.71;;1011 Right 30 Caudate AAVl—eGFP rCD11" 18 + 20 (PCL) Right 60 Putamen Left 60 Right 56.4 + 31 Caudate rCD11" 33.6 + 31 Right 62 Putamen Left 60 Right 30 + 20 Caudate AAVZ—eGFP CD11" 1.71;;1011 r 30 (PCL) Right 20 + 35 Putamen Left 42 + 19.6 Right Caudate rCD11" t—‘t—‘Nwt—‘Nt—‘t—‘NNHt—‘Nt—‘t—‘t—‘t—‘t—‘Nt—‘Nt—‘t—‘Nt—‘t—‘NNHt—‘NN 30 The first cohort of animals received 1.7 X 1011 vg of AAVl—eGFP (TT) (n=3), or AAVZ—eGFP (TT) (n22) per hemisphere. The second cohort of animals ed 1.7 X 1011 vg of AAVl—eGFP (PCL) (n=2), or 1.3 x 1011 vg of AAVZ—eGFP (PCL) (n22). Serial MRI was acquired to monitor infusate distribution within each target site and provide real—time feedback to the team. Immediately after the intraparenchymal dosing procedure, animals were transferred to the operating room, the guide cannula d, and wound site closed in anatomical layers. All experiments were med in accordance with National Institutes of Health guidelines and with protocols approved by the institutional Animal Care and Use Committee. Immediately after surgery, the s were transferred to the MRI suite for AAV dosing procedures. Comments regarding the dosing procedure for each of the subjects depicted in Table 2 are ed below.

Dosing Procedure—Treatment Group 2 (AAVl-eGFP TT) Subject Number 1. Approximately 60uL of infusate was administered per hemisphere via two trajectories (30uL/deposit) into each putamen. Coronal images from infusions into the anterior putamen showed a majority of gadolinium signal within each of the targeted structures. In the right hemisphere, slight perivascular transport was seen in the ventral putamen and distribution into the anterior commissure.

Subject Number 2. T1 MRI was acquired after 0.l27mL infusion into the putamen and 0.207mL into the thalamus. Post infusion Tl MRI showed extensive infusate distribution within the right thalamus measuring approximately lcm in the anterior—posterior direction and lcm in the dorso—ventral direction. Coronal Tl showed gadolinium distribution within the target site that extended ly towards the internal capsule and superiorly toward the dorsal putamen. A majority of the infusate was contained within the putaminal margins; r, transport via the perivascular space was also present in white matter tracts of the internal capsule and anterior commissure. Analysis revealed that the ratio of gadolinium infusion volume (Vi) to distribution volume (Vd) in the thalamus and putamen was 1:2.

In-Life ations Detailed observations of animal health and neurological symptoms were performed on a daily basis for a period of 5 days after dosing; subsequently, detailed observations were performed once per week until study ation. Observations and daily mortality checks were performed. Body s assessments were med before ranial dosing, at time of blood collection procedures, and at necropsy. No significant difference in body weight was observed between the ent groups prior to y or at the time of necropsy (. Whole blood, blood serum and cerebrospinal ?uid (CSF) were collected for hematology, serum chemistry, AAVl and AAV2 capsid antibody assay and eGFP mRNA level analysis.

In-Life Blood Collection and Processing Blood (approximately 5 mL) was ted prior to injection, approximately 72 hours post—injection, and at necropsy according to the Blood Sample Collection Schedule (see Table 3 below). imately 0.5—1.0 mL of whole blood was collected into EDTA tubes for logy analysis. Approximately 2.0 mL of whole blood was collected into serum separator tubes (with gel, BD ainer) and processed to serum to obtain imately serum for chemistry analysis and AAVl and AAV2 Capsid dy analysis.

Table 3. Blood sample collection schedule Time Point Hematology Serum Chemistry AAVl and AAV2 Antibody Analysis EDTA —— —-samples were collected N/A— not applicable EDTA — Ethylenediaminetetraacetic acid CSF Collection and Processing CSF was collected at two separate time points: prior to intracranial dosing and at necropsy. CSF collection was performed under anesthesia by cerVical spinal tap with the animal placed in a prone position. Prior to test article administration, l—2 mL of CSF was collected, frozen on dry ice, and stored at 360°C. At necropsy (prior to PBS perfusion) 2—4 mL of CSF were collected, filtered h a 0.8 micron syringe filter into a labeled collection tube, and transferred in duplicate (lmL t for Capsid Antibody analysis and 2— 4 mL aliquot for GFP analysis) into eppendorf tubes, immediately frozen on dry ice and stored at 360°C.

Necropsy and Tissue Collection All animals were euthanized at approximately 30 days after the intracranial dosing procedure. Each animal was euthanized using intravenous stration of sodium pentobarbital. Following euthanasia and blood and CSF collection, the body was transcardially perfused with PBS (under RNAse free conditions), followed by perfusion with 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 that were collected (Table 4). During PBS perfusion (prior to tion of 4% PFA perfusion) biopsy samples (0.5 — 1.0 cm3) of select tissues (also listed in the Tissue Collection Table) were collected under RNAse free conditions with able sterile scalpels (a new scalpel for each individual biopsy) into RNAse free tubes, and stored frozen at 360°C.

Table 4. Tissue Collection Tissues Collected into PFA s Collected Frozen y punch) Brain Heart Spinal Cord Kidney Liver.

Spleen Testes Cervical lymph nodes Quadricep Sciatic Nerve Optic Nerve Brain and Spinal Cord Processing The entire brain was carefully removed from the animal and photographed along— side a ruler for scale. Once removed from the skull the brain was placed into a brain matrix and coronally sliced into 6 mm blocks. Coronal blocks were stored in PFA and processed for histology. Relevant blocks ning the frontal cortex and midbrain regions were sectioned into free ?oating 40 micron sections. The entire spinal cord was lly removed from the animal. Spinal cord segments were stored in PFA and processed for histology. A representative segment from the cervical, thoracic, and lumbar region were sectioned into free ?oating 40 micron sections.

After perfusion with PBS—heparin ed by 4% buffered paraformaldehyde (PFA), the brain from each animal was cut into 6—mm blocks (coronal plane) using disposable blades and monkey brain matrix. The sequential blocks (1 1—12 brain slabs per animal) were placed horizontally on a white board and photographed with sequentially assigned letters. All brain blocks were then post—fixed in 4% buffered PFA for 24 hours. The quality of fixation for each block was inspected visually (no pink color was observed within blocks). After PFA—postfixation, each brain block was processed for oating sections by rinsing 3X in PBS and immersion in 30% sucrose (cryopreservation) before cutting into 40—um free— ?oating sections.

Production of AAV Vectors Prior to clinical evaluation, AAV vectors are typically produced via the rd triple ection method (TT) in which HEK293 cells are co—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 er al., (2009) Mol. Ther. 17:144—152). Since input of plasmid DNA may be easily and rapidly modified, this method allows evaluation of vectors based on diverse serotypes and harboring a variety of sion cassettes. Despite its ?exibility and relatively fast tum—around time, the transfection method presents a nge with regard to scalability, which limits the suitability of this method for large—scale rAAV vector production for clinical use.

At the present time, al—grade rAAV is generated at large scale via the helper virus—free transient transfection method, the recombinant baculovirus or herpes simplex virus—based production systems, or ing/producer cell lines (Ayuso er al., (2010) Curr.

Gene Ther. 10:423—436). Adeno—associated virus producer cell lines (PCL) are an effective method for large—scale production of clinical grade AAV vectors. In this , a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. However, it is desirable to determine whether AAV vector derived from producer cell lines is etprivalent in potency to vector generated via other s, for example, the standard transient transfection .

AAV viral vectors were generated for this study using two different production methods: triple transfection (TT) and producer cell line (PCL). Recombinant AAV vectors AAVl—GFP (TT) and AAV2—GFP (TT) were produced by triple transfection (using calcium phosphate) of human embryonic kidney carcinoma 293 cells (HEK— 293) (referenced in Xiao et al., (1998) Journal of Virology 72:2224—2232). Briefly, for the production of AAV s by transient transfection, HEK293 cells were transfected using polyethyleneimine (PEI) and a l:l:l ratio of the three ds (ITR vector, AAV2rep/cap2 or AAV2rep/capl, and pAd helper plasmid). The ITR vector plasmid d the cDNA for EGFP downstream of the cytomegalovirus enhancer / chicken beta actin — hybrid promoter (CBA). The pAd helper used was pHelper (Stratagene/Agilent Technologies, Santa Clara, CA). inant AAV vectors AAVl—GFP (PCL) and FP (PCL) were produced using an AAV er cell process (referenced in Thome et al., (2009) Human Gene Therapy 20:707—7l4 and Martin et al., (2013) Human Gene Therapy Methods 24:253— 269). Brie?y, product—specific er cell lines were generated by stable transfection of Hela—S3 cells (ATCC CCL—2.2) with a plasmid containing the rep gene from serotype 2 and a capsid gene from either serotype 1, or 2, the promoter—heterologous nucleic acid sequence, the vector genome ?anked by AAV2 inverted terminal s (ITRs), and a Puromycin resistance gene. The vector genome harbored the cDNA for EGFP downstream of the cytomegalovirus enhancer / chicken beta actin—hybrid promoter, CBA. Transfected cells were grown in the presence of puromycin to isolate stable integrants. The cell lines generated were screened to select a lead clone. The product—specific cell clone was subsequently expanded to a production bioreactor, and infected with a wild type Adenovirus as helper to initiate AAV production. Virus was harvested 72 hours post—infection, the adenovirus was inactivated by heat and removed by anion exchange methods.

Purification of AAV from both production platforms was performed as previously described (Qu, G. et al. (2007) J. Virol. s 140:183—192). The resulting titers of all AAVl and AAV2—GFP vectors are shown on Table 5. All vectors were prepared in water ning 180 mM sodium chloride; 10 mM sodium phosphate (5 mM NaHzPO4-2 H20 + 5 mM NazHPO4-HZO); and 0.001% Poloxamer 188 (Lutrol F68), pH 7.5.

Table 5. Study design table and test articles Group No. of Test Article Drug Vector Dose per Animals concentration here (vg) ssAAV2/1-CBA-GFP (TT) 1.90;;1012 vg/mL 1.7;;1011 ssAAV2/2—CBA—GFP (TT) 1.90;;1012 vg/mL 011 ssAAV2/1-CBA-GFP (PCL) 2.30x1012 vg/mL 1.7;;1011 ssAAV2/2—CBA—GFP (PCL) 1.43;;1012 vg/mL 1.3;;1011 Immunohistochemistry Immunostaining with antibodies against GFP (l:500, AB3080; Chemicon) was performed on Zamboni fixed 40—um coronal sections ng the entire frontal cortex and extending in a posterior direction to the level of the striatum. The localization of GFP positive s was analyzed with reference to The Rhesus Monkey Brain in tactic Coordinates (Paxinos, G.H.X. and Toga, AW. (2000) San Diego, CA: ic Press) to identify specific areas of immunostaining in the cortex and striatum.

GFP staining by 3,3 '-diaminobenzidine (DAB): Sections (3 per each 6—mm block: tion of 2 mm) were washed 3 times in PBST for 5 min each followed by treatment with 1% H202 for 20 min. Sections were incubated in Sniper blocking solution able online at biocare.net/product/background—sniper/) for 30 min at room temperature followed by overnight incubation with the primary anti—GFP antibody (available online at www.lifetechnologies.com/) diluted 1:1000 in Da Vinci Green Diluent (available online at biocare.net/). After 3 rinses in PBS containing 0.1% Tween—20 (PBST) for 5 min each, sections were ted in Mach 2 HRP polymer (http://biocare.net/) for l h, followed by 3 washes and colorimetric development (DAB). Immunostained sections were counterstained with cresyl violet and mounted on slides and sealed with Cytoseal® (available online at www.thermoscientific.com/).

Calculation ofcoverage of GFP expression in the man primate (NHP) brain: GFP staining from matching IHC—stained serial sections was projected onto individual corresponding MRI scans of each monkey brain (Tl—weighted MR images in the coronal plane). Distribution/coverage of GFP expression was med with OsiriX Imaging Software version 3.1 (The OsiriX Foundation, Geneva, Switzerland).

Double-immunofluorescence for vector tropism and e?‘iciency of neuronal transduction: For double ?uorescence immunostaining of different cellular markers (NeuN, S—100, Ibal) with GFP, a combination of primary antibodies was d to sections as a il of primary antibodies by overnight incubation at room temperature in PBST with % horse serum. Primary dies used were as follows: 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—Ibal (1:500, available online at biocare.net/); anti—Olig2 (l:50, available online at www.emdmillipore.com/). After 3 washes in PBST, primary antibodies were visualized by incubation in the dark for 2 hours with appropriate secondary ?uorochrome—conjugated antibodies: goat anti—mouse DyLight 549 and goat abbit DyLight 488 (available online at www.biocare.net/). All secondary antibodies were diluted l:l,000 in Fluorescence Antibody Diluent (available online at biocare.net/). In on, to quench autofluorescence, ns were incubated in 0.1% Sudan Black solution (70% ethanol). After final washes in PBS, sections were cover—slipped with Vectashield Hard Set Mounting Medium for Fluorescence (available online at www.vectorlabs.com/). l sections were processed without primary antibodies, and no significant immunostaining was observed under these conditions.

Zeiss Axioskop fluorescence microscope (available online at www.zeiss.com/) equipped with CCD color video camera and image analysis system (Axiovision Software, ble online at www.zeiss.com/) was used to ine the presence of -labeled cells (positive in both red and green channels). Photomicrographs for double—labeled sections were obtained by merging images from two separate channels (red and green; co—localization appears yellow) without altering the position of the sections or focus (objective X 20). For GFP/NeuN double—staining, 3 sections from each monkey at ~ 4—mm als were selected from the sites of vector infusion. For evaluation of efficiency of al uction by GFP or AAV2—eGFP vectors within the targeted brain areas (caudate and putamen), counting frames (700 um X 550 um) were placed randomly in the GFP+ area. The primary area of transduction (PAT) was defined as GFP—positive area ("cloud") that covered more than 40% of the targeted structure. Similarly, to evaluate the efficiency of neuronal transduction, outside PAT (OPAT), 5 counting frames (700 um X 550 um) from each n were chosen beyond the clear margins of GFP—positive "cloud" in the targeted structures (caudate and putamen) or in the cortex (C). To determine the proportion of GFP/NeuN—positive cells, each counting frame was counted twice, first with the red channel for the number of NeuN+ cells and second with a combined red and green channel for the number of co—stained cells (GFP+ and NeuN+). At least 1,500 NeuN+ cells were counted for each of the 3 chosen sections (5 counting frames per section). Finally, the percentage of GFP+/NeuN+ to total NeuN+ was determined. All of the calculations for the striatum were made by adding results from both heres of each animal and combining values from putamen and caudate s since the mean transduction efficiencies were identical in both ures of each animal.

Quantitative real-time PCR (TaqMan) GFP mRNA levels were measured by tative real—time PCR. Liver, heart, lung, kidney, and spleen samples were used for all RT—PCR analysis. Total RNA was extracted using the QIAGEN sy mini kit and then reverse transcribed and amplified using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) ing to the manufacturer’s instructions. Quantitative RT—PCR reactions were conducted and analyzed on a QuantStudiolZK Flex ime PCR System (Applied Biosystems). Each sample was run in duplicate and the relative gene expression was determined using a standard curve.

MR imaging data analysis Semi-quantitative Analyses (Digital MRIs Vd/Vi): Distribution volume (Vd) analysis was performed with OsiriX Imaging Software n 3.1 (The OsiriX Foundation, Geneva, Switzerland). Infusion sites, cannula tracks and cannula tip were identified on Tl—weighted MR images in the coronal plane. Regions—of—interest (ROIs) were delineated to outline Tl gadolinium signals and target sites (i.e. putamen and caudate nucleus). Three—dimensional volumetric reconstructions of the image series and ROI were ed to estimate volume of distribution (Vd) of infusions and ratio to volume of infusate (Vi).

Histological is of transgene expression: To assess transgene sion, brain sections were processed for immunohistochemical analysis (IHC). Animals were deeply anesthetized with sodium pentobarbital (25 mg/kg iv.) and euthanized 4 weeks after administration of the vectors. The brains were removed and sectioned coronally into 6—mm blocks. The blocks were post—fixed in buffered paraformaldehyde (4%) for 24 h, washed y in PBS and adjusted in a 30% sucrose/PBS solution for cryopreservation. The formalin—fixed brain blocks were cut into 40—um l sections in a cryostat. Free—?oating sections spanning the entire brain were collected in series and were kept in antifreeze solution for r IHC analysis.

Results Both AAVl— GFP and AAV2—GFP vectors drove abundant expression of GFP from transduced neurons as Visualized by immunohistochemistry. After infusion of AAVl into the caudate and n by CED, extensive GFP immunostaining was ed in the caudate and putamen (FIGS. 2C&D), as well as the substantia nigra (). In addition to the striatum a large number of cortical regions of the Rhesus monkey brain were also transduced (FIGS. 2A-D). Cortical GFP expression was most evident in prefrontal ation cortical areas, the premotor cortex, primary somatosensory cortical areas, and the primary motor cortex, as well as extensive s of the occipital cortex (FIGS. 2A-D).

A large majority of GFP—positive neurons within the cortex were identified morphologically as pyramidal neurons located in cortical lamina IV, with axonal projections into the overlying layers. The density of GFP—positive neurons was particularly high in the frontal (FIGS. 3A&B) and occipital cortex (FIGS. 3C&D), where large numbers of neurons (FIGS. 3B&D) in addition to ytes (FIGS. 3A&C) were transduced.

Example 2: Widespread GFP Expression after Intrastriatal AAV2 Vector Delivery.

The ability of AAV2 to efficiently target both striatal and cortical structures in the Rhesus monkey brain when delivered via convection—enhanced ry (CED) was evaluated. AAV vectors containing GFP cDNA under the control of cytomegalovirus enhancer/chicken beta—actin (CBA) er were infused into the caudate and putamen of 8 adult Rhesus monkeys using CED according to the methods described in Example 1 above.

Infusion of AAV2 into the striatum by CED resulted in GFP expression in the injected regions te and putamen) (), substantia nigra (), and a large number of cortical regions of the Rhesus monkey brain (FIGS. 4A-D). Expression of GFP in the striatum of AAV2 injected animals appeared slightly more cted and localized when compared to striatal coverage with AAV1 vectors. The expression of GFP within the NHP striatum was comprehensive but relatively ned within the gray matter bounds of the ed region, with no evidence of significant infusion related leakage or re?ux of the AAV2—GFP vector into nt non—targeted areas. Cortical GFP sion was evident in the same regions seen for AAV1. Prefrontal association cortical areas, the premotor cortex, primary somatosensory cortical areas, and the primary motor cortex, as well as extensive s of the occipital cortex were well transduced (FIGS. 4A-D).

Example 3: Comparability of GFP Expression after Intrastriatal AAV1 and AAV2 Vectors made by triple transfection or producer cell line process.

To date the majority of preclinical studies utilize AAV s made by Triple Transfection followed by purification using cesium chloride gradients or column chromatography. Thus, to evaluate the impact of vector production on biodistribution in vivo, two methods of vector production Triple Transfection (TT) or Producer Cell line (PCL), were compared. AAVl and AAV2 vectors generated by these two ent manufacturing platforms were administered via CED, and their distribution within the Rhesus monkey brain was compared.

Infusion of AAVl—GFP vectors made by triple transfection yielded equivalent GFP bution and coverage when compared to AAVl—GFP vectors made by the producer cell line process. GFP distribution was comparable between AAVl—GFP (TT) (FIGS. 5C&D) and AAVl—GFP (PCL) (FIGS. 5A&B) vectors 30 days ing injection into the striatum of Rhesus monkeys.

Similar results were seen with the AAV2—GFP vectors. GFP bution was similar and comparable n AAV2—GFP (TT) (FIGS. 6C&D) and AAV2—GFP (PCL) (FIGS. 6A&B) injected .

To measure on performance, AAVl—eGFP (TT); AAV2—eGFP (TT); AAVl— eGFP (PCL); and AAV2—eGFP (PCL) was infused into each striatum (60 ul into putamen and ul into caudate nucleus), using 90 ul of each vector mixed with nium contrast agent (2 mM Prohance; Bracco Diagnostics, Inc.). Magnetic resonance images (MRI) from each infusion confirmed that positioning of each cannula was accurate and infusate covered the target area. All infusions were well contained in the target structure. Three—dimensional reconstructions of the infusate distribution ted from gadolinium signal on MR images showed that both ent and distribution of infusate were very consistent throughout all the animals. In addition, the ratio (Vd/Vi) between volume of distribution (Vd) and volume of infusion (Vi) was calculated for each delivery and data were consistent across all ons.

Vd was approximately 3—fold larger (range of 2.1 — 4.6) than the Vi (Tables 6 and 7).

Table 6. Vector infusion and extent of distribution within the brain 4 weeks after transduction (mean i st. dev.).

Vd/Vi a Gadolinium coverage ' Cortical coverage of GFP expression c 2.79 i 0.44 Putamen: 29.5 i 10.9 % 62.2 i 19.1 % Caudate: 18.3 i 5.2 % 3.29 i 0.75 Putamen: 23.5 i 9.3% 61.3 i 14.8 % Caudate: 24.6 i 8.0% a Ratio of volume of distribution (Vd) to volume of infusion (Vi) was calculated (OsiriX Imaging software, V. 3.1) by dividing the volume of vector distribution within the injected brain parenchyma (based on the Gadolinium signal from MRI scans) by the volume of the ed vector. Values from left and right hemispheres were added to determine the average Vd/Vi for each animal.

Gadolinium coverage within targeted structures was calculated (OsiriX Imaging software, v. 3.1) by dividing Vd by the volume of Putamen (600 mm3) or Caudate (500 mm3).

C Cortical GFP coverage was calculated by projecting GFP signal from matching IHC—stained sections onto corresponding MRI scans of each monkey (BrainLab software).

Table 7. Vector infusion and extent of distribution within the brain 4 weeks after transduction (individual values).

Vd/Via Gadolinium coverage ' Cortical GFP Putamen: Putamen L 34.8% Putamen R 30.2% AAVl— 3.2 Caudate L 14.8 % Caudate R 16.2 % eGFP (TT) Caudate: 2 n: n L 35.7% n R 18.3% AAVl— 3.1 Caudate L 18.2% Caudate R 13.8% eGFP (TT) Caudate: 3 n: Putamen L 52.7% Putamen R 37.8% AAVl— 3.3 e L 19.4% e R 21.2% eGFP (TT) Caudate: Putamen: Putamen L 23.3% Putamen R 17.7% 3.0 Caudate L 23.8% Caudate R 20.6% Caudate: Putamen: n L 22.3% Putamen R 22.2% 2.7 Caudate L 26.8% Caudate R 8.6% Caudate: u amen. u amen L . u amen R . 0 AAV2— 2.7 Caudate L 20.0% Caudate R 23.0% eGFP (TT) Caudate: 7 Putamen: Putamen L 11.3% Putamen R 16.3% AAV2— 2.9 Caudate L 20.6% Caudate R 15.2% eGFP (TT) Caudate: Putamen: Putamen L 36.0% Putamen R 19.8% 2.4 Caudate L 34.0% Caudate R 39.4% Caudate: Putamen: Putamen L 35.2% Putamen R 30.5% 4.6 Caudate L 21.2% Caudate R 23.4% Caudate: a Ratio of volume of bution (Vd) to volume of infusion (Vd) was ated (OsiriX Imaging software, V. 3.1) by dividing the volume of vector distribution within the injected brain parenchyma (based on the Gadolinium signal from MRI scans) by the volume of the injected vector. Values from left and right heres were added to determine the average Vd/Vi for each animal.

Gadolinium coverage within ed structures was calculated (OsiriX Imaging software, v. 3.1) by dividing Vd by the volume of Putamen (600 mm3) or Caudate (500 mm3).

C Cortical GFP coverage was calculated by ting GFP signal from matching IHC—stained sections onto corresponding MRI scans of each monkey.

After bilateral injection of both AAVl—eGFP and GFP (prepared by both methods of productions, TT and PCL) into the striatum of NHP, robust expression of eGFP was evident throughout both the target structures (putamen and caudate nucleus) as well as projection regions (external and internal globus pallidus — GPe and GPi, substantia nigra — SN, subthalamic nucleus — STN, cortical regions — neuronal layers IV and V) s (.

To evaluate role of Gadolinium (Gd) as a marker of vector distribution, the ratio of the area of GFP expression (from histological sections) to the area of Gadolinium signal on corresponding MR scans was calculated. For monkeys infused with serotype AAVl, this ratio was 1.21 i 0.10 whereas for AAV2 it was 0.74 i 0.04 (. The ratio of 1.0 indicates a perfect match between GFP expression and vector distribution as determined by MRI. This ence indicated that AAVl vector buted beyond the Gd signal and achieved better spread in the primary area of transduction than AAV2.

To evaluate the distribution of the infused AAV vectors within the brain, representative free—?oating brain sections (3 per each block; 40—um thick) from each animal were stained with a rabbit anti—GFP dy (Millipore; Cat. No. 3850, dilution 1:500). histochemical assessment revealed dark brown DAB signal within the injected targets (right and left ns and caudates) as well as le areas projected from the injected areas (cortical regions).

By ting the extent of GFP expression onto MRI scans of each monkey brain, the percentage of ge for cortical regions (brain areas relevant in HD) was calculated (see Table 6 for summary and Table 7 for further details). In all monkeys, 24% of striatum was transduced on average, which resulted in substantial expression of GFP in the cortex (. The extent of GFP expression in the cortex did not ate with the AAV serotype used (AAVl vs. AAVZ) or the method of vector production (TT vs. PCL). It seems that broader distribution of infusate within the infusion site (striatum) was a key driver of the extent of transduction in the cortex. One NHP (Subject No. l; AAVl—eGFP [TT]) showed a particularly robust spread of GFP expression into cortical regions (layer IV and V) of the entire brain (both frontal and occipital — see . Other s showed ility in al expression associated with variations both in the extent and in localized anatomical regions within caudate and putamen. Since pre— and commissural regions of the striatum were targeted, GFP was detected more in frontal and parietal cortical regions and less in the occipital cortex. Histological analysis for each animal is summarized below (grouped by treatment .

Treatment Group 2 (ssAAVZ/I -CBA-eGFP TT) Immunohistochemical evaluation of eGFP expression in the whole brain revealed robust signal in the targeted sites (both putamens and caudate nuclei) and projected structures (globus us, substantia nigra, thalamus, subthalamic nucleus, and cortical regions).

Subject number 1. Subject showed a particularly robust spread of GFP expression to cortical regions (layer 4 and 5) of the entire brain (both frontal and occipital). The morphology of the GFP—positive cells implied both neuronal and astrocytic transduction, which was later confirmed by double immuno?uorescence staining (see below). The calculation of GFP expression coverage showed that 91% of the entire cortex (see Table 7) was transduced (this calculation was done by projecting the extent of GFP signal onto MRI scans of the ed monkey brain).

Subject number 2. Subject showed robust transduction in putamens and caudate nuclei as well as globus us and substantia nigra of both hemispheres. The projection of GFP expression to cortex was less pronounced than in subject number 1 and was observed mainly in the l regions of the cortex. Although GFP signal was also detected in occipital cortex, the y of GFP—positive cells was icantly lower. The al GFP expression coverage was accounted for 50% (Table 7). Similarly as in subject number 1, GFP—positive cells had both neuronal and astrocytic logy, which was confirmed by double immuno?uorescence.

Subject number 3. Subject showed strong GFP expression in ns and caudate nuclei as well as all projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus, and cortex). Although GFP signal was y detected in some regions of cortex, GFP expression was accounted for only 41% of its overall al coverage (the lowest in all tested monkeys; see Table 7). Both neurons and astrocytes were transduced. A large part of the right anterior corona radiate also showed GFP—positive signal, most likely as a result of vector spillage from the cannula penetrating to the striatum.

Treatment Group 3 (ssAAVZ/Z-CBA-eGFP TT) Subject number 6. Subject showed a strong GFP signal in both ed structures (putamen and caudate nucleus). Densely scattered positive cells were detected in both of those regions. The GFP expression spread also to frontal cortical regions, globus pallidus, substantia nigra, subthalamic nucleus, and some parts of thalamus. The GFP sion coverage in the cortex accounted for 75% (Table 7). GFP—positive cells had mostly neuronal morphology, which was later confirmed by double immuno?uorescence. GFP—positive cells of astrocyte shape were detected in the internal capsule as well as in a few cortical spots and closely neighboring white matter areas with clearly visible tracks of the infusion cannulas.

Subject number 7. Subject showed sitive signal within the right and left striatum (both putamen and caudate nucleus). Its distribution was rather poor and pattern appeared "spotty" rather than uniform when compared to other infused monkeys.

Consequently, the GFP signal in all projected brain structures appeared weaker as well. The GFP sion coverage in the cortex accounted for 47% (Table 7). sitive cells had mostly neuronal morphology, which was later med by double immuno?uorescence.

GFP—positive cells of astrocyte shape were detected in the internal capsule as well as in a few cortical spots and closely neighboring white matter areas with clearly visible tracks of the infusion cannulas.

Treatment Group 4 (ssAAVZ/I-CBA-eGFP PCL) Subject number 4. Subject showed very robust GFP expression in targeted structures, putamen and e nucleus. GFP—positive signal was also detected in globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions. The GFP expression coverage in the cortex accounted for 61% (Table 7). Anterior part of the corona radiata also showed GFP—positive signal, mostly likely as a result of vector spillage from the cannulas penetrating to the striatum. Positive cells had both neuronal and astrocytic morphology, which was later confirmed by double immuno?uorescence. t number 5. Subject showed robust GFP expression in the striatum (both putamen and caudate s). GFP—signal was also detected in projected structures (globus pallidus, ntia nigra, subthalamic nucleus, thalamus and cortical regions). The GFP expression coverage in the cortex accounted for 68% (Table 7). Anterior part of the corona radiata also showed GFP—positive signal, mostly likely as a result of vector spillage from the cannulas penetrating to the striatum. Positive cells had both neuronal and astrocytic morphology.

Treatment Group 5 (ssAAVZ/Z-CBA-eGFP PCL) Subject number 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, lamic nucleus, thalamus and cortical regions). The GFP expression coverage in the cortex accounted for 73% (Table 7). GFP—positive cells had mostly neuronal morphology although GFP—positive cells of astrocyte shape were also detected within white matter tracts (internal e) and immediate vicinity of cannula tracks. t number 8. Subject showed robust GFP expression in the striatum and projected ures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions). The GFP expression coverage in the cortex ted for 50% (Table 7).

GFP—positive cells had mostly neuronal morphology although GFP+ cells of astrocyte shape were also detected within white matter tracts (internal capsule, corona radiata) and in the immediate vicinity of cannula tracks.

Double immuno?uorescence For both groups of NHPs transduced with AAVl—eGFP vectors (TT and PCL), the morphology of GFP—positive cells suggested both neuronal and ytic transduction (FIGS. . This was confirmed by double immuno?uorescence staining with a combination of antibodies against GFP and NeuN (neuronal marker) or GFP and S—100 (astrocytic marker) (FIGS. 10A-10C). In contrast, AAV2—eGFP (both TT and PCL) directed predominantly neuronal transduction (FIGS. 9E-9G and 10D). GFP—positive cells of astrocytic lineage were also ed in the internal capsule () as well as in cortical regions of white matter where the infusion cannula tracks were Visible.

Based on double fluorescence, the efficiency of neuronal transduction in the striatum and cortical regions was calculated (at the coronal plane of the infusion site) for all NHPs. A summarizes the findings in the striatum. Individual calculations for each animal are shown in Table 8 below.

Table 8. Efficiency of al transduction by AAVl—eGFP and AAV2—eGFP vectors within the striatal primary areas of transduction (PAT) and the cortex of the man primate brain.

Subject No. 1 2 Targeted AAVI-eGFP AAVI-eGFP GFP AAVZ-eGFP AAVZ-7eGFP region (TT) (TT) (TT) (TT) (TT) Left uutamen 57.0 i 7.75 % 64.4 i 11.75 % 54.7 i 11.4 % 36.5 i 8.8 % 59.6 i 12.8 % Right 68.0 i 15.9 % 70.1i 14.4 % 66.2 i 20.0 % 33.1i11.7% 56.3i7.7% I utamen Left caudate 70.1 i 7.4 % 72.6 i 13.3 % 56.4 i 6.02 % 33.7ill.4% 61.4il3.1% Riht e 65.6 i 7.75 % 65.1 i 13.9 % 60.1 i 5.65 % 42.7 i 10.5 % 49.4 i 13.5 % C0rtex* 24.8 i 3.04 % 4.04 i 2.77 % 6.75 i 3.38 % 8.56+82.81% 3.36+1.83% Subject N0. 5 4 9 Targeted AAVI-eGFP AAVI-eGFP AAVZ-eGFP AAVZ-eGFP region (PCL) (PCL) (PCL) Left putamen 58.6 i 7.61 % 57.1 i 10.5% 53.1 i 12.7% (_PCL)_2.-4+11.8% Right 50.5 i 9.78 % 73.2 i 9.88% 52.1 i 10.6 % putamen -0.1+4.5% Left caudate 57.0 i 7.13 % 51.8 i 9.42 % 43.0 i 14.5 % Right caudate 59.1 i9.11 % 70.5 i 8.38 % 52.1 i 7.22 % 4__.4+8.5%_1.2+12.9% 16.3 i 5.09% 16.1 i 6.59 % 23.0 i 7.55 % * Neuronal transduction by AAV vectors was also detected in cortical regions projected from the striatum (target structure). The efficiency of cortical transduction was calculated in coronal sections of the striatal plane with injection sites.

Striatal neuronal transduction in the regions of primary transduction, ted by MRI, ranged from 50 % to 65%. The highest efficiency of transduction was observed in NHPs infused with AAVl—eGFP (TT) with the mean of 64.2 i 5.9% and the lowest in group AAV2—eGFP (TT) with the mean of 46.6 i 11.7% (p < 0.05; 2—way ANOVA). This suggests that serotype AAVl has ~ 18 % higher efficiency in transducing neurons than AAV2. AAVl— eGFP produced by PCL evinced a weaker trend (p> 0.05; 2—way ANOVA) toward transducing more neurons (59.7i 8.1%) than AAV2—eGFP (50.1i 5.8%).

The above calculations were derived from areas of strong GFP transduction as defined by MRI, (primary area of transduction — PAT). In addition, the efficiency of al striatal transduction in s outside the PAT was calculated to see if GFP—positive cells could also be detected outside the clear boundary of strong GFP signal ("outside the primary area of transduction"— OPAT), suggesting perhaps that all tested vectors spread in the same . The scheme of how these areas were chosen (random selection of 5 counting frames) is illustrated in B. A dramatic difference in the estimation of transduction efficiency in OPAT between serotypes AAVl and AAV2 was ed (C), with AAVl transducing many more neurons than AAV2 (8.1 i 3.8% vs. 0.74 i 0.25% for TT groups and 7.2 i 3.5% vs. 2.16 i 1.8% for PCL groups; p<0.05 in both isons 2—way ANOVA;).

Individual calculations for each animal are shown in Table 9 below.

Table 9. ency of neuronal transduction by AAVl—eGFP and AAV2—eGFP vectors within the striatum but outside the primary areas of uction (OPAT) of the non—human primate brain.

Subject N0. 1 2 3 6 7 Targeted region AAVI-eGFP GFP AAV]-eGFP AAVZ-eGFP AAVZ-eGFP (TT) (TT) (TT) (TT) (TT) Left putamen 14.2 i 9.49 % 6.88 i 6.48% 14.2 i 7.87% 0.57 i 0.53 % 1.28 i 0.69 % Right putamen 4.36 i 2.06 % 4.04 i 4.36 % 5.35 i 3.68% 0.51 i 0.61 % 0.80 i 0.55 % Left e 10.1 i 7.42 % 2.01 i 2.54 % 9.11 i 5.24 % 0.86 i 0.95 % 0.71 i 0.36 % Right caudate 10.8 i 6.37 % 6.89 i 5.62 % 8.76 i 3.50 % 0.51 i 0.91 % 0.71 i 0.93 % Subject N0. 5 4 9 8 Targeted region GFP AAVI-eGFP AAV2-eGFP AAV2-eGFP (PCL) (PCL) (PCL) (PCL) Left putamen 2.82 i 1.99% 12.3 i 6.27% 1.16 i 0.87% 1.98 i 2.20% Right n 3.59 i 3.01% 9.83 i 5.64% 1.17 i 1.18% 2.68 i 0.90 % Left caudate 7.11 i 4.61% 10.7 i 5.53 % 0.85 i 0.63 % 3.61 i 3.22 % Right caudate 6.95 i 5.82% 4.18 i 2.94% 1.13 i 0.65 % 1.99 i 1.26 % In addition, the efficiency of transduction in cortical s projecting to the striatum was calculated. Since the degree of cortical coverage varied among animals, random cortical areas were d in sections with adjacent GFP—positive striatum. There was an evident discrepancy observed among the animals (Table 8) with no clear ation with the serotype used. The mean neuronal transduction efficiency for AAVl was 13.6 i 8.3 % vs. 13.4 i 9.0 % for AAV2 (p > 0.97).

As mentioned above, AAVl—eGFP transduced many more astrocytes (S—lOO marker) than neurons. GFP—positive astrocytes were detected in the sites of primary transduction (striatum) as well as in cortical regions projecting to striatum. Examples of GFP— transduced astrocytes are shown in C. For AAV2—eGFP vectors, only sporadic GFP+ astrocytes could be detected surrounding the track of the infusion cannulas. To determine whether the vectors transduced other antigen—presenting cells in the brain, representative brain sections from all s were co—stained with antibodies t GFP and Iba—l, specific for microglia. None of the animals showed double—labeled cells, excluding this possibility (E). In turn, in all tested monkeys, staining against Olig—2, the marker for oligodendrocytes, showed only a few cells ve for both GFP and Olig—2. Those sparse cells were detected mainly in the vicinity of the cannula tracks (data not shown).

Brain sections were stained with xylin—eosin (H&E) to ine whether these vectors triggered neuroinflammation. Sections were examined mainly for the presence of perivascular g — the accumulation of lymphocytes or plasma cells in a dense mass around blood vessels. Although varying degrees of such infiltrates were detected in all s, no other vector/transgene—related histological findings were observed. However, AAVl was observed to cause slightly more pronounced infiltration of macrophages and lymphocytes within the primary areas of transduction than did AAV2 (FIGS. 12A and 12B).

Also, vectors produced by Triple Transfection seemed to cause more extensive scular cuffing than vectors generated by Producer Cell Line process. Of note, no infiltrates were detected in projecting areas of transduction (cortical regions).

Example 4: Absence of detectable GFP expression in peripheral tissue outside of the central nervous system (CNS).

Peripheral organ tissues, including kidney, liver, lung, heart, and spleen, were collected at sy to evaluate whether CED administered P transgene expression could be detected outside the CNS.

Results No detectable levels of GFP were detected in any of the organs collected. Both AAV—GFP (TT) (A) and AAV—GFP (PCL) (B) vectors showed no detectable peripheral expression of GFP. Mouse brain tissue injected with AAV2/l—GFP (TT) vectors were used as ve controls for this assay.

Conclusions Efficient delivery of therapeutic proteins to the brain remains a serious obstacle to achieving clinical efficacy while minimizing adverse effects. Developments in gene delivery have provided an opportunity to establish production of biologics within the brain parenchyma. These advances have led to the initiation of multiple al trials in which AAV vectors have become a preferred vector system for treating neurologic ers. gh focal targeting of a specific nucleus can be reliably accomplished by tactic neurosurgical infusion, the extensive convoluted arrangement of the human cortex is not easily targeted by direct infusion of viral vectors. The difficulties in safely achieving widespread gene expression in the brain have hindered the development of potential treatments for ogic diseases which require cortical delivery.

As described herein, AAV vectors (e.g., AAVl and AAVZ) are e of providing ive delivery to the entire primate striatum (caudate and n), as well as delivering to significant numbers of cells within the al cortex (including frontal cortex, occipital cortex, and layer IV), thalamus, and hippocampus. GFP, a reporter protein with no known function in the cerebral cortex, was utilized in the studies discussed herein. AAVl and AAVZ—GFP infused into the caudate and putamen using a CED delivery method resulted in a high level of GFP expression in both caudate and putamen as well as several regions of the cortex. GFP—positive neurons in the frontal cortex were located >20 mm from the AAV— GFP on site, thereby demonstrating axonal transportation of the GFP protein and AAV vector. Without wishing to be bound by theory, because GFP remains cytoplasmic and is not a secreted protein, the presence of GFP in the cortex is thought to indicate direct cellular transduction and active transportation of AAV2 vector along single axonal projections.

Huntington’s disease is an exemplary disease for which striatal ry of AAVs (e.g., CED striatal delivery) may be useful. Huntington’s disease affects both striatal and cortical regions and thus a therapeutic gy that targets both areas is ideal.

The findings sed herein underscore the potential for ry of AAV vectors (e.g., AAVl and AAVZ) to transduce s located a considerable distance from the striatal infusion site. Intrastriatal administration of AAV vectors (e.g., AAVl and AAVZ) is therefore ideal for use in treating CNS disorders that require delivery of therapeutic molecules to the striatum and cortex, including but not limited to, Huntington’s disease. In addition, as AAV vectors ted by the triple transfection method and producer cell line method show comparable transgene expression patterns and levels of transduction, triple ection and producer cell line methods of generating AAV vectors are suitable for use in the present invention.

Claims

1. Use of a recombinant adeno-associated viral (rAAV) particle in the manufacture of a medicament for treating a disorder of the central nervous system (CNS) in a t, wherein the rAAV particle is to be administered to the striatum by convection enhanced ry (CED), wherein the rAAV particle ses a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the occipital cortex and/or layer IV of the cerebral cortex and striatum of the subject, wherein the rAAV particle comprises an AAV 2 capsid, and n the rAAV particle is to be administered to at least one site in the caudate nucleus and two sites in the putamen in each hemisphere of the striatum.

2. The use of claim 1, wherein the heterologous c acid is expressed in the subject’s frontal cortex, occipital cortex, and/or layer IV.

3. The use of claim 1 or 2, wherein the heterologous nucleic acid is further expressed in the subject’s thalamus, subthalamic nucleus, globus pallidus, substantia nigra and/or hippocampus.

4. The use of any one of claims 1-3, wherein the rAAV vector comprises the heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.

5. The use of claim 4, wherein the ITR and the capsid of the rAAV particle are derived from a same AAV serotype; or wherein the ITR and the capsid of the rAAV viral particles are derived from different AAV pes.

6. The use of any one of claims 1-5, wherein the heterologous nucleic acid is operably linked to a promoter. 20263358_1 (GHMatters) P43229NZ00 02/