US20190111157A1 - Enhanced delivery of viral particles to the striatum and cortex - Google Patents

Enhanced delivery of viral particles to the striatum and cortex Download PDF

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US20190111157A1
US20190111157A1 US15/549,962 US201615549962A US2019111157A1 US 20190111157 A1 US20190111157 A1 US 20190111157A1 US 201615549962 A US201615549962 A US 201615549962A US 2019111157 A1 US2019111157 A1 US 2019111157A1
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raav
nucleic acid
aav
striatum
heterologous nucleic
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Lisa M. STANEK
Lamya Shihabuddin
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Genzyme Corp
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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Definitions

  • the present invention relates to the delivery of AAV gene therapy vectors to the brain, e.g., the striatum and/or cortex.
  • Adeno-associated virus (AAV)-based vectors have become the preferred vector system for neurologic gene therapy, with an excellent safety record established in multiple clinical trials (Kaplitt et al., (2007) Lancet 369:2097-2105; Eberling et al., (2008) Neurology 70:1980-1983; Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27-35).
  • Effective treatment of neurologic disorders has been hindered 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, relying on diffusion, which is effective only within a 1- to 3-mm radius.
  • CED convection-enhanced delivery
  • a reflux-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 cannula reflux and leakage of infusate into ventricles (Eberling et al., (2008) Neurology 70:1980-1983; Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27-35; Saito et al., (2011) Journal of Neurosurgery Pediatrics 7:522-526).
  • aberrant events such as cannula reflux and leakage of infusate into ventricles
  • the invention provides a method for delivering a recombinant adeno-associated viral (rAAV) particle to the central nervous system of a mammal comprising administering the 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.
  • rAAV adeno-associated viral
  • 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 striatum of the mammal and wherein the rAAV particle comprises an AAV serotype 1 (AAV1) capsid.
  • AAV1 AAV serotype 1
  • 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 striatum of the mammal and wherein the rAAV particle comprises an AAV serotype 2 (AAV2) capsid.
  • the mammal is a human.
  • 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 administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal cortex, occipital cortex, and/or layer IV of the mammal.
  • 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 cortex, occipital cortex, and/or primary motor cortex.
  • the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex.
  • the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and/or hippocampus.
  • the rAAV particle is administered to the caudate nucleus and the putamen at a rate of greater than 1 ⁇ L/min to about 5 ⁇ L/min.
  • the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the rAAV vector comprises the heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.
  • the heterologous nucleic acid is flanked by two AAV ITRs.
  • the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • the ITR and the capsid of the rAAV particle are derived from the same AAV serotype.
  • the ITR and the capsid are derived from AAV2.
  • the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes.
  • the ITR is derived from AAV2 and the capsid is derived from AAV1.
  • the heterologous nucleic acid is operably linked to a promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • the promoter expresses the heterologous nucleic acid in a brain cell.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or a glial cell.
  • 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.
  • the glial cell is an astrocyte.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible.
  • 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.
  • 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 length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid.
  • the heterologous nucleic acid encodes a therapeutic polypeptide.
  • 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 anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylgluco
  • the heterologous nucleic acid encodes a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.
  • the disorder 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.
  • LSD lysosomal storage disease
  • CBD corticobasal degeneration
  • CBGD corticogasal ganglionic degeneration
  • FTD frontotemporal dementia
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • the disorder is a lysosomal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, a mannosidosis disease, 13 mannosidosis disease, Maroteaux-Lamy, metachromatic 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
  • the rAAV particle is in a composition.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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 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.
  • 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.
  • 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 delivery system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a reflux-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.
  • 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 is administered to the striatum at a rate of greater than 1 ⁇ L/min to about 5 ⁇ L/min.
  • 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.
  • the poloxamer is poloxamer 188.
  • 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 0.001%. In some embodiments, the composition further comprises sodium chloride, 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 embodiments, the composition further comprises sodium phosphate, wherein the concentration of sodium phosphate in the composition ranges from about 5 mM to about 20 mM and the pH is about 7.0 to about 8.0.
  • 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.
  • the composition is administered to the caudate nucleus and the putamen at a rate of greater than 1 ⁇ L/min to about 5 ⁇ L/min.
  • the amount of the composition delivered to the putamen is about twice the volume delivered to the caudate nucleus.
  • about 20 ⁇ L to about 50 ⁇ L of the composition is administered to the caudate nucleus of each hemisphere and about 40 ⁇ L to about 100 ⁇ L of the composition is administered to the putamen of each hemisphere.
  • about 30 ⁇ L of the composition is administered to the caudate nucleus of each hemisphere and about 60 ⁇ L of the composition is administered to the putamen of each hemisphere.
  • the invention provides a method of treating 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.
  • the invention provides a method of treating Huntington's Disease 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.
  • the rAAV particle comprises an AAV1 capsid or an AAV2 capsid.
  • the invention provides a method of treating Parkinson's disease in a mammal comprising administering an effective amount of 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.
  • the rAAV particle comprises an AAV2 capsid.
  • the mammal is a human.
  • 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 administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1. In some embodiments, the heterologous nucleic acid is expressed in at least the frontal cortex, occipital cortex, and/or layer IV of the mammal.
  • 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 cortex, occipital cortex, and/or primary motor cortex.
  • the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex.
  • the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and/or hippocampus.
  • the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the rAAV vector comprises the heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.
  • the heterologous nucleic acid is flanked by two AAV ITRs.
  • the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • the ITR and the capsid of the rAAV particle are derived from the same AAV serotype.
  • the ITR and the capsid are derived from AAV2.
  • the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes.
  • the ITR is derived from AAV2 and the capsid is derived from AAV1.
  • the heterologous nucleic acid is operably linked to a promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • the promoter expresses the heterologous nucleic acid in a brain cell.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or a glial cell.
  • 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.
  • the glial cell is an astrocyte.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible.
  • 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.
  • 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 length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid.
  • 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.
  • the heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the heterologous nucleic acid encodes a miRNA that targets huntingtin.
  • the huntingtin comprises a mutation associated with Huntington's disease.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for treating Huntington's disease.
  • 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.
  • the heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the heterologous nucleic acid encodes a miRNA that targets huntingtin.
  • the huntingtin comprises a mutation associated with Huntington's disease.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for treating Parkinson's disease.
  • 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).
  • the rAAV particle is in a composition.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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 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.
  • 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.
  • 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 delivery system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a reflux-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.
  • the invention provides a system for expression of a heterologous 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 heterologous nucleic acid; and b) a device for delivery of the rAAV particles to the striatum.
  • the rAAV particle comprises an AAV1 capsid or an AAV2 capsid.
  • the mammal is a human.
  • the rAAV particle is administered to the putamen and the caudate nucleus 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 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.
  • 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 cortex, occipital cortex, and/or primary motor cortex.
  • the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex.
  • the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra and/or hippocampus.
  • the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the rAAV vector comprises the heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.
  • the heterologous nucleic acid is flanked by two AAV ITRs.
  • the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • the ITR and the capsid of the rAAV particle are derived from the same AAV serotype.
  • the ITR and the capsid are derived from AAV2.
  • the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes.
  • the ITR is derived from AAV2 and the capsid is derived from AAV1.
  • the heterologous nucleic acid is operably linked to a promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • the promoter expresses the heterologous nucleic acid in a brain cell.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or a glial cell.
  • 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.
  • the glial cell is an astrocyte.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible.
  • 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.
  • 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 length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • 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 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 anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucos
  • the heterologous nucleic acid encodes a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.
  • the disorder 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.
  • LSD lysosomal storage disease
  • CBD corticobasal degeneration
  • CBGD corticogasal ganglionic degeneration
  • FTD frontotemporal dementia
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • the disorder is a lysosomal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, a mannosidosis disease, ⁇ mannosidosis disease, Maroteaux-Lamy, metachromatic 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
  • the rAAV of the invention comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid for treating Huntington's disease.
  • 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.
  • the heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the heterologous nucleic acid encodes a miRNA that targets huntingtin.
  • the huntingtin comprises a mutation associated with Huntington's disease.
  • the rAAV particle of the invention comprises a heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid for treating Parkinson's disease.
  • 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).
  • the rAAV particle is in a composition.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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 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.
  • 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.
  • 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 delivery system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a reflux-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.
  • 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.
  • the rAAV particles comprise an AAV serotype 1 (AAV1) capsid.
  • the rAAV particles comprise an AAV serotype 2 (AAV2) capsid.
  • 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 expressed in at least the cerebral cortex and striatum of the mammal.
  • 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 comprise a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum of the mammal.
  • the rAAV particles of the kits comprise an AAV serotype 1 (AAV1) capsid or an AAV serotype 2 (AAV2) capsid.
  • the kit further comprising a device for delivery of the rAAV particles to the striatum.
  • the rAAV particles of the kit are in a composition.
  • the composition comprises a buffer and/or a pharmaceutically acceptable excipient.
  • the kit comprises instructions for delivery of the composition of rAAV particles to the striatum.
  • the invention provides a rAAV particle for use in any of the methods described above.
  • 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.
  • rAAV recombinant adeno-associated viral
  • 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 comprises an AAV serotype 1 (AAV1) capsid.
  • rAAV adeno-associated viral
  • 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 wherein the rAAV particle further comprises an AAV serotype 1 (AAV2) capsid.
  • rAAV2 adeno-associated viral
  • the invention provides a rAAV particle for use in treating Huntington'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 mammal.
  • 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 mammal.
  • the rAAV particle comprises an AAV2 capsid.
  • the mammal is a human.
  • the rAAV particle of the invention 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 administered to the putamen to rAAV particles administered to the caudate nucleus is at least about 2:1.
  • 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 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.
  • the rAAV particle of the invention comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the rAAV vector comprises the heterologous nucleic acid flanked by one or more AAV inverted terminal repeat (ITR) sequences.
  • the heterologous nucleic acid is flanked by two AAV ITRs.
  • the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • the ITR and the capsid of the rAAV particle are derived from the same AAV serotype.
  • the ITR and the capsid are derived from AAV2.
  • the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes.
  • the ITR is derived from AAV2 and the capsid is derived from AAV1.
  • the heterologous nucleic acid is operably linked to a promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • the promoter expresses the heterologous nucleic acid in a brain cell.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or a glial cell.
  • 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.
  • the glial cell is an astrocyte.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible.
  • 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.
  • 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 length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid.
  • the heterologous nucleic acid encodes a therapeutic polypeptide.
  • 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 anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylgluco
  • the heterologous nucleic acid encodes a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.
  • the disorder 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.
  • LSD lysosomal storage disease
  • CBD corticobasal degeneration
  • CBGD corticogasal ganglionic degeneration
  • FTD frontotemporal dementia
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • the disorder is a lysosomal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, a mannosidosis disease, ⁇ mannosidosis disease, Maroteaux-Lamy, metachromatic 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
  • the rAAV particle is in a composition.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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 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.
  • 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.
  • 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 delivery system. In some embodiments, the CED system comprises a cannula. In some embodiments, the cannula is a reflux-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.
  • FIG. 1 shows Rhesus monkey body weights, 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.
  • TT triple transfection
  • PCL producer cell line
  • 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. 2A-2D extend through the brain in the rostral to caudal direction. Sections from three representative animals are displayed in each panel.
  • AAV1-GFP AAV1-GFP
  • FIGS. 3A-3D show representative brain sections demonstrating cortical 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 infusion of AAV1-GFP (TT) into Rhesus monkey caudate and putamen.
  • AAV1-GFP AAV1-GFP
  • FIGS. 4A-4D show representative brain sections stained 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 rostral to caudal direction. Sections from three representative animals are displayed in each panel.
  • AAV2-GFP AAV2-GFP
  • 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.
  • PCL producer cell lines
  • TT triple transfection
  • FIGS. 6A-6D show representative 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 ) processes into Rhesus monkey caudate and putamen.
  • PCL producer cell line
  • TT triple transfection
  • FIG. 7 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.
  • IHC immunohistochemistry
  • 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).
  • FIG. 8 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 >1.0 indicate that the extent of GFP expression exceeds the boundaries of Gadolinium signal after infusion.
  • the results from monkeys infused with AAV vectors showed that AAV1 spreads better in the brain parenchyma than AAV2 (1.21 ⁇ 0.1 vs. 0.74 ⁇ 0.04; p ⁇ 0.007 with 2-tailed unpaired t-test).
  • FIGS. 9A-9H show GFP expression in the NHP brain transduced with AAV1-eGFP and AAV2-eGFP.
  • FIG. 9A High magnification (40 ⁇ ) of the target structure caudate nucleus transduced with AAV1-eGFP (TT) of subject number 1. Dark-brown GFP+ neurons stained by DAB are visible against densely stained network of positive neuronal fibers. Such a robust signal was detected in all monkeys injected with AAV1-eGFP vector produced by both TT and PCL methods.
  • FIG. 9B Fragment of prefrontal cortex of subject number 1 ( FIG. 7 ) demonstrating massive transport of vector AAV1-eGFP from the sites of injection (striatum) to cortical regions.
  • FIG. 9C Higher magnification (40 ⁇ ) of the frame indicated in FIG. 9B showing numerous cortical neurons expressing GFP.
  • FIG. 9D High (40 ⁇ ) magnification of the cortex from subject number 1 showing GFP+ cells of astrocytic morphology.
  • FIG. 9E High magnification (40 ⁇ ) of the target structure putamen transduced with AAV2-eGFP (TT) of subject number 6. Dark-brown DAB signal show expression of GFP in neurons and their densely stained network of fibers.
  • FIG. 9F Fragment of prefrontal cortex of subject number 6 ( FIG.
  • FIG. 9G Higher magnification (40 ⁇ ) of the frame indicated in FIG. 9F showing numerous cortical neurons expressing GFP.
  • FIG. 9H Higher magnification (20 ⁇ ) of internal capsule of subject number 6 showing GFP+ cells with astrocytic morphology.
  • FIGS. 10A-10E show the cellular tropism of AAV1-eGFP and AAV2-eGFP injected into the monkey brain.
  • Monkey brain sections were processed for double immunofluorescence staining against GFP and various cellular markers to determine cellular tropism of the injected vectors.
  • FIG. 10A Section from caudate 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 20 ⁇ ; right column) from both channels show numerous neurons expressing GFP, verifying neuronal tropism of AAV1-eGFP.
  • FIG. 10A Section from caudate 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 20
  • FIG. 10B Section from caudate nucleus (target structure) from subject number 1 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and astrocytic marker S-100 (red channel for DyLightTM 549 dye; middle column). Merged pictures (magnification 20 ⁇ ; right column) from both channels show numerous astrocytes expressing GFP, verifying that AAV1-eGFP also transduces astrocytes.
  • FIG. 10C Section from caudate nucleus (target structure) from subject number 1 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and astrocytic marker S-100 (red channel for DyLightTM 549 dye; middle column). Merged pictures (magnification 20 ⁇ ; right column) from both channels show numerous astrocytes expressing GFP, verifying that AAV1-eGFP also transduces astrocytes.
  • FIG. 10C Section from caudate nucleus (target structure) from subject number 1 stained with antibodies against GFP
  • FIG. 10D 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 20 ⁇ ; right column) from both channels show numerous neurons expressing GFP, verifying neuronal tropism of AAV2-eGFP.
  • FIG. 10E Section from caudate nucleus (target structure) from subject number 3 stained with antibodies against GFP (green channel for DyLightTM 488 dye; left column) and microglia marker Iba-1 (red channel for DyLightTM 549 dye; middle column). The lack of co-staining of both markers in merged picture (magnification 20 ⁇ ; right column) indicates that AAV1 does not transduce microglia, and this was also the case for AAV2 (data not shown).
  • FIGS. 11A-11C show the efficiency of neuronal transduction in the striatum of NHP injected with AAV1-eGFP and AAV2-eGFP.
  • Double immunofluorescence staining against GFP and neuronal marker NeuN of monkey brain sections was performed to calculate the efficiency of neuronal transduction within the striatum (target structure) and cortical regions.
  • the efficiency of transduction was calculated in the primary area of GFP transduction (PAT) where signal was robust with densely distributed GFP+ neurons ( FIG. 11A ).
  • Neurons were also detected in regions outside the primary areas of GFP transduction (OPAT; FIG. 11C ).
  • Scheme for the technique of counting GFP+ neurons in PAT (inner shading) and OPAT (outer shading) is shown in FIG. 11B .
  • Data from individual counts for each monkey and brain structure are shown in Table 8 (PAT) and Table 9 (OPAT).
  • FIGS. 12A & 12B show vector-related histological findings. Independent evaluation of hematoxylin and eosin (H&E) staining of coronal sections from areas of primary transduction (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 AAV1, especially when the vector was prepared by the TT method.
  • FIG. 12A H&E-stained section from subject number 3 shows numerous perivascular cuffs in the left putamen transduced with AAV1-eGFP (TT).
  • FIG. 12B H&E-stained section from subject number 5 shows only a few localized perivascular cuffs in the left caudate nucleus transduced with AAV1-eGFP (PCL). A few blood vessels are magnified (5 ⁇ ) in the left bottom corner.
  • FIGS. 13A & 13B show quantitative PCT (QPCR) analysis of eGFP mRNA expression in liver, spleen, heart, kidney, and lung samples 1 month following injection of AAV1-eGFP into Rhesus monkey caudate and putamen.
  • FIG. 13A AAV1 and AAV2-eGFP vectors made by a triple transfection (TT) process.
  • FIG. 13B AAV1 and AAV2-eGFP vectors made by a producer cell line (PCL) process.
  • PCL producer cell line
  • AAV vectors e.g., AAV1 and AAV2 vectors
  • CED convection enhanced delivery
  • the methods in the invention may also utilize a delivery device (e.g., a CED device) 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 striatum of a mammal.
  • a delivery device e.g., a CED device
  • the systems and kits of the invention may further include a device for delivery of the rAAV particle to the striatum of a mammal.
  • 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.
  • polynucleotide or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. 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 groups.
  • the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P—NH 2 ) or a mixed phosphoramidate-phosphodiester oligomer.
  • a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
  • 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, peptides, oligopeptides, dimers, trimers, and multimers 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, sialylation, acetylation, phosphorylation, and the like.
  • 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 modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • a “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin).
  • the recombinant nucleic acid is flanked by at least one inverted terminal repeat sequences (ITRs).
  • ITRs inverted terminal repeat sequences
  • the recombinant nucleic acid is flanked by two ITRs.
  • a rAAV vector When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), 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.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a cellular sequence e.g., a gene or portion thereof
  • 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.
  • “Chicken ⁇ -actin (CBA) promoter” refers to a polynucleotide sequence derived from a chicken ⁇ -actin gene (e.g., Gallus gallus beta actin, represented by GenBank Entrez Gene ID 396526).
  • “chicken ⁇ -actin promoter” may refer to a promoter containing a cytomegalovirus (CMV) early enhancer element, the promoter and first exon and intron of the chicken ⁇ -actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequences described in Miyazaki, J. et al. (1989) Gene 79(2):269-77.
  • CAG promoter may be used interchangeably.
  • the term “CMV early enhancer/chicken beta actin (CAG) promoter” may be used interchangeably.
  • genome particles refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality.
  • the number of genome particles in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.
  • vector genome 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.
  • 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.
  • a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a sequence of interest (e.g., a heterologous nucleic acid), and a polyadenylation sequence.
  • a complete vector genome may include a complete set of the polynucleotide sequences of a vector.
  • 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).
  • infection unit (iu), infectious particle, or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.
  • transducing unit (tu) refers to the number of infectious recombinant AAV vector particles that result in the production of a functional heterologous nucleic acid product as measured in functional assays such as described in Examples herein, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).
  • ITR inverted terminal repeat
  • An “AAV inverted terminal repeat (ITR)” sequence 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 between 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 base-pairing to occur within this portion of the ITR.
  • a “terminal resolution sequence” 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 terminal resolution sequence is refractory to cleavage by AAV rep proteins.
  • AAV helper functions 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.
  • 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.
  • helper viruses have been identified, including adenoviruses, herpesviruses and, poxviruses such as vaccinia and baculovirus.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus 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 viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • adenovirus helper functions for the replication of AAV include E1A functions, E1B functions, E2A functions, VA functions and E4orf6 functions.
  • Baculoviruses available 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 infectious helper virus particles is at least about 10 2 :l; at least about 10 4 :l, at least about 10 6 :l; or at least about 10 8 :l or more.
  • preparations are also free of equivalent amounts of helper virus proteins (i.e., 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 contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2 and VP3).
  • AAV helper functions 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.
  • helper viruses have been identified, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.
  • Ad5 Adenovirus type 5 of subgroup C
  • 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 viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Percent (%) sequence identity with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides 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 sequence 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 Biology (Ausubel et al., eds., 1987), Supp.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues 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 identity of A to B will not equal the % amino acid sequence identity of B to A.
  • the % nucleic acid sequence identity of a given nucleic acid sequence C 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 alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
  • An “isolated” molecule e.g., nucleic acid or protein
  • cell means it has been identified and separated and/or recovered from a component of its natural environment.
  • 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 administrations.
  • an effective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease.
  • CED tissue enhanced delivery
  • the term “convection enhanced delivery” may refer to delivery of a therapeutic agent to the CNS by infusion at a rate in which hydrostatic pressure leads to convective distribution.
  • the infusion is done at a rate greater than 0.5 ⁇ L/min.
  • any suitable flow rate can be used such that the intracranial 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 reflux-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).
  • the cannula 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 maintained 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 technique and/or delivered by standard stereotaxic injection equipment and techniques (e.g., the ClearPoint® system from MRI Interventions, Memphis, Tenn.).
  • iMRI intraoperative MRI
  • MRI real-time MRI technique
  • standard stereotaxic injection equipment and techniques e.g., the ClearPoint® system from MRI Interventions, Memphis, Tenn.
  • polystyrene resin may refer to a block copolymer made of a chain of polyoxypropylene flanked 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).
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, 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 prolonging survival as compared to expected survival if not receiving treatment.
  • 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 IT15). It may be characterized by symptoms including abnormal movements (termed chorea), gradual loss of motor function, emotional or psychiatric illnesses, and progressively impaired 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 disease. The normal function of huntingtin is not fully understood. However, mutations in the huntingtin gene are known to cause HD. These mutations are typically inherited in an autosomal dominant fashion and involve expansion of trinucleotide CAG repeats in the HTT gene, leading to a polyglutamine (polyQ) tract in the Htt protein.
  • polyQ polyglutamine
  • a “therapeutic” agent e.g., a therapeutic polypeptide, nucleic acid, transgene, or the like
  • a therapeutic agent is one that provides a beneficial or desired clinical result, such as the exemplary clinical results described above.
  • a therapeutic agent may be used in a treatment as described above.
  • references 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.”
  • 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 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.
  • rAAV adeno-associated viral
  • 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 (AAV1) capsid.
  • AAV1 AAV serotype 1
  • 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 2 (AAV2) capsid.
  • AAV2 AAV serotype 2
  • the invention provides methods for treating Huntington's disease 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.
  • the mammal is a human.
  • the rAAV particle 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 outputs to the basal ganglia (the striatum is also referred to as the striate nucleus and the neostriatum).
  • the striatum controls both motor movements and emotional control/motivation and has been implicated in many neurological diseases, such as Huntington's disease.
  • spiny projection neurons also known as medium spiny neurons
  • GABAergic interneurons GABAergic interneurons
  • cholinergic interneurons GABAergic interneurons
  • GABAergic interneurons GABAergic interneurons
  • cholinergic interneurons Several cell types of interest are located in the striatum, including without limitation spiny projection neurons (also known as medium spiny neurons), GABAergic interneurons, and cholinergic interneurons.
  • 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.
  • the rAAV particle is administered to the caudate nucleus (the term “caudate” may be used interchangeably herein).
  • the caudate nucleus is known as a structure of the dorsal striatum.
  • the caudate nucleus has been implicated in control 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.
  • the rAAV particle is administered to the putamen.
  • the putamen is known as a structure of the dorsal striatum.
  • 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 contains a putamen.
  • rAAV particles may be administered to one or more sites of the striatum.
  • the rAAV particle is administered to the putamen and the caudate nucleus of the striatum.
  • the rAAV particle is administered to the putamen and the caudate nucleus of each hemisphere of the striatum.
  • the rAAV particle is administered to at least one site in the caudate nucleus and two sites in the putamen.
  • the rAAV particle is administered 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 containing 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.
  • the composition containing rAAV particles is administered to any combination of the caudate nucleus of the left hemisphere, the caudate nucleus of the right hemisphere, the putamen of the left hemisphere and the putamen of the right hemisphere.
  • the composition containing rAAV particles is administered to more than one location simultaneously or sequentially. In some embodiments, multiple injections 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 particles are more than about 24 hours apart.
  • a composition of the invention can be delivered (e.g., from about 100 ⁇ L to about 500 ⁇ L of a composition).
  • 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 1 ⁇ , 1.25 ⁇ , 1.5 ⁇ . 1.75 ⁇ , 2 ⁇ , 2.25 ⁇ , 2.5 ⁇ .
  • 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 ⁇ L of the composition is administered to the caudate nucleus of each hemisphere and about 60 ⁇ L of the composition is administered to the putamen of each hemisphere).
  • the volume of the composition administered to the caudate nucleus of each hemisphere is less than about any of the following volumes (in ⁇ L): 50, 45, 40, 35, 30, or 25. In some embodiments, the volume of the composition administered to the caudate nucleus of each hemisphere is greater than about any of the following volumes (in ⁇ L): 20, 25, 30, 35, 40, or 45.
  • 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 selected lower limit of 20, 25, 30, 35, 40, or 45, wherein the lower limit is less than the upper limit.
  • the volume of the composition administered to the putamen of each hemisphere is less than about any of the following volumes (in ⁇ L): 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45.
  • the volume of the composition administered to the putamen of each hemisphere is greater than about any of the following volumes (in ⁇ L): 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 volumes having an upper limit of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or 45 and an independently selected lower limit of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, wherein the lower limit is less than the upper limit.
  • the composition is administered to the striatum at a rate of greater than 1 ⁇ L/min to about 5 ⁇ L/min. In some embodiments, the composition is administered to the caudate nucleus and the putamen at a rate of greater than 1 ⁇ L/min to about 5 ⁇ L/min.
  • the composition is administered to the striatum (the caudate nucleus and/or the putamen) at a rate of greater than about any of 1 ⁇ L/min, 2 ⁇ L/min, 3 ⁇ L/min, 4 ⁇ L/min, 5 ⁇ L/min, 6 ⁇ L/min, 7 ⁇ L/min, 8 ⁇ L/min, 9 ⁇ L/min, or 10 ⁇ L/min.
  • the composition is administered to the striatum (the caudate nucleus and/or the putamen) at a rate of any of about 1 ⁇ L/min to about 10 ⁇ L/min, about 1 ⁇ L/min to about 9 ⁇ L/min, about 1 ⁇ L/min to about 8 ⁇ L/min, about 1 ⁇ L/min to about 7 ⁇ L/min, about 1 ⁇ L/min to about 6 ⁇ L/min, about 1 ⁇ L/min to about 5 ⁇ L/min, about 1 ⁇ L/min to about 4 ⁇ L/min, about 1 ⁇ L/min to about 3 ⁇ L/min, about 1 ⁇ L/min to about 2 ⁇ L/min, about 2 ⁇ L/min to about 10 ⁇ L/min, about 2 ⁇ L/min to about 9 ⁇ L/min, about 2 ⁇ L/min to about 8 ⁇ L/min, about 2 ⁇ L/min to about 7 ⁇ L/min, about 2 ⁇ L/min to about
  • administration of the rAAV particle is performed once. In other embodiments, administration 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 patient response to treatment.
  • the methods comprise 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 striatum.
  • the viral titer of the rAAV particles is at least about any of 5 ⁇ 10 12 , 6 ⁇ 10 12 , 7 ⁇ 10 12 , 8 ⁇ 10 12 , 9 ⁇ 10 12 , 10 ⁇ 10 12 , 11 ⁇ 10 12 , 15 ⁇ 10 12 , 20 ⁇ 10 12 , 25 ⁇ 10 12 , 30 ⁇ 10 12 , or 50 ⁇ 10 12 genome copies/mL.
  • the viral titer of the rAAV particles is about any of 5 ⁇ 10 12 to 6 ⁇ 10 12 , 6 ⁇ 10 12 to 7 ⁇ 10 12 , 7 ⁇ 10 12 to 8 ⁇ 10 12 , 8 ⁇ 10 12 to 9 ⁇ 10 12 , 9 ⁇ 10 12 to 10 ⁇ 10 12 , 10 ⁇ 10 12 to 11 ⁇ 10 12 , 11 ⁇ 10 12 to 15 ⁇ 10 12 , 15 ⁇ 10 12 to 20 ⁇ 10 12 , 20 ⁇ 10 12 to 25 ⁇ 10 12 , 25 ⁇ 10 12 to 30 ⁇ 10 12 , 30 ⁇ 10 12 to 50 ⁇ 10 12 , or 50 ⁇ 10 12 to 100 ⁇ 10 12 genome copies/mL.
  • the viral titer of the rAAV particles is about any of 5 ⁇ 10 12 to 10 ⁇ 10 12 , 10 ⁇ 10 12 to 25 ⁇ 10 12 , or 25 ⁇ 10 12 to 50 ⁇ 10 12 genome copies/mL. In some embodiments, the viral titer of the rAAV particles is at least about any of 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 10 ⁇ 10 9 , 11 ⁇ 10 9 , 15 ⁇ 10 9 , 20 ⁇ 10 9 , 25 ⁇ 10 9 , 30 ⁇ 10 9 , or 50 ⁇ 10 9 transducing units/mL.
  • the viral titer of the rAAV particles is about any of 5 ⁇ 10 9 to 6 ⁇ 10 9 , 6 ⁇ 10 9 to 7 ⁇ 10 9 , 7 ⁇ 10 9 to 8 ⁇ 10 9 , 8 ⁇ 10 9 to 9 ⁇ 10 9 , 9 ⁇ 10 9 to 10 ⁇ 10 9 , 10 ⁇ 10 9 to 11 ⁇ 10 9 , 11 ⁇ 10 9 to 15 ⁇ 10 9 , 15 ⁇ 10 9 to 20 ⁇ 10 9 , 20 ⁇ 10 9 to 25 ⁇ 10 9 , 25 ⁇ 10 9 to 30 ⁇ 10 9 , 30 ⁇ 10 9 to 50 ⁇ 10 9 or 50 ⁇ 10 9 to 100 ⁇ 10 9 transducing units/mL.
  • the viral titer of the rAAV particles is about any of 5 ⁇ 10 9 to 10 ⁇ 10 9 , 10 ⁇ 10 9 to 15 ⁇ 10 9 , 15 ⁇ 10 9 to 25 ⁇ 10 9 , or 25 ⁇ 10 9 to 50 ⁇ 10 9 transducing units/mL. In some embodiments, the viral titer of the rAAV particles is at least any of about 5 ⁇ 10 10 , 6 ⁇ 10 10 , 7 ⁇ 10 10 , 8 ⁇ 10 10 , 9 ⁇ 10 10 , 10 ⁇ 10 10 , 11 ⁇ 10 10 , 15 ⁇ 10 10 , 20 ⁇ 10 10 , 25 ⁇ 10 10 , 30 ⁇ 10 10 , 40 ⁇ 10 10 , or 50 ⁇ 10 10 infectious units/mL.
  • the viral titer of the rAAV particles is at least any of about 5 ⁇ 10 10 to 6 ⁇ 10 10 , 6 ⁇ 10 10 to 7 ⁇ 10 10 , 7 ⁇ 10 10 to 8 ⁇ 10 10 , 8 ⁇ 10 10 to 9 ⁇ 10 10 , 9 ⁇ 10 10 to 10 ⁇ 10 10 , 10 ⁇ 10 10 to 11 ⁇ 10 10 , 11 ⁇ 10 10 to 15 ⁇ 10 10 , 15 ⁇ 10 10 to 20 ⁇ 10 10 , 20 ⁇ 10 10 to 25 ⁇ 10 10 , 25 ⁇ 10 10 to 30 ⁇ 10 10 , 30 ⁇ 10 10 to 40 ⁇ 10 10 , 40 ⁇ 10 10 to 50 ⁇ 10 10 , or 50 ⁇ 10 10 to 100 ⁇ 10 10 infectious units/mL.
  • the viral titer of the rAAV particles is at least any of about 5 ⁇ 10 10 to 10 ⁇ 10 10 , 10 ⁇ 10 10 to 15 ⁇ 10 10 , 15 ⁇ 10 10 to 25 ⁇ 10 10 , or 25 ⁇ 10 10 to 50 ⁇ 10 10 infectious units/mL.
  • the methods comprise 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 striatum.
  • the dose of viral particles administered to the individual is at least about any of 1 ⁇ 10 8 to about 1 ⁇ 10 13 genome copies/kg of body weight. In some embodiments, the dose of viral particles administered to the individual is about 1 ⁇ 10 8 to 1 ⁇ 10 13 genome copies/kg of body weight.
  • the methods comprise 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 striatum.
  • the total amount of viral particles administered to the individual is at least about 1 ⁇ 10 9 to about 1 ⁇ 10 14 genome copies. In some embodiments, the total amount of viral particles administered to the individual is about 1 ⁇ 10 9 to about 1 ⁇ 10 14 genome copies.
  • compositions of the invention 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 administration can be in terms of at least (or, alternatively, less than) minutes, hours, or days.
  • the invention provides methods for delivering rAAV particles to the CNS of a mammal by administering the rAAV particles to the striatum.
  • 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.
  • the rAAV vector encodes a heterologous nucleic acid.
  • a heterologous nucleic acid may encode a therapeutic polypeptide or therapeutic nucleic acid.
  • a therapeutic polypeptide or therapeutic nucleic acid may be used, for example, to ameliorate a symptom, prevent or delay progression, and/or provide a treatment of a disorder (e.g., a disorder described herein).
  • 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 nucleic acid may be expressed in one or more regions of interest within the CNS.
  • the heterologous nucleic acid is expressed in at least the cerebral cortex and striatum.
  • the heterologous nucleic acid may be capable of expression ubiquitously throughout the CNS, or it may be expressed in a subset of CNS cells.
  • the heterologous 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 complex functions. It may be divided into left and right hemispheres. In addition, 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 functions.
  • Occipital cortex may refer to the occipital lobe of the cortex and is known to be involved 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 language, memory, and emotional association.
  • 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 planning, memory, attention, and other higher cognitive tasks and assume the majority of the human cortex
  • sensor processing higher order areas
  • primary sensory areas e.g., auditory, visual, and somatosensory
  • 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.
  • the cerebral cortex may be divided into different cortical layers (moving from superficial to deep), each containing a characteristic pattern of neuronal connectivities and cell types. These layers may be divided into supragranular layers (layers internal granular (IV), and infragranular (V and VI). Supragranular 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 ganglia. Layer V neurons in the primary motor cortex also form the corticospinal tract that is critical for voluntary motor control.
  • Layer IV receives inputs from the thalamus and connects to the rest of the column, thereby providing critical functions related to integration of the thalamus and cortex.
  • Characteristic cells of layer IV include stellate cells (e.g., spiny stellate cells) and pyramidal neurons.
  • the rAAV particle undergoes retrograde or anterograde transport in the cerebral cortex.
  • Retrograde transport refers to the phenomenon by which cargo (e.g., rAAV particles) is moved from a neuronal process (e.g., an axon) to the cell body.
  • Anterograde transport refers to movement from the cell body to the cell membrane (e.g., a synapse).
  • Retrograde transport of AAV particles is thought to occur via receptor-mediated internalization at the axon terminal, followed by microtubule-mediated transport to the nucleus (see, e.g., Kaspar et al., (2002) Mol. Ther. 5:50-56; Boulis et al., (2003) Neurobiol.
  • striatum contains projections from other brain regions, such as regions of the cortex. Both anterograde and retrograde transport may allow rAAV particles to be distributed throughout the brain, such as between the cortex and thalamus (see, e.g., Kells, A. P. et al. (2009) Proc. Natl. Acad. Sci. 106:2407-2411).
  • AAV particles may be 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) through retrograde transport.
  • one brain region e.g., the striatum, caudate nucleus, and/or putamen
  • the AAV particles may be delivered to other areas of the brain (e.g., the cortex) through retrograde transport.
  • the heterologous nucleic acid is further expressed in the thalamus, substantia nigra and/or hippocampus.
  • 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 regions of the brain, particularly those that connect to the cortex.
  • the thalamus is between the cortex and midbrain, 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.
  • the substantia 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 provides dopamine to the striatum that is critical for proper striatal function.
  • 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 alterations, and the like) in a mammal, such as for example, a gene defect that results in a polypeptide deficiency or polypeptide excess in a subject, or for treating or reducing the severity or extent of deficiency in a subject manifesting a CNS-associated disorder linked to a deficiency in such polypeptides in cells and tissues.
  • gene defects e.g., heritable gene defects, somatic gene alterations, and the like
  • methods involve administration of a rAAV vector that encodes one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, microRNAs, 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 disorders: neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxlase (TM, GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase (SOD1) and amino acid decarboxylase (AADC).
  • NAIP neuronal apoptosis inhibitory protein
  • NEF nerve growth factor
  • GDNF glial-derived growth factor
  • BDNF brain-derived growth factor
  • CNTF ciliary neurotrophic factor
  • TM tyrosine hydroxlase
  • GTPCH GTP-cycl
  • a useful transgene 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 TII cofactor tetrahydrobiopterin, may also be used in the treatment of Parkinson's disease.
  • a transgene encoding GDNF or BDNF, or AADC which facilitates conversion of L-Dopa to DA, may also be used for the treatment of Parkinson's disease.
  • a useful transgene may encode: GDNF, BDNF or CNTF.
  • a useful transgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibits the expression of SOD1.
  • a useful transgene may encode NAIP or NGF.
  • a transgene encoding Beta-glucuronidase (GUS) may be useful for the treatment of certain lysosomal storage diseases (e.g., Mucopolysacharidosis type VII (MPS VII)).
  • a transgene encoding 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 combination with the prodrug.
  • a transgene encoding an endogenous opioid, such a ⁇ -endorphin may be useful for treating pain.
  • Other examples of transgenes that may be used in the rAAV vectors of the invention will be apparent to the skilled artisan (See, e.g., Costantini L C, et al., Gene Therapy (2000) 7, 93-109).
  • the heterologous nucleic acid may encode a therapeutic nucleic acid.
  • a therapeutic nucleic acid may include without limitation an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • a therapeutic nucleic acid may encode an RNA that when transcribed from the nucleic acids of the vector can treat a disorder of the invention (e.g., a disorder of the CNS) by interfering with translation or transcription of an abnormal or excess protein associated with a disorder of the invention.
  • 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 include RNAi, small inhibitory RNA (siRNA), micro RNA (miRNA), and/or ribozymes (such as hammerhead and hairpin ribozymes) that can treat disorders by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins.
  • 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.
  • a therapeutic polypeptide may supply a polypeptide and/or enzymatic activity that indirectly counteracts an imbalance in a cell or organism.
  • a therapeutic polypeptide for a disorder related 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.
  • a polypeptide e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated
  • the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.
  • a therapeutic polypeptide or therapeutic nucleic acid may be used to reduce or eliminate the expression and/or activity of a polypeptide whose gain-of-function has been associated with a disorder, or to enhance the expression and/or activity of a polypeptide to complement a deficiency that has been associated with a disorder (e.g., a mutation in a gene whose expression shows similar or related activity).
  • Non-limiting examples of CNS disorders of the invention that may be treated by a therapeutic 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, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, and/or any of the genes described in Fukuda, A. M. and Badaut, J. (2013) Genes ( Basel ) 4:435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and/or any of the genes described in Boison, D.
  • stroke e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, and/or any of the genes described in Fukuda, A. M. and Badaut, J. (2013) Genes ( Basel ) 4:435-456
  • Huntington's disease mutant HTT
  • epilepsy e
  • Parkinson's disease alpha-synuclein
  • Lou Gehrig's disease also known as amyotrophic lateral sclerosis; SOD1
  • Alzheimer's disease tau, amyloid precursor protein
  • corticobasal degeneration or CBD corticobasal degeneration or CBD
  • corticogasal ganglionic degeneration or CBGD corticogasal ganglionic degeneration or CBGD (tau)
  • frontotemporal dementia or FTD tau
  • progressive supranuclear palsy or PSP progressive supranuclear palsy or PSP (tau)
  • MSA alpha-synuclein
  • cancer of the brain e.g., a mutant or overexpressed oncogene implicated in brain cancer
  • lysosomal 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 include 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 without limitation leukodystrophies (including Canavan's disease) and any of the lysosomal storage diseases described below.
  • the therapeutic polypeptide or therapeutic nucleic acid is used to treat a lysosomal storage disease.
  • 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 therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary 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), GM1 gangliosidosis (beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), a mannosidosis disease (a mannosidase, such as alpha-D-mannosidase, MAN2B1), ⁇ mannosidosis disease (beta-mannosidase, MANBA), metachromatic leukodystrophy disease (pseudoarylsulfatase A, ARSA), mucolipidosisII/III disease (N-acetylglucosamine-1-
  • lysosomal storage diseases as well as the defective enzyme associated with each disease, are listed in Table 1 below.
  • 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.
  • Lysosomal storage diseases Defective enzyme Aspartylglusoaminuria Aspartylglucosaminidase Fabry Alpha-galactosidase A Infantile Batten Disease (CNL1) Palmitoyl protein thioesterase Classic Late Infantile Batten Tripeptidyl peptidase Disease (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 Protective protein/cathepsin A Gaucher types 1, 2, and 3 Acid beta-glucosidase GM1 gangliosidosis Acid beta-galactosidase Hunter Iduronate-2-sulfatase Hurler-Scheie Alpha-L-iduronidase Kra
  • the therapeutic polypeptide is caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sul
  • the therapeutic polypeptide may increase or decrease the function of the target polypeptide in the subject (e.g., it may supply the missing function in a lysosomal storage disease, or reduce the level of alpha-synuclein in MSA, such as by blocking its function or dysfunction).
  • the therapeutic nucleic acid is caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfo
  • the therapeutic nucleic acid may increase or decrease the function of the target polypeptide in the subject (e.g., it may supply the missing function in a lysosomal storage disease, or reduce the level of alpha-synuclein in MSA, such as by RNAi).
  • HD Huntington's disease
  • polyQ polyglutamine
  • mHTT mutant huntingtin protein
  • AAV vectors provide an ideal delivery system for nucleic acid therapeutics 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.
  • certain aspects of the invention relate to methods for treating Huntington's disease 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.
  • HD is characterized by progressive 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.
  • CAG repeats in the HTT gene are 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 expansion is associated with cellular aggregates or inclusion bodies, perturbations to protein homeostasis, and transcriptional dysregulation. While these toxic phenotypes may be associated with several parts of the body, they are most typically associated with neuronal cell death.
  • HD patients often display cortical thinning and a striking, progressive loss of striatal neurons.
  • the striatum appears to be the most vulnerable region of the brain to HD (particularly the striatal medium spiny neurons), with early effects seen in the putamen 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, hypothalamus, and cerebellum.
  • Animal models of HD may be used to test potential therapeutic strategies, 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 R6/1 and N171-82Q HD mice (Harper et al., (2005) Proc. Natl. Acad. Sci. USA 102:5820-5825, Rodriguez-Lebron et al., (2005) Mol. Ther. 12:618-633, Machida et al., (2006) Biochem. Biophys. Res. Commun. 343:190-197).
  • Another example of a mouse HD model described herein is the YAC128 mouse model.
  • This model bears a yeast artificial chromosome (YAC) expressing a mutant human HTT gene with 128 CAG repeats, and YAC128 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. Mol. Genet. 21:2219-2232).
  • yeast artificial chromosome expressing a mutant human HTT gene with 128 CAG repeats
  • transgenic rat von Horsten, S. et al. (2003) Hum. Mol. Genet. 12:617-24
  • rhesus monkey Yang, S. H. et al. (2008) Nature 453:921-4
  • 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. 48:356-73).
  • the invention provides methods for ameliorating a symptom of HD, comprising administration of a rAAV particle comprising a rAAV vector encoding a heterologous nucleic acid that is expressed in at least the cerebral cortex and striatum to the striatum.
  • the symptoms of HD include, but are not limited 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 personality, aggression, compulsive behavior, obsessive-compulsive behavior, hypersexuality, psychosis, apathy, irritability, suicidal thoughts, weight loss, muscle atrophy, heart failure, reduced glucose tolerance, testicular atrophy, and osteoporosis.
  • chorea e.g., 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
  • the invention provides methods to prevent or delay progression of HD.
  • Autosomal dominant HD is a genetic disease that can be genotyped.
  • the number of CAG repeats in HTT may be determined by PCR-based 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 clinical symptoms), prenatal screening or prenatal exclusion testing (e.g., by chorionic villus sampling or amniocentesis), or preimplantation screening of embryos.
  • HD may be diagnosed by brain imaging, looking for shrinkage of the caudate nuclei and/or putamen and/or enlarged ventricles. These symptoms, combined with a family history of HD and/or clinical symptoms, may indicate HD.
  • UHDRS Unified Huntington's Disease Rating Scale
  • This rating scale was developed to provide a uniform, comprehensive test for multiple facets of the disease 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 (HDMRS), the HD functional capacity scale (HDFCS), and the quantitated neurological exam (QNE).
  • test useful for determining 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.
  • the methods are used for the treatment of humans with HD.
  • HD is inherited in an autosomal 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 inherited from the paternal side.
  • Huntington disease-like phenotypes have also been correlated with other genetic loci, such as HDL1, PRNP, HDL2, HDL3, and HDL4. It is thought that other genetic loci may modify the manifestation of HD symptoms, including mutations in the GRIN2A, GRIN2B, MSX1, GRIK2, and APOE genes.
  • delivery of recombinant viral particles is by injection of viral particles to the striatum.
  • Intrastriatal administration delivers recombinant viral particles to an area of the brain, the striatum (including the putamen and caudate nucleus), that is highly affected by HD.
  • 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 without limitation projection areas (e.g., the cerebral cortex).
  • the recombinant viral particles are delivered by convection enhanced delivery (e.g., convection enhanced delivery to the striatum).
  • 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 promoter (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 ⁇ -globin promoter (CAG promoter; Niwa cyto
  • the promoter comprises a human ⁇ -glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken ⁇ -actin (CBA) promoter.
  • the promoter can be a constitutive, inducible or repressible promoter.
  • the invention provides a recombinant vector comprising nucleic acid encoding a heterologous nucleic acid of the present disclosure operably linked to a CBA promoter.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter.
  • constitutive promoters 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., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen].
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available 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.
  • 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 ecdysone 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.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (
  • inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • the native promoter, or fragment thereof, for the transgene 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.
  • other native expression control elements such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
  • the regulatory sequences impart tissue-specific gene expression capabilities.
  • the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • Exemplary tissue-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., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci.
  • the tissue-specific promoter is a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adapter molecule 1 (Iba-1).
  • the promoter is a chicken Beta-actin promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • a therapeutic polypeptide or a therapeutic nucleic acid of the invention may be used to treat a disorder of the CNS.
  • 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.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or glial cell.
  • 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.
  • promoters that express transcripts e.g., a heterologous transgene
  • Such promoters can comprise control sequences normally associated with the selected gene or heterologous control sequences.
  • useful heterologous control sequences include those derived from sequences encoding mammalian or viral genes.
  • Examples include, without limitation, the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, may also be used.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).
  • CNS-specific promoters and inducible promoters may be used.
  • CNS-specific promoters include without limitation those isolated from CNS-specific genes such as myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).
  • MBP myelin basic protein
  • GFAP glial fibrillary acid protein
  • NSE neuron specific enolase
  • inducible promoters include DNA responsive elements for ecdysone, tetracycline, metallothionein, and hypoxia, inter alia.
  • the present invention contemplates the use of a recombinant viral genome for introduction of one or more nucleic acid sequences encoding for a heterologous 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 nucleic acid, an ITR, a ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal, and/or origin of replication.
  • the rAAV vector comprises one or more of an enhancer, a splice donor/splice acceptor pair, a matrix attachment site, or a polyadenylation signal.
  • the administration of an effective amount of rAAV particles comprising 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.
  • cells e.g., CNS cells, brain cells, neurons, and/or glial cells
  • the site of administration e.g., the striatum and/or cortex
  • 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.
  • about 5% to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30% to about 50% of the neurons are transduced.
  • Methods to identify neurons transduced by recombinant viral particles expressing miRNA are known in the art; for example, immunohistochemistry, RNA detection (e.g., qPCR, Northern blotting, RNA-seq, in situ hybridization, and the like) or the use of a co-expressed marker such as enhanced green fluorescent protein can be used to detect expression.
  • the invention provides viral particles comprising a recombinant self-complementing genome (e.g., a self-complementary rAAV vector).
  • a recombinant self-complementing genome e.g., a self-complementary rAAV vector.
  • AAV viral particles with self-complementing vector genomes and methods of use of self-complementing AAV genomes are described in U.S. Pat. Nos. 6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which are incorporated herein by reference in its entirety.
  • a rAAV comprising a self-complementing genome will quickly form a double stranded DNA molecule by virtue of its partially complementing sequences (e.g., complementing coding and non-coding strands of a heterologous nucleic acid).
  • 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 nucleic acid sequence along most or all of its length.
  • the first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., the right ITR).
  • the ITR comprises the polynucleotide sequence 5′-CACTCCCTCTCTGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3′ (SEQ ID NO:1).
  • the mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence.
  • 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 sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide and a third AAV ITR.
  • the rAAV particle comprises a rAAV vector.
  • the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a heterologous nucleic acid flanked by one or two AAV inverted terminal repeats (ITRs).
  • the nucleic acid is encapsidated in the AAV particle.
  • the AAV particle also comprises capsid proteins.
  • the nucleic acid comprises the coding sequence(s) of interest (e.g., a heterologous nucleic acid) operatively linked components in the direction of transcription, control sequences including transcription initiation and termination sequences, thereby forming an expression cassette.
  • the expression cassette is flanked on the 5′ and 3′ end by at least one functional AAV ITR sequence.
  • functional AAV ITR sequence it is meant that the ITR sequence functions as intended for the rescue, replication and packaging of the AAV virion. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein in their entirety by reference.
  • the recombinant vectors comprise at least all of the sequences of AAV essential for encapsidation and the physical structures 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 currently known, and new serotypes and variants of existing serotypes continue to be identified.
  • a rAAV vector is a vector derived from an AAV serotype, including without limitation, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV or the like.
  • the nucleic acid in the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.
  • the nucleic acid in the AAV comprises an AAV2 ITR.
  • a vector may include a stuffer nucleic acid.
  • the stuffer nucleic acid may encode a green fluorescent protein.
  • the stuffer nucleic acid may be located between the promoter and the nucleic acid encoding the RNAi.
  • the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid such as ShH10, as described in U.S. PG Pub. 2012/0164106), an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAVrh8R capsid, an AAV9 capsid (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S.
  • AAV9 capsid e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S.
  • an AAV10 capsid an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, a tyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2/2-7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in U.S. PG Pub.
  • a mutant capsid protein maintains the ability to form an AAV capsid.
  • the rAAV particle comprises AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832.
  • the rAAV particle comprises capsid proteins of an AAV serotype from Clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381).
  • the rAAV particle comprises an AAV1 capsid protein or mutant thereof.
  • the rAAV particle comprises an AAV2 capsid protein or mutant thereof.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the rAAV particle comprises an AAV serotype 1 (AAV1) capsid.
  • the rAAV particle comprises an AAV serotype 2 (AAV2) capsid.
  • a rAAV particle can comprise viral proteins and viral nucleic acids derived from the same serotype or different serotypes (e.g., a mixed serotype).
  • a rAAV particle can comprise AAV1 capsid proteins and at least one AAV2 ITR or it can comprise AAV2 capsid proteins and at least one AAV1 ITR. Any combination of AAV serotypes for production of a rAAV particle is provided herein as if each combination had been expressly stated herein.
  • the invention provides rAAV particles comprising an AAV1 capsid and a rAAV vector of the present disclosure (e.g., an expression cassette comprising a heterologous nucleic acid), flanked by at least one AAV2 ITR.
  • the invention provides rAAV particles comprising an AAV2 capsid.
  • the ITR and the capsid are derived from AAV2.
  • the ITR is derived from AAV2 and the capsid is derived from AAV1.
  • rAAV vectors Numerous 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, J E et al., (1997) J. Virology 71(11):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 insect-derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (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) flanked by at least one AAV ITR sequences; and 5) suitable media and media components to support rAAV production.
  • suitable host cells including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems
  • suitable helper virus function provided by wild-type or mutant
  • the AAV rep and cap gene products may be from any AAV serotype.
  • 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 vectors. 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. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No.
  • the AAV helper functions are provided by adenovirus or HSV.
  • the AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).
  • rAAV particles may be produced by a triple transfection method, such as the exemplary triple transfection method provided infra.
  • a triple transfection method such as the exemplary triple transfection method provided infra.
  • 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.
  • 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 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.
  • 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).
  • a cell line e.g., a HeLa cell line
  • a cell line may be stably transfected with a plasmid 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.
  • adenovirus e.g., a wild-type adenovirus
  • Virus may subsequently be harvested, adenovirus may be inactivated (e.g., by heat) and/or removed, and the rAAV particles may be purified.
  • 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.
  • a method for producing any rAAV particle as disclosed herein comprising (a) culturing a host cell under a condition that rAAV particles 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 flanked by at least one AAV ITR, and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell.
  • said at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.
  • said encapsidation protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid proteins or mutants thereof.
  • the encapsidation protein is an AAV5 capsid protein including AAV5 capsid proteins having tyrosine capsid mutations. In some embodiments, the encapsidation protein is an AAV5 capsid protein including AAV5 capsid proteins having tyrosine capsid mutations and the ITR is an AAV2 ITR. In further embodiments, the rAAV particle comprises capsid proteins of an AAV serotype from Clades A-F.
  • the rAAV particles comprise an AAV1 capsid and a recombinant genome comprising AAV2 ITRs, a mutant AAV2 ITR and nucleic acid encoding a therapeutic transgene/nucleic acid.
  • the AAV ITRs are AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV5, AAVrh8, AAVrh8R, AAV5, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • Suitable rAAV production 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).
  • rAAV vectors may be produced in serum-free conditions which may also be referred to as media with no animal-derived products.
  • 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 production 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) suitable to the particular host cell being utilized.
  • 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 fluidized-bed bioreactors.
  • rAAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, 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 production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in U.S. Pat. No. 6,566,118).
  • Suitable methods of lysing cells include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.
  • the rAAV particles are purified.
  • purified 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.
  • isolated rAAV particles may be prepared 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.
  • DNase-resistant particles DNase-resistant particles
  • gc genome copies
  • the rAAV production culture harvest is clarified to remove host cell debris.
  • the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 ⁇ m Filter Opticap XL1O Millipore Express 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 cellulose acetate filter of 0.2 ⁇ m or greater pore size known in the art.
  • the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture.
  • 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 purified 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 inactivation 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.
  • TFF tangential flow filtration
  • SEC size exclusion chromatography
  • nanofiltration nanofiltration
  • the rAAV particle is in a pharmaceutical composition.
  • the pharmaceutical compositions may be suitable for any mode of administration described herein.
  • a pharmaceutical composition of a recombinant viral particle comprising a nucleic acid encoding a therapeutic transgene/nucleic acid can be introduced to the CNS (e.g., the striatum and/or cerebral cortex).
  • the rAAV particle is in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, pH buffering substances, and buffers.
  • 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, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • the administered composition includes rAAV particles and poloxamer.
  • the term “poloxamer” may encompass many compounds because different lengths for the polyoxypropylene and polyoxyethylene chains may be used in combination.
  • a poloxamer may have the chemical formula of HO(C 2 H 4 O) n (C 3 H 6 O) m (C 2 H 4 O) n H, 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.
  • the poloxamer is poloxamer 188 (e.g., CAS No. 9003-11-6).
  • Poloxamers may be described by a numbering system that designates their approximate 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 molecule in the composition. Under this methodology, 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.
  • 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 physical 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 polyoxyethylene block.
  • PLURONIC® 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.
  • the concentration of poloxamer in the composition ranges from about 0.0001% to about 0.01%. In some embodiments, the concentration of poloxamer in the composition is less than about any of the following percentages: 0.01, 0.005, 0.001, or 0.0005. In some embodiments, the concentration of poloxamer in the composition is greater than about any of the following percentages: 0.0001, 0.0005, 0.001, or 0.005. That is, the concentration of poloxamer in the composition 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 embodiments, the concentration of poloxamer in the composition is about 0.001%.
  • the composition further comprises sodium chloride.
  • 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 less than about any of the following concentrations (in mM): 250, 225, 200, 175, 150, or 125. In some embodiments, the concentration of sodium chloride in the composition is greater than about any of the following concentrations (in mM): 100, 125, 150, 175, 200, or 225.
  • 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, 200, or 225, wherein the lower limit is less than the upper limit. In certain embodiments, the concentration of sodium chloride in the composition is about 180 mM.
  • the composition further comprises sodium phosphate.
  • Sodium phosphate may refer to any single species of sodium phosphate (e.g., monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, and so forth), or it may refer to sodium phosphate buffer, a mixture of monobasic and dibasic sodium phosphate solutions. Recipes for sodium phosphate buffers across a range of pH may be found in a variety of standard molecular biology protocols, such as the Promega Protocols & Applications Guide, “Buffers for Biochemical Reactions,” Appendix B part C.
  • the concentration of sodium phosphate in the composition ranges from about 5 mM to about 20 mM. In some embodiments, the concentration of sodium phosphate in the composition is less than about any of the following concentrations (in mM): 20, 15, or 10. In some embodiments, the concentration of sodium phosphate in the composition is greater than about any of the following concentrations (in mM): 5, 10, or 15. That is, the concentration of sodium phosphate in the composition 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.
  • the pH of sodium phosphate in the composition is about 7.0 to about 8.0.
  • the pH of sodium phosphate in the composition is about 7.0, about 7.2, about 7.4, about 7.5, about 7.6, about 7.8, or about 8.0.
  • the pH of sodium phosphate in the composition is about 7.5. Any of the pH values for sodium phosphate described herein may be combined with any of the concentration values for sodium phosphate described above.
  • the concentration of sodium phosphate in the composition is about 10 mM, and the pH is about 7.5.
  • 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).
  • the pharmaceutical composition further comprises a poloxamer (e.g., poloxamer 188, such as PLURONIC® or LUTROL® F68).
  • 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 carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the pharmaceutical composition may further comprise additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, 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.
  • systems for expression of a heterologous 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 heterologous nucleic acid; and (b) a device for delivery of the rAAV particles to the striatum.
  • the systems and devices of the invention may be used to deliver any of the rAAV particles described herein to the CNS (e.g., the striatum) of a mammal.
  • a rAAV particle delivered to the striatum may be used to introduce a rAAV vector encoding a heterologous nucleic acid for expression in the cerebral cortex and striatum.
  • the rAAV particle 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 pressure in which the hydrostatic pressure of the interstitial fluid is overcome. This brings the infusate into contact with the CNS perivasculature, 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.
  • infusate e.g., a composition containing a rAAV particle
  • an advantage of using CED is the enhanced distribution of the infusate throughout the brain.
  • CED may result in improved delivery at the site of injection within the brain (e.g., the striatum, caudate nucleus, and/or putamen).
  • delivery to other regions of the brain e.g., the cerebral cortex, frontal cortex, prefrontal association cortical areas, premotor cortex, primary somatosensory cortical areas, and/or primary motor cortex
  • CED may be achieved through CED.
  • 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 without limitation projection areas (e.g., the cortex).
  • the rAAV particle is delivered using a CED delivery system.
  • AAV particles may be delivered by CED (see, e.g., WO 99/61066).
  • CED may be accomplished using any of the systems described herein.
  • Devices for CED e.g., for delivery of a composition including rAAV particles
  • a pump e.g., an osmotic and/or infusion pump, as described below
  • an injection device e.g., a catheter, cannula, etc.
  • 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.
  • positioning is accomplished through an anatomical map obtained for example by CT and/or MRI imaging of the subject's brain to guide the injection device to the target CNS tissue.
  • iMRI and/or real-time imaging of the delivery may be performed.
  • the device is used to administer rAAV particles to a mammal by the methods of the invention.
  • intraoperative magnetic resonance imaging (iMRI) and/or real-time imaging of the delivery may be performed.
  • 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 imaging 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 et al., (2009) Neuroimage 47 Suppl. 2:T27-35).
  • a tracing agent e.g., an MRI contrast enhancing agent
  • the infusate e.g., a composition including rAAV particles
  • 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.
  • rAAV particles may be administered by standard stereotaxic injection using devices and methods known in the art for delivery of rAAV particles.
  • these methods may use an injection device, a planning system for translating a region of the tissue targeted for delivery into a series of coordinates (e.g., parameters along the latero-lateral, dorso-ventral, and rostro-caudal axes), and a device for stereotaxic localization according to the planned coordinates (a stereotactic device, optionally including the probe and a structure for fixing the head in place in alignment with the coordinate system).
  • 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, Memphis, Tenn.).
  • the device for convection enhanced delivery 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., Hamilton Corp., ALZA Inc. in Palo Alto, Calif.).
  • Pump systems may be implantable. Exemplary pump systems may be found, e.g., in U.S. Pat. Nos. 7,351,239; 7,341,577; 6,042,579; 5,735,815; and 4,692,147. in some embodiments, the pump is a manual pump. Exemplary devices for CED, including reflux-resistant and stepped cannulae, may be found in WO 99/61066 and WO 2006/042090, which are hereby incorporated by reference in its entirety.
  • the device for convection enhanced delivery comprises a reflux-resistant cannula (e.g., a reflux-free step design cannula).
  • a reflux-resistant cannula e.g., a reflux-free step design cannula
  • Further descriptions and exemplary reflux-resistant cannulae may be found, for example, in Krauze et al., (2009) Methods Enzymol. 465:349-362; U.S. PG Pub 2006/0135945; U.S. PG Pub 2007/0088295; and PCT/US08/64011.
  • only one cannula is used. In other embodiments, more than one cannula is used.
  • the device for convection enhanced delivery comprises a reflux-resistant cannula joined with a pump that produces enough pressure to cause the infusate to flow through the cannula to the target tissue at controlled rates. Any suitable flow rate can be used such that the intracranial pressure is maintained at suitable levels so as not to injure the brain tissue.
  • the cannula is a stepped cannula.
  • a stepped cannula has a number of steps (e.g., four in FIG. 1 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 (e.g., the striatum) will depend on the depth of penetration needed to reach that target in the subject.
  • the operator can readily determine the appropriate depth of penetration, taking 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 infusate may be delivered through this tubing.
  • the tubing may be flush with the distal end of the cannula.
  • the tubing extends from the distal end of the cannula, in such embodiments, the amount which the tubing extends may vary depending on the application.
  • the tubing will extend from about 1 mm to about 1 cm from the cannula (or any length therebetween), e.g., from about 1 to about 50 mm (or any length therebetween), or from about 1 mm to about 25 mm (or any length therebetween, including, but not limited to, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 min, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, mm, 23 mm, 24 mm or 25 mm), such as 10 mm beyond the distal end thereof.
  • tubing extending through the cannula may have a coating or surrounding material in one or more regions, for example to protect the tubing in contact with the infusate.
  • tubing e.g., FEP (Teflon) tubing
  • FEP Teflon
  • the fused silica tubing may be connected to the syringe by any suitable means, including, but not limited to, a Luer compression fitting, and the syringe 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 deliver the appropriate amount of product(s). Thus, 1 mL, 2.5 mL, 5 mL, or even larger syringes may be used.
  • Stepped cannulae may be made out of the variety of materials that are physiologically acceptable, including without limitation stainless steel (e.g. 316SS or 304SS), metal, metal alloys, polymers, organic fibers, inorganic fibers and/or combinations thereof.
  • stainless steel e.g. 316SS or 304SS
  • metal e.g. 316SS or 304SS
  • metal alloys e.g. 316SS or 304SS
  • polymers e.g., polymers, organic fibers, inorganic fibers and/or combinations thereof.
  • an infusate-contact surface may extend through the lumen of the cannula.
  • materials may also be used for the o infusate-contact surface, including but not limited to metals, metal alloys, polymers, organic fibers, inorganic fibers and/or combinations thereof.
  • the product-contact surface is not stainless steel.
  • 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 compatible with the biologically active agent or product formulation.
  • 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 (e.g., CNS target tissue).
  • target tissue e.g., CNS target tissue.
  • a non-limiting example of a facilitating agent is low molecular weight heparin (see, e.g., U.S. Pat. No. 7,922,999).
  • 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, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.
  • a disorder of the CNS in a mammal comprising administering a rAAV particle to the mammal according to the methods described herein.
  • methods for treating Huntington's disease in a mammal comprising administering a rAAV particle to the mammal according to the methods described herein using a system as described herein.
  • kits for administering a rAAV particle described herein to a mammal may comprise any of the rAAV particles or rAAV particle compositions of the invention.
  • 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.
  • the kits further comprise any of the devices or systems described above.
  • 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 include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
  • Suitable packaging materials may also be included and may be any packaging materials known in the art, including, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.
  • 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 include a pharmaceutically 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 mammal.
  • 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., N.J. 1991).
  • the kits include one or more pharmaceutically acceptable excipients, carriers, solutions, and/or additional 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.
  • AAV1 The ability of AAV1 to efficiently target both striatal and cortical structures in the Rhesus monkey brain when delivered via convection-enhanced delivery (CED) was evaluated.
  • AAV vectors containing GFP cDNA under the control of cytomegalovirus enhancer/chicken beta-actin (CBA) promoter were infused into the caudate and putamen of 9 adult male Rhesus monkeys using CED (see, e.g., Bankiewicz et al., (2000) Exp. Neurol. 164:2-14 and WO 2010/088560).
  • the head was mounted onto a stereotaxic frame, and the animal transported to the MRI (Siemens 3.0 T Trio MR unit) for a T1-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.
  • TT Triple Transfection
  • PCL Producer Cell Line
  • Prohance (2 mM gadoteridol) was added to the virus.
  • CED convection enhanced delivery
  • Serial MRI was acquired to monitor infusate distribution within each target site and provide real-time feedback to the team.
  • animals were transferred to the operating room, the guide cannula removed, and wound site closed in anatomical layers.
  • T1 MRI was acquired after 0.127 mL infusion into the putamen and 0.207 mL into the thalamus.
  • Post infusion T1 MRI showed extensive infusate distribution within the right thalamus measuring approximately 1 cm in the anterior-posterior direction and 1 cm in the dorso-ventral direction.
  • Coronal T1 showed gadolinium distribution within the target site that extended medially towards the internal capsule and superiorly toward the dorsal putamen. A majority of the infusate was contained within the putaminal margins; however, 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.
  • Blood (approximately 5 mL) was collected prior to injection, approximately 72 hours post-injection, and at necropsy according to the Blood Sample Collection Schedule (see Table 3 below). Approximately 0.5-1.0 mL of whole blood was collected into EDTA tubes for hematology analysis. Approximately 2.0 mL of whole blood was collected into serum separator tubes (with gel, BD Microtainer) and processed to serum to obtain approximately serum for chemistry analysis and AAV1 and AAV2 Capsid antibody analysis.
  • 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, 1-2 mL of CSF was collected, frozen on dry ice, and stored at ⁇ 60° C. At necropsy (prior to PBS perfusion) 2-4 mL of CSF were collected, filtered through a 0.8 micron syringe filter into a labeled collection tube, and transferred in duplicate (1 mL aliquot for Capsid Antibody analysis and 2-4 mL aliquot for GFP analysis) into eppendorf tubes, immediately frozen on dry ice and stored at ⁇ 60° C.
  • mice All animals were euthanized at approximately 30 days after the intracranial dosing procedure. Each animal was euthanized using intravenous administration 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).
  • 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 containing the frontal cortex and midbrain regions were sectioned into free floating 40 micron sections. The entire spinal cord was carefully 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 floating 40 micron sections.
  • each brain block was processed for free-floating sections by rinsing 3 ⁇ in PBS and immersion in 30% sucrose (cryopreservation) before cutting into 40- ⁇ m free-floating sections.
  • AAV vectors Prior to clinical evaluation, AAV vectors are typically produced via the standard triple transfection 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 et 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 expression cassettes. Despite its flexibility and relatively fast turn-around time, the transfection method presents a challenge with regard to scalability, which limits the suitability of this method for large-scale rAAV vector production for clinical use.
  • TT triple transfection method
  • Adeno-associated virus producer cell lines are an effective method for large-scale production of clinical grade AAV vectors.
  • 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.
  • AAV viral vectors were generated for this study using two different production methods: triple transfection (TT) and producer cell line (PCL).
  • Recombinant AAV vectors AAV1-GFP (TT) and AAV2-GFP (TT) were produced by triple transfection (using calcium phosphate) of human embryonic kidney carcinoma 293 cells (HEK-293) (referenced in Xiao et al., (1998) Journal of Virology 72:2224-2232).
  • HEK293 cells were transfected using polyethyleneimine (PEI) and a 1:1:1 ratio of the three plasmids (ITR vector, AAV2rep/cap2 or AAV2rep/cap1, and pAd helper plasmid).
  • the ITR vector plasmid encoded the cDNA for EGFP downstream of the cytomegalovirus enhancer/chicken beta actin—hybrid promoter (CBA).
  • CBA cytomegalovirus enhancer/chicken beta actin—hybrid promoter
  • the pAd helper used was pHelper (Stratagene/Agilent Technologies, Santa Clara, Calif.).
  • AAV1-GFP PCL
  • AAV2-GFP PCL
  • product-specific producer 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 flanked by AAV2 inverted terminal repeats (ITRs), and a Puromycin resistance gene.
  • 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.
  • Sections (3 per each 6-mm block: separation of 2 mm) were washed 3 times in PBST for 5 min each followed by treatment with 1% H 2 O 2 for 20 min. Sections were incubated in Sniper blocking solution (available 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/).
  • GFP staining from matching IHC-stained serial sections was projected onto individual corresponding MRI scans of each monkey brain (T1-weighted MR images in the coronal plane). Distribution/coverage of GFP expression was performed with OsiriX Imaging Software version 3.1 (The OsiriX Foundation, Geneva, Switzerland).
  • 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 hemispheres of each animal and combining values from putamen and caudate nucleus since the mean transduction efficiencies were identical in both structures of each animal.
  • GFP mRNA levels were measured by quantitative real-time PCR. Liver, heart, lung, kidney, and spleen samples were used for all RT-PCR analysis. Total RNA was extracted using the QIAGEN miRNeasy mini kit and then reverse transcribed and amplified using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative RT-PCR reactions were conducted and analyzed on a QuantStudio12K Flex Real-Time PCR System (Applied Biosystems). Each sample was run in duplicate and the relative gene expression was determined using a standard curve.
  • Vd Distribution volume analysis was performed with OsiriX Imaging Software version 3.1 (The OsiriX Foundation, Geneva, Switzerland). Infusion sites, cannula tracks and cannula tip were identified on T1-weighted MR images in the coronal plane. Regions-of-interest (ROIs) were delineated to outline T1 gadolinium signals and target sites (i.e. putamen and caudate nucleus). Three-dimensional volumetric reconstructions of the image series and ROI were analyzed to estimate volume of distribution (Vd) of infusions and ratio to volume of infusate (Vi).
  • ROI Three-dimensional volumetric reconstructions of the image series and ROI were analyzed to estimate volume of distribution (Vd) of infusions and ratio to volume of infusate (Vi).
  • mice were deeply anesthetized with sodium pentobarbital (25 mg/kg i.v.) 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 briefly in PBS and adjusted in a 30% sucrose/PBS solution for cryopreservation.
  • the formalin-fixed brain blocks were cut into 40- ⁇ m coronal sections in a cryostat. Free-floating sections spanning the entire brain were collected in series and were kept in antifreeze solution for further IHC analysis.
  • AAV1-GFP and AAV2-GFP vectors drove abundant expression of GFP from transduced neurons as visualized by immunohistochemistry. After infusion of AAV1 into the caudate and putamen by CED, extensive GFP immunostaining was detected in the caudate and putamen ( FIGS. 2C &D), as well as the substantia nigra ( FIG. 2D ). 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 association cortical areas, the premotor cortex, primary somatosensory cortical areas, and the primary motor cortex, as well as extensive regions of the occipital cortex ( FIGS. 2A-D ).
  • 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 astrocytes ( FIGS. 3A &C) were transduced.
  • AAV2 The ability of AAV2 to efficiently target both striatal and cortical structures in the Rhesus monkey brain when delivered via convection-enhanced delivery (CED) was evaluated.
  • AAV vectors containing GFP cDNA under the control of cytomegalovirus enhancer/chicken beta-actin (CBA) promoter were infused into the caudate and putamen of 8 adult Rhesus monkeys using CED according to the methods described in Example 1 above.
  • FIG. 4C Infusion of AAV2 into the striatum by CED resulted in GFP expression in the injected regions (caudate and putamen) ( FIG. 4C ), substantia nigra ( FIG. 4D ), and a large number of cortical regions of the Rhesus monkey brain ( FIGS. 4A-D ).
  • the expression of GFP within the NHP striatum was comprehensive but relatively contained within the gray matter bounds of the targeted region, with no evidence of significant infusion related leakage or reflux of the AAV2-GFP vector into adjacent non-targeted areas.
  • Cortical GFP expression 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 regions of the occipital cortex were well transduced ( FIGS. 4A-D ).
  • AAV1-GFP vectors made by triple transfection yielded equivalent GFP distribution and coverage when compared to AAV1-GFP vectors made by the producer cell line process.
  • GFP distribution was comparable between AAV1-GFP (TT) ( FIGS. 5C &D) and AAV1-GFP (PCL) ( FIGS. 5A &B) vectors 30 days following injection into the striatum of Rhesus monkeys.
  • FIGS. 6C &D Similar results were seen with the AAV2-GFP vectors. GFP distribution was similar and comparable between AAV2-GFP (TT) ( FIGS. 6C &D) and AAV2-GFP (PCL) ( FIGS. 6A &B) injected brains.
  • TT AAV2-GFP
  • PCL AAV2-GFP
  • AAV1-eGFP TT
  • AAV2-eGFP TT
  • AAV1-eGFP PCL
  • AAV2-eGFP PCL
  • Gadolinium coverage within targeted structures was calculated (OsiriX Imaging software, v. 3.1) by dividing Vd by the volume of Putamen (600 mm 3 ) or Caudate (500 mm 3 ).
  • Cortical GFP coverage was calculated by projecting GFP signal from matching IHC-stained sections onto corresponding MRI scans of each monkey.
  • Gadolinium 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 AAV1, this ratio was 1.21 ⁇ 0.10 whereas for AAV2 it was 0.74 ⁇ 0.04 ( FIG. 8 ). The ratio of 1.0 indicates a perfect match between GFP expression and vector distribution as determined by MRI. This difference indicated that AAV1 vector distributed beyond the Gd signal and achieved better spread in the primary area of transduction than AAV2.
  • AAV1-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 FIG. 7 ).
  • Other animals showed variability in cortical 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 group).
  • Treatment Group 2 (ssAAV2/1-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 pallidus, substantia nigra, thalamus, subthalamic nucleus, and cortical regions).
  • 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 immunofluorescence 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 analyzed monkey brain).
  • Subject showed robust transduction in putamens and caudate nuclei as well as globus pallidus 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 frontal regions of the cortex.
  • GFP signal was also detected in occipital cortex, the density of GFP-positive cells was significantly lower.
  • the cortical GFP expression coverage was accounted for 50% (Table 7).
  • GFP-positive cells had both neuronal and astrocytic morphology, which was confirmed by double immunofluorescence.
  • Subject showed strong GFP expression in putamens and caudate nuclei as well as all projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus, and cortex). Although GFP signal was clearly detected in some regions of cortex, GFP expression was accounted for only 41% of its overall cortical 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 (ssAAV2/2-CBA-eGFP TT)
  • Subject showed a strong GFP signal in both targeted 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 expression coverage in the cortex accounted for 75% (Table 7). GFP-positive cells had mostly neuronal morphology, which was later confirmed by double immunofluorescence. 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.
  • GFP-positive 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 expression coverage in the cortex accounted for 47% (Table 7).
  • GFP-positive cells had mostly neuronal morphology, which was later confirmed by double immunofluorescence.
  • 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 (ssAAV2/1-CBA-eGFP PCL)
  • Subject showed very robust GFP expression in targeted structures, putamen and caudate 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 immunofluorescence.
  • Subject showed robust GFP expression in the striatum (both putamen and caudate nucleus). GFP-signal was also detected in projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions). 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 (ssAAV2/2-CBA-eGFP PCL)
  • GFP-positive cells had mostly neuronal morphology although GFP-positive cells of astrocyte shape were also detected within white matter tracts (internal capsule) and immediate vicinity of cannula tracks.
  • Subject showed robust GFP expression in the striatum and projected structures (globus pallidus, substantia nigra, subthalamic nucleus, thalamus and cortical regions).
  • the GFP expression coverage in the cortex accounted 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.
  • FIGS. 9A-9D For both groups of NHPs transduced with AAV1-eGFP vectors (TT and PCL), the morphology of GFP-positive cells suggested both neuronal and astrocytic transduction ( FIGS. 9A-9D ). This was confirmed by double immunofluorescence staining with a combination of antibodies against GFP and NeuN (neuronal marker) or GFP and S-100 (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 detected in the internal capsule ( FIG. 9H ) as well as in cortical regions of white matter where the infusion cannula tracks were visible.
  • FIG. 11A summarizes the findings in the striatum. Individual calculations for each animal are shown in Table 8 below.
  • TT AAV1-eGFP
  • TT AAV2-eGFP
  • AAV1-eGFP transduced many more astrocytes (S-100 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 FIG. 10C .
  • For AAV2-eGFP vectors only sporadic GFP+astrocytes could be detected surrounding the track of the infusion cannulas.
  • representative brain sections from all monkeys were co-stained with antibodies against GFP and Iba-1, specific for microglia. None of the animals showed double-labeled cells, excluding this possibility ( FIG.
  • perivascular cuffing the accumulation of lymphocytes or plasma cells in a dense mass around blood vessels. Although varying degrees of such infiltrates were detected in all monkeys, no other vector/transgene-related histological findings were observed. However, AAV1 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 perivascular cuffing than vectors generated by Producer Cell Line process. Of note, no infiltrates were detected in projecting areas of transduction (cortical regions).
  • Peripheral organ tissues including kidney, liver, lung, heart, and spleen, were collected at necropsy to evaluate whether CED administered AAV-GFP transgene expression could be detected outside the CNS.
  • AAV vectors are capable of providing extensive delivery to the entire primate striatum (caudate and putamen), as well as delivering to significant numbers of cells within the cerebral 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.
  • AAV1 and AAV2-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 infusion site, thereby demonstrating axonal transportation of the GFP protein and AAV vector.
  • 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 delivery of AAVs (e.g., CED striatal delivery) may be useful. Huntington's disease affects both striatal and cortical regions and thus a therapeutic strategy that targets both areas is ideal.
  • AAV vectors e.g., AAV1 and AAV2
  • intrastriatal administration of AAV vectors 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.
  • AAV vectors generated by the triple transfection method and producer cell line method show comparable transgene expression patterns and levels of transduction, triple transfection and producer cell line methods of generating AAV vectors are suitable for use in the present invention.

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