WO2023183594A2 - Methods and compositions for the treatment of parkinson's disease - Google Patents

Methods and compositions for the treatment of parkinson's disease Download PDF

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Publication number
WO2023183594A2
WO2023183594A2 PCT/US2023/016270 US2023016270W WO2023183594A2 WO 2023183594 A2 WO2023183594 A2 WO 2023183594A2 US 2023016270 W US2023016270 W US 2023016270W WO 2023183594 A2 WO2023183594 A2 WO 2023183594A2
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subject
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months
raav
mds
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French (fr)
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WO2023183594A3 (en
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Krystof S. Bankiewicz
Adrian P. Kells
Amber VAN LAAR
Massimo S. FIANDACA
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AskBio Inc
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Asklepios Biopharmaceutical Inc
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Priority to IL315725A priority patent/IL315725A/en
Priority to KR1020247032559A priority patent/KR20250006358A/ko
Priority to CA3255025A priority patent/CA3255025A1/en
Priority to JP2024556470A priority patent/JP2025510150A/ja
Priority to EP23775736.4A priority patent/EP4499155A4/en
Priority to CN202380042769.7A priority patent/CN119256090A/zh
Priority to US18/850,414 priority patent/US20250222072A1/en
Publication of WO2023183594A2 publication Critical patent/WO2023183594A2/en
Publication of WO2023183594A3 publication Critical patent/WO2023183594A3/en
Priority to MX2024011436A priority patent/MX2024011436A/es
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the technology described herein relates to methods for treating neurological diseases or disorders, e.g., Parkinson’s disease.
  • Parkinson’s disease is a progressive neurodegenerative disease that advances inexorably over a period of 10 to 30 years to disability and death.
  • Medications generally those aimed at ameliorating the known striatal dopamine deficiency, can provide substantial clinical benefits for the cardinal motor signs of PD, namely rest tremor, rigidity, bradykinesia and postural instability.
  • disease progression continues since dopamine replacement and other medical therapies do not impact the underlying neurodegenerative process.
  • Clinical responses to anti-parkinsonian medications wane over time and a variety of drug-related complications ensue, including motor fluctuations, dyskinesias, and neuropsychiatric manifestations.
  • DBS Deep brain stimulation
  • Duopa is a levodopa / carbidopa intestinal gel administered via a gastrostomy tube connected to an external portable pump to provide consistent dosing.
  • Duopa requires the need to maintain stoma site and the inconvenience of carrying external components. Due to oxidation of Duopa, this therapy is approved for 16 hr/day and therefore leaves some patients inadequately treated overnight.
  • PD is a progressive, multicentric neurodegenerative disease characterized by tremor at rest, rigidity, bradykinesia and postural instability.
  • the majority of PD is an idiopathic disease and the second most common neurodegenerative disorder after Alzheimer's disease. Patients struggle with emotional symptoms including depression and anxiety and with characteristic motor features and movement disturbances. There is currently no cure for PD; therapeutic options are limited to ameliorating disease symptoms.
  • One aspect provided herein describes a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene (sometimes referred to as a transgene), and wherein the subject does not exhibit an increase in PD-associated symptoms for a least 6 months following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • the rAAV is introduced via systemic introduction. [0010] In one embodiment of any aspect herein, the rAAV is introduced via local introduction.
  • local introduction is introduction directly to the subject’s putamen.
  • the local introduction comprises directly introducing the rAAV to each of the subject’s putamen.
  • the local introduction is performed in simultaneously with non-invasive imaging.
  • the non-invasive imaging techniques include intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED), ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); positron emission tomography (PET); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof.
  • iMRI intraoperative magnetic resonance image
  • CED computed tomography
  • fMRI functional magnetic resonance imaging
  • PET positron emission tomography
  • EEG electroencephalography
  • MEG magnetoencephalography
  • fNIRS functional near-infrared spectroscopy
  • the local introduction comprises introducing about half of the total delivered dose of rAAV vector to each putamen via intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED).
  • iMRI intraoperative magnetic resonance image
  • CED convection enhanced delivery
  • local introduction further comprises introducing an MRI contrast agent at substantially the same time as the AAV vector.
  • the MRI contrast agent is gadoteridol.
  • the MRI contrast agent is introduced to the subject in the same composition as the rAAV. In one embodiment of any aspect herein, the MRI contrast agent is introduced to the subject in a different composition as the rAAV.
  • the rAAV is introduced via systemic (e.g., intravenous) introduction.
  • the transduction and/or coverage of the putamen is assessed via Magnetic-resonance imaging.
  • at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the volume of the subject’s putamen is transduced with the GDNF gene.
  • the subject does not exhibit a substantial increase in PD-associated symptoms for at least 12 months immediately following the introducing as compared to prior to the introducing.
  • the subject exhibits a decrease in PD-associated symptoms for at least 6 months or more immediately following the introducing as compared to prior to introducing.
  • the subject exhibits a decrease in PD-associated symptoms for a least 12 months or more immediately following the introducing as compared to prior to introducing.
  • the subject has an initial Movement Disorder Society-Unified Parkinson Disease Rating Scale (MDS-UPDRS) score, prior to introduction, that is less than 32.
  • MDS-UPDRS Movement Disorder Society-Unified Parkinson Disease Rating Scale
  • the slowing or inhibiting the progression of Parkinson’s’ disease in the subject is characterized by a second MDS-UPDRS score 6 months immediately following the introducing that is not substantially higher than the initial MDS-UPDRS score.
  • the slowing or inhibiting the progression of Parkinson’s’ disease in the subject is characterized by a second MDS-UPDRS score about 12 months immediately following the introducing that is not substantially higher than the initial MDS-UPDRS score.
  • the subject has an initial MDS-UPDRS score, prior to introduction, that is greater than or equal to 32.
  • the subject exhibits a decrease in the initial MDS- UPDRS score for at least 6 months immediately following the introducing as compared to prior to introducing.
  • the slowing or inhibiting the progression of Parkinson’s’ disease in the subject is characterized by a second MDS-UPDRS score about 6 months immediately following the introducing that is at least about 20% lower than the initial MDS-UPDRS score.
  • the slowing or inhibiting the progression of Parkinson’s’ disease in the subject is characterized by a second MDS-UPDRS score about 12 months immediately following the introducing that is at least about 30% lower than the initial MDS-UPDRS score
  • the method further comprises, prior to introducing, determining an initial MDS-UPDRS score for the subject.
  • the method further comprises, prior to introducing, receiving results of an assay that provides an initial MDS-UPDRS score for the subject.
  • slowing or inhibiting the progression of PD in the subject is characterized by a reduction of an initial MDS-UPDRS score following introduction.
  • the reduction is an at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater reduction of the initial MDS-UPDRS score 6 months following introduction.
  • the reduction is an at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater reduction of the initial MDS-UPDRS score 12 months following introduction.
  • slowing or inhibiting the progression of PD in the subject is characterized stabilization of an initial MDS-UPDRS score following introduction.
  • the stabilization is characterized by no more than a 10% increase or decrease of the initial MDS-UPDRS score. In one embodiment of any aspect herein, stabilization occurs for at least 6 months or longer.
  • the subject is mildly affected by PD.
  • the subject mildly affected by PD has an initial MDS-UPDRS score less than 32 prior to the introduction of rAAV and was diagnosed with PD less than 5 years prior to the introduction.
  • the method further comprises, prior to the introduction, diagnosing the subject as being mildly affected by PD.
  • the method further comprises, prior to the introduction, receiving the results of an assay that diagnoses the subject as being mildly affected by PD.
  • the subject is moderately affected by PD.
  • the subject moderately affected by PD has an initial MDS-UPDRS score equal to or greater than 32 prior to the introduction of rAAV and was diagnosed with PD less than 4 years prior to the introduction.
  • the method further comprises, prior to the introduction, diagnosing the subject as being moderately affected by PD.
  • the method further comprises, prior to introduction, receiving the results of an assay that diagnoses the subject as being moderately affected by PD.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the promoter is a nervous system (NS) or central nervous system (CNS) specific promoter.
  • NS nervous system
  • CNS central nervous system
  • the NS specific promoter is selected from the NS specific promoters in Table 1.
  • the CNS specific promoter is selected from the CNS specific promoters in Table 2.
  • the nucleic acid comprises a sequence of SEQ ID NO: 1, or a functional variant that is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO: 1.
  • the rAAV is AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or a rational haploid thereof. In one embodiment of any aspect herein, the rAAV is AAV2.
  • the rAAV exhibits brain-specific tropism. In one embodiment of any aspect herein, the rAAV comprises a modification that increases its brain-specific tropism. In one embodiment of any aspect herein, brain-specific tropism is increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater as compared to an unmodified AAV.
  • the rAAV is introduced at a total dose within the range of 5x10 12 vg to about 1.5x10 13 vg.
  • about one half of the total dose is administered to each of the subject’s putamen.
  • introducing is performed at a flow rate of from about 1 ⁇ L/min to about 30 ⁇ L/min.
  • the rAAV is introduced as a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier.
  • the liquid composition has an rAAV concentration of from about 3x10 12 vg/mL to about 4x10 12 vg/mL.
  • the subject is administered at least one anti-PD therapeutic prior to the introduction of the rAAV.
  • the subject is administered at least one anti-PD therapeutic prior to and following the introduction of the rAAV.
  • the at least one anti-PD therapeutic is selected from the group consisting of levodopa, Sinemet, Rytary, Stalevo, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, entacapone.
  • the subject maintains or decreases the dose of the at least one anti-PD therapeutic following introduction. In one embodiment of any aspect herein, the dose of the at least one anti-PD therapeutic is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.
  • Another aspect provided herein describes a method of slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject in need thereof comprising locally introducing to the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene.
  • rAAV recombinant adeno-associated virus
  • Another aspect provided herein describes a method of slowing or inhibiting a progression of PD in a subject in need thereof comprising transducing greater than or equal to about 30% of the volume of the subject’s putamen with a glial cell line-derived neurotrophic factor (GDNF) gene, wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months following the transducing.
  • the transducing is performed by administering a rAAV comprising the GDNF gene to each of the subject’s putamen.
  • Another aspect provided herein describes a method of reducing or stabilizing an initial Movement Disorder Society-Unified Parkinson’s Disease Rating Scale Part (MDS-UPDRS) score in a subject having Parkinson’s disease (PD) comprising administering to the subject’s putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the subject has a second MDS- UPDRS score at 6 months following the administration is decreased or stabilized as compared to the initial MDS-UPDRS score of the subject prior to administering.
  • MDS-UPDRS Movement Disorder Society-Unified Parkinson’s Disease Rating Scale Part
  • the method further comprises the step of, prior to administering, obtaining or receiving an initial MDS-UPDRS score from the subject.
  • the second MDS-UPDRS score is decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater as compared to the initial MDS-UPDRS score 12 months following administering.
  • stabilization is no more than a 10% increase or decrease of the initial MDS-UPDRS score.
  • Another aspect provided herein describes a method of treating a subject mildly affected by Parkinson’s disease (PD) comprising administering to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with GDNF, and wherein the subject has a second MDS-UPDRS score at 6 months post-administering that is stabilized as compared to the initial MDS-UPDRS score.
  • the subject has a MDS-UPDRS score at 12 month post-administering that is stabilized as compared to the initial MDS-UPDRS score prior to administering.
  • Another aspect provided herein describes a method of treating a subject moderately affected by Parkinson’s disease (PD) comprising administering to each of the subject’s putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with the transgene, and wherein the subject has a second MDS-UPDRS score at 6 months post-administering that is at least about 20% lower than the initial MDS-UPDRS score.
  • the reduction is an at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater as compared to the initial MDS-UPDRS score.
  • Another aspect provided herein describes a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising locally introducing to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing an MRI contrast agent to each of the subject’s putamen at substantially the same time as the rAAV, wherein at least 30% of the volume of the subject’s putamen is transduced with the nucleic acid, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • Another aspect provided herein describes a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • compositions for slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject comprising a recombinant adeno-associated virus (rAAV) comprising a genome comprising a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter; and a pharmaceutically acceptable carrier.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • the composition has a rAAV concentration of 3x10 12 vg to 4x10 12 vg per mU.
  • the composition comprises an rAAV concentration of 3.3x10 12 vg per mU.
  • Another aspect provided herein describes a formulation for slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject comprising an adeno-associated virus (AAV) at a concentration of 3x10 12 vg to 4x10 12 vg per mU of a pharmaceutically acceptable carrier, wherein the rAAV comprises a genome comprising a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter.
  • GDNF glial cell line-derived neurotrophic factor
  • Fig. 1 shows a schematic of the clinical study schedule.
  • the subject will cycle “ON” and “OFF” their prescribed anti -Parkinson’s therapeutic as indicated by “OFF” (off medication) and “ON” (on medication) arrows.
  • MRIs, FDG, and DaT scans are administered as indicated by crosses.
  • Blood work is taken as indicated by the droplet.
  • the subject’s activity is monitored as indicated by the hexagon. For visits requiring evaluation in the defined OFF medication state, participants are asked to stop all PD medications (e.g.
  • Fig. 2 shows a summary of the subject’s included in Cohort A (mildly affected by PD).
  • Fig. 3 shows a summary of the subject’s included in Cohort B (moderately affected by PD).
  • Fig. 4 shows a summary of the percent volume of a subject’s putamen that is transduced with GDNF following local administration of AAV2-GDNF. The average volume of putamen transduced with GDNF is 63%.
  • Fig. 5 presents a bar graph showing the percent volume of a subject’s putamen that is transduced with GDNF following local administration of AAV2-GDNF operatively linked to CMV promoter. The average volume of putamen transduced with GDNF is -63%.
  • Figs 6A and 6B show representative post-surgical MRI of subjects in Cohort A.
  • Fig. 6A shows MRI T1 (pre-contrast) images a period of time following administration of the therapeutic.
  • Fig. 6B shows MRI T2 images a period of time following administration of the therapeutic. Variable appearance of putaminal hyperintensities are observed.
  • Figs 7A and 7B show representative post-surgical MRI of subjects in Cohort B.
  • Fig. 7A shows MRI T1 (pre-contrast) images a period of time following administration of the therapeutic.
  • Fig. 7B shows MRI T2 images a period of time following administration of the therapeutic. Variable appearance of putaminal hyperintensities are observed.
  • Figs 8A-8H present data that assess PD progression at various time points post administration.
  • Fig. 8A-8D present line graphs showing the change in MDS-UPDRS aggregate motor skills either on an anti-Parkinson’s therapeutic (Fig. 8A) or off an anti -Parkinson’s therapeutic (Fig. 8B).
  • a stabilized MDS-UPDRS score is observed in cohort A (represented by “mild PD cohort”) for 12 months.
  • a marked decrease in MDS-UPDRS score is observed in cohort B (represented by “moderate PD cohort”).
  • MDS-UPDRS scores were assessed when screening prior to administration, to establish a baseline, and then 3, 6, 9 and 12 months post-surgery.
  • Total UPDRS scores Fig.
  • FIGs 8E-8H show stable motor measures over 18 months post AAV2-GDNF dosing in the mild PD cohort (FIGs. 8E and 8F) and motor improvement over 18 months post AAV2-GDNF dosing in moderate PD cohort (Figs 8G and 8H).
  • FIGs 8E and 8F (A) indicates stability demonstrated over 18 months in mild PD.
  • (B) indicates limited window to measure large magnitude of functional improvements in the mild PD.
  • (C) indicates one outlier identified as having a TH mutation.
  • FIG. 9A-9D present data showing PD motor diary data.
  • FIG. 9A-9B Bar graphs showing PD motor diary data for cohort A (Fig. 9A) and cohort B (Fig. 9B). The diary “on/off’ times have been normalized to 16-hour waking times.
  • FIG. 9C Pie charts illustrating marked improvement of subjects in cohort B 12-months and 18-months post administration. Good ON time was improved by 27% from baseline and OFF time was improved by 52% from baseline.
  • FIG. 9D Pie charts illustrating marked improvement of subjects in cohort A 12-months and 18-months post administration. Good ON time was decreased by 12% from baseline and OFF time was increased by 46% from baseline.
  • Fig. 10A and 10B present plot graphs showing NMSS (circles) and PDG-39 (squares) for cohort A (mildly affected; Fig. 10A) and cohort B (moderately affected; Fig. 10B).
  • the NMSS and PDQ-39 scores were assessed when screening prior to administration, 6 months post-surgery, 12 months post-surgery, 18 months post-surgery, and 24 months post-surgery.
  • Fig. 11 presents a line graph showing the dose response of AAV2-GDNF in cohort B (moderately affected) versus phase 1 of the trial.
  • the data indicate that there is a dose and putamen coverage (>50%) correlation with clinical response in moderate-stage PD.
  • the magnitude of functional motor improvement in moderate -stage PD exceeded expectations of anticipated placebo effect (-5pt).
  • Fig. 12 presents a graph showing volumetric distribution of putaminal infusions, including for gene therapy, are highly dependent on the infusion volume delivered, as predicted in animal models.
  • the average unilateral putaminal volume is approximately 4200 cubic mm.
  • early gene therapy products infused within the putamen provided limited volumes of distribution, much less than 50% of the total putaminal volume.
  • Initial limitations in distribution volumes were primarily a result of the small infusion volumes delivered and utilizing the standard bi-frontal trajectories to the putamen.
  • the standard bi- frontal approach provides trajectories that are nearly perpendicular to the long axis of the putamen; such trajectories volumetric coverage of the putamen is limited by the small dorsoventral putaminal dimension, requiring multiple trajectories to expand volumetric coverage.
  • the evolution of gene therapy infusions that parallel the long axis of the putamen has provided for much larger infusion volumes (up to 1800 microliters/putamen) and achieving putaminal coverage of >50%.
  • FIGs 13A and 13B show schematics of bi-frontal and bi-occipital trajectory techniques.
  • FIG. 13A Bi-frontal Trajectories — One or more frontal burr hole(s) is made bilaterally. Minimum of 2 trajectories per putamen to cover pre- and post-commissural putamen. Trajectories are nearly perpendicular to long axis of putamen and volumetric coverage primarily limited by short dorsoventral dimension of putamen and number of trajectories used. Putaminal volumetric coverage typically achieved is ⁇ 50%.
  • FIG. 13B Bi-occipital Trajectories — A single occipital burr hole is made per putamen.
  • This technique requires a single trajectory per putamen to cover pre- and post- commissural putamen. Trajectories parallel to long axis of putamen and volumetric coverage primarily limited by perivascular leakage from within putamen. Putaminal volumetric coverage typically achieved is >50%.
  • FIG. 14A and 14B present a summary of a previous, completed trial and the current ongoing trial.
  • FIG. 14A presents a chart showing the clinical experience with both the bi-frontal and bi- occipital delivery methods for AAV2-GDNF gene therapy to the putamen in Parkinson’s disease.
  • FIG. 14A provides details from previous, completed Phase 1 and ongoing clinical trials testing the safety and tolerability of differing vector doses and putaminal coverage in advanced, moderate, and earlier stages of PD.
  • the current clinical trial (as described in Examples 1-3 herein below) is the first human gene therapy trial to be approved for testing the safety of a gene therapy product in participants with earlier stage PD.
  • the clinical study has enrolled and treated 11 of the 12 planned participants.
  • the Phase 1 study delivered 450 microliters of infusion volume (at 9x10 10 vg to 9x10 11 vg) to each putamen of 13 participants, resulting in a mean putaminal coverage of 26%.
  • FIG. 14B presents a summary of putaminal coverage achieved in the previous, completed Phase 1 trial and the current clinical trial (Phase lb; as described in Examples 1-3 herein below) for indicated cohort.
  • FIG. 15 presents a schematic of AAV-GDNF.
  • CMV cytomegalovirus
  • hGDNF human glial cell line-dervied neurotrophic factor
  • hGH human growth hormone
  • ITR inverted terminal repeat.
  • FIG. 16 presents a schematic of the study design.
  • AAV2-GDNF was administered via onetime, MRI-monitored CED to bilateral putamina (up to 1.8 mL per putamen with maximum dose of 1.2 x 10 13 vg) and contrast agent to visualize distribution (2mM gadoteridol).
  • Fig. 17 present a summary of postoperative adverse effects (i.e., treatment emergent adverse events (TEAEs)) observed more than 1 month after surgery.
  • TEAEs treatment emergent adverse events
  • FIG. 19A MRI image of participant of intraputaminal administration of AAV2- GDNF.
  • FIG. 19B-19D Tyrosine hydroxylase staining of putamen biopsy sample showing enrichment of dopaminergic neurons in the putamen.
  • FIG. 19B shows area in FIG. 19A as indicated by arrow.
  • FIG. 19C shows enhanced, zoomed-in image of area in FIG. 19B as indicated by arrow.
  • FIG. 19A MRI image of participant of intraputaminal administration of AAV2- GDNF.
  • FIG. 19B-19D Tyrosine hydroxylase staining of putamen biopsy sample showing enrichment of dopaminergic neurons in the putamen.
  • FIG. 19B shows area in FIG. 19A as indicated by arrow.
  • FIG. 19C shows enhanced, zoomed-in image of area in FIG. 19B as indicated by arrow.
  • FIG. 19A MRI image of participant of intraputaminal administration of AAV2- GDNF.
  • FIG. 19B-19D Tyrosine
  • FIG. 19D shows enhanced, zoomed-in image of area in FIG. 19C as indicated by arrow.
  • FIG. 19E Locations of 6 biopsies performed in sample. Biopsy locations #1 and 5 are the infusion sites used during surgery. Biopsy location #6 is located outside the putamen in white matter tract.
  • FIG. 19F Level of GDNF transgene (pg GDNF/mg protein) in indicated biopsy location. The highest levels of GDNF were found in locations #1 and 5. No expression of GDNF identified in biopsy location #6.
  • Fig. 20 presents data showing longitudinal MRI monitoring for safety reads.
  • T1 (top row) and T2 (bottow row) weighted MRI brain scans in the left column show gadoteridol distribution (bright white signal from T1 image) following bilateral infusion into the putamen (outlines).
  • Matched MRI scans acquired at 6 and 18 month time points demonstrate no remaining gadoteridol signal or tissue abnormalities in the putamen or other brain structures.
  • Fig. 21 presents a chart depiciting response of moderate PD cohort. A strong and more progressive restoration and motor function was found as compared to previous CGTs. 18 months clinical data shows (1) stronger improvements than previous neurotrophic CGTs, (2) AAV2- GDNF effects are more progressive than previous neurotrophic factor GTx with continuous improvement after six months, unlike brief improvement in other CGTs, and (3) clinically meaningful improvements beyond six months consistent with anticipated Mechanism of action, e.g., terminal sprouting and progressive restoration of dopamine function.
  • Figs 22A and 22B present charts showing unified dyskinesia rating scale historical, objective, and total scores up to 18 months post treatment for mild (Fig. 22A) and moderate (Fig. 22B) cohorts.
  • Figs 23A and 23B present charts showing levadopa equivalent daily dose (LEDD) average values for mild (Fig. 23A) and moderate (Fig. 23B) cohorts up to 18 months post treatment.
  • LEDD levadopa equivalent daily dose
  • Figs 24A-24C present data showing preliminary analysis of functional imaging with DaT Scan in mild and moderate cohorts.
  • Fig. 24A present bar graph of values.
  • Figs 24B and 24C show tables presenting values depicted in Fig. 24A for mild (Fig. 24B) and moderate (Fig. 24C) cohorts.
  • Preliminary analysis of change in DAT binding overtime is shown. Reductions in binding in caudate in both mild and moderate cohorts is observed. Relatively stable or increased put him in DaT signal in both cohorts is shown.
  • Figs 25A-25D present data showing change in F-dopa uptake at the infusion site 6 and 18 months after gene therapy administration.
  • Figs 25A-25C MRI images show gadoteridol distribution in the axial (left column) and coronal (right column) planes following bilateral infusion into the interior (precommissural) and posterior (postcommissural) putamina (Fig. 25A).
  • F-dopa Ki parametric maps in axial and coronal planes from one patient at baseline (Fig. 25B) and 18 months after surgery (Fig. 25C) showing increased Ki in the areas corresponding to the infusion sites as visualized as gadoteridol signal in the MRIs.
  • FIG. 26 presents a schematic of a plasmid used to generate the AAV2-GDNF vector, e.g., SEQ ID NO: 64.
  • aspects of the technology disclosed herein relate to administration, e.g., local or systemic, of the glial cell line-derived neurotrophic factor (GDNF) gene such that at least 30% of the subject’s putamen is covered and/or transduced with the gene.
  • This level of coverage and/or transduction is shown to be effective for reducing, slowing, or inhibiting the progression of symptoms related to PD.
  • methods and compositions described by the disclosure are useful, in some embodiments, for the treatment of PD.
  • Parkinson’s disease refers to a neurodegenerative disease characterized by progressively worsening shaking and stiffness and increasing problems with balance, walking, and coordination.
  • PD idiopathic in nature, typically a combination of genetic predisposition and environmental influences acting on epigenetic contols, and a number of genes mutations can contribute to or increase the risk of PD, including those mutations with the synuclein alpha (SNCA; NCBI Gene ID: 6622), leucine rich repeat kinase 2 (LRRK2/PARK8; NCBI Gene ID 120892), glucosylceramidase beta (GBA1; NCBI Gene ID 2629), parkin RBR E3 ubiquitin (PRKN; NCBI Gene ID 5071), PTEN induced kinase 1 (PINK1; NCBI Gene ID 65018), Parkinsonism associated deglycase (DJ1/PARK7; NCBI Gene ID 11315), VPS35 retromer complex component (VPS35; NCBI Gene ID 55737), eukaryotic translation initiation factor 4 gamma 1 (EIF4G1; NCBI Gene ID 1981), DnaJ heat shock protein
  • SNCA
  • PD-associated genes are known in a number of species, e.g., human mRNAs and protein sequences are available in the NCBI database using the provided Gene ID numbers.
  • These PD-associated genes and others, as well as PD-associated alleles thereof are known in the art and described further in, e.g., D’Souza et al. Acta Neuropsychiatrica 2020 32: 10-22; Sardi et al. Parkinsonism & Related Disorders 2019 59:32-38; Hardy et al. Current Opinion in Genetics & Development 2009 19:254-65; Ferreria et al.
  • Risk factors for developing PD include, but are not limited to, age, heredity, exposure to certain toxins, and sex. Diagnosis of PD as a juvenile and young adult is rare. The risk of developing Parkinson’s increases with age, beginning at middle to late age; subjects typically develop the disease around age 60 or older. Having a close relative (e.g., an immediate family member, uncle, aunt, or grandparent) with PD increases the chances that a subject will develop the disease. However, the risk is still considered small unless multiple relatives have been diagnosed as having PD. Ongoing exposure to certain herbicides and pesticides has been shown to slightly increase the risk of PD in a subject. And finally, males are more likely to develop PD than females.
  • Symptoms of PD are well documented and known to one skilled in the art. Early symptoms of PD include, but are not limited to, tremors (e.g., shaking that usually begins in a limb, often in hands or fingers, when one’s body is at rest); pilling-rolling tremor (e.g., rubbing a thumb and forefinger back and forth when one’s body is at rest); slowed movement (bradykinesia); rigid muscles (i.e., muscle stiffness in any part of the body that can be painful and limit one’s range of motion); impaired posture and balance (e.g., posture may become stooped, or one may have balance problems); loss of automatic movements (e.g., decreased ability to perform unconscious movements, including blinking, smiling or swinging arms when walking); speech changes (e.g., one may speak more softly and quickly, slur or hesitate before talking; or change in tone and loss of inflections); and writing changes (e.g., writing may appear smaller
  • Complications of PD include, but are not limited to, cognitive problems (dementia) and thinking difficulties in the later stages of PD; depression and emotional changes (i.e., fear, anxiety or loss of motivation) in early and late stages of Parkinson’s; swallowing problems as the condition progresses (e.g., difficulties with swallowing, saliva accumulation and drooling); chewing and eating problems in late stage PD that can lead to choking and poor nutrition; sleep problems and sleep disorders (i.e., frequent waking, waking up early, and falling asleep during the day); rapid eye movement sleep behavior disorder; bladder problems (i.e., inability to control urine or having difficulty urinating); constipation; orthostatic hypotension (i.e., sudden drop in blood pressure); smell dysfunction (e.g., loss of smell or difficulty identifying certain odors or the difference between odors); fatigue; pain, i.e., either in specific areas of their bodies or throughout their bodies; and sexual dysfunction.
  • cognitive problems disementia
  • SPECT single-photon emission computerized tomography
  • DaTscan dopamine transporter scan
  • Non-invasive imaging e.g., MRI, ultrasound of the brain, and PET scans, can be performed to rule out other neurological disorders, but are not helpful in diagnosing PD.
  • a subject suspected of having Parkinson’s can be administered a sufficient (i.e., high) dose of an antiParkinson’s therapeutic (e.g., carbidopa-levodopa) and monitor for improvement of symptom(s); an improvement following administration would indicate/confirm a diagnosis of PD.
  • an antiParkinson’s therapeutic e.g., carbidopa-levodopa
  • Treatment for PD include, but are not limited to, therapeutics designed to treat the ongoing symptoms of the disease. These therapeutics include, but are not limited to, carbidopa-levodopa; Inhaled carbidopa-levodopa; Carbidopa-levodopa infusion; Dopamine agonists; MAO B inhibitors; catechol O-methyltransferase (COMT) inhibitors; anticholinergics; and amantadine.
  • therapeutics include, but are not limited to, carbidopa-levodopa; Inhaled carbidopa-levodopa; Carbidopa-levodopa infusion; Dopamine agonists; MAO B inhibitors; catechol O-methyltransferase (COMT) inhibitors; anticholinergics; and amantadine.
  • Levodopa the most effective PD medication, is a natural chemical that passes into the brain and is converted to dopamine.
  • Levodopa is typically combined with carbidopa (e.g., Lodosyn® carbidopa), which protects levodopa from early conversion to dopamine outside the brain, preventing or lessening side effects such as nausea.
  • carbidopa e.g., Lodosyn® carbidopa
  • the benefit from levodopa may become less stable, with a tendency to wax and wane (i.e., “wearing off’).
  • Involuntary movements (dyskinesia) is associated with higher doses of levodopa.
  • Inbrija® levodopa inhalation powder is a therapeutic drug delivering levodopa in an inhaled form.
  • DuopaTM carbidopa/levodopa suspension is a brand-name medication made up of carbidopa and levodopa administered via a feeding tube such that the medication is delivered via a gel form directly to the small intestine.
  • DuopaTM carbidopa/levodopa suspension is for patients with more-advanced Parkinson's who still respond to carbidopa-levodopa, but who have significant fluctuations in their response. Because DuopaTM is continually infused, blood levels of the two drugs (carbidopa and levodopa) remain constant.
  • dopamine agonists do not change into dopamine, but rather mimic dopamine effects in the patient’s brain. Dopamine agonists are less effective than levodopa in treating PD symptoms; however, they last longer and may be used with levodopa to support the off-and-on effect of levodopa.
  • Exemplary dopamine agonists include pramipexole (e.g., Mirapex® pramipexole), ropinirole (e.g., Requip® ropinirole), rotigotine (e.g., Neupro® rotigotine transdermal system, given as a patch), and apomorphine (e.g., Apokyn® apomorphine), which is a short-acting injectable dopamine agonist.
  • pramipexole e.g., Mirapex® pramipexole
  • ropinirole e.g., Requip® ropinirole
  • rotigotine e.g., Neupro® rotigotine transdermal system, given as a patch
  • apomorphine e.g., Apokyn® apomorphine
  • MAO B inhibitors help prevent the breakdown of brain dopamine by inhibiting the brain enzyme monoamine oxidase B (MAO B), which metabolizes brain dopamine.
  • MAO B inhibitors include selegiline (e.g., Zelapar® selegiline hydrochloride), rasagiline (e.g., Azilect® rasagiline) and safmamide (e.g., Xadago® safinamide).
  • selegiline e.g., Zelapar® selegiline hydrochloride
  • rasagiline e.g., Azilect® rasagiline
  • safmamide e.g., Xadago® safinamide
  • COMT inhibitors mildly prolongs the effect of levodopa therapy by blocking an enzyme that breaks down dopamine.
  • COMT inhibitors include entacapone (e.g., Comtan® entacapone), opicapone (e.g., Ongentys® opicapone), and tolcapone (e.g., Tasmar® tolcapone). Tolcapone is rarely prescribed due to a risk of serious liver damage and liver failure.
  • Anticholinergics were previously administered to be help control the tremor associated with PD.
  • Exemplary anticholinergic include Antipsychotics (clozapine, quetiapine); Atropine; Benztropine (e.g., Cogentin® benztropine mesylate); Biperiden; Chlorpheniramine; Certain SSRIs (Paroxetine); Dicyclomine (Dicycloverine); Dimenhydrinate; Diphenhydramine; Doxepi; Doxylamine; Flavoxate; Glycopyrrolate; Glycopyrronium; Hyoscyamine; Ipratropium; Orphenadrine; Oxitropium;
  • Oxybutynin Promethazine; Propantheline bromide; Scopolamine; Solifenacin; Tolterodine; Tiotropium; Tricyclic antidepressants; Trihexyphenidyl; Tropicamide; and Umeclidinium.
  • Amantadine e.g., Gocovri® amantadine
  • Gocovri® amantadine is an anti-dyskinesia medication prescribed as a mono-therapy to provide short-term relief of symptoms of mild, early-stage PD. It is further prescribed with carbidopa-levodopa therapy during the later stages of PD to control involuntary movements (dyskinesia) induced by carbidopa-levodopa.
  • DBS deep brain stimulation
  • a deep brain stimulation involves implanting electrodes into a specific part of a patient’s brain; the electrodes are connected to a generator implanted in the patient’s chest near the collarbone and the generator sends electrical pulses to the patient’s brain.
  • DBS is effective in controlling erratic and fluctuating responses to levodopa or for controlling dyskinesia that doesn't improve with medication adjustments.
  • DBS is more commonly used in later stage patients that exhibit unstable responses to medication, e.g., levopoda.
  • Methods for delivering a nucleic acid and/or a transgene (e.g., a nucleic acid encoding GDNF) to a subject are provided by the disclosure.
  • the methods typically involve administering to a subject an effective amount of a nucleic acid encoding GDNF.
  • administration is systemic administrations.
  • administration is local administration.
  • the nucleic acid is provided in a viral vector and/or in a viral particle, e.g., a rAAV.
  • One aspect provided herein relates to a method of slowing or inhibiting progression of PD in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene, and wherein the subject does not exhibit an increase in PD- associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • One aspect provided herein relates to a method of slowing or inhibiting progression of PD in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the introducing the rAAV results in at least 30% coverage of the the subject’s putamen with the rAAV, and wherein the subject does not exhibit an increase in PD- associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • the putamen comprises two bilaterally symmetrical, oblong, ovular subcortical lobes that extend longitudinally about an anterior-posterior (A-P) axis.
  • A-P anterior-posterior
  • the term “putamen” can refer to either a single putamen (i.e., the left putamen or right putamen) or both putamen collectively.
  • the putamen are located within the paraventricular deep white matter of the forebrain of each brain hemisphere (telencephalon) and comprise a plurality of nerve cell (neuronal) bodies. The putamen form the striatum together with the adjacent caudate nucleus.
  • the striatum is additionally one component of many that form the basal ganglia of each brain hemisphere.
  • the putamen are connected to the substantia nigra (including the pars compacta and pars reticulata), the globus pallidus, the claustrum, and the thalamus, in addition to many regions of the cerebral cortex.
  • a primary function of the putamen is to regulate the preparation and execution of physical movements and plays a role in various types of learning.
  • the putamen also plays a role in the development of degenerative neurological disorders, such as PD.
  • Retrograde axonal transport of the GDNF protein and/or AAV2 vector from the putamen to substantia nigra is possible; however, anterograde axonal transport of the GDNF protein and/or AAV2 vector to the pars reticulata is more probable in a PD state.
  • the direction of axonal transport can be determined by the vector used to deliver the GDNF transgene.
  • One aspect provided herein relates to a method of slowing or inhibiting progression of PD in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • the rAAV is introduced via local introduction.
  • the AAV capsid is a rational haploid, e.g., the capsid is AAV8, AAV9, and contains at least one capsid protein from a Rhesus AAV strain.
  • local introduction is introduction directly to the subject’s putamen.
  • local introduction can comprise directly introducing the rAAV to one or both of the subject’s putamen.
  • local introduction is performed simultaneously with non-invasive imaging.
  • the local introduction comprises introducing about half of the total rAAV vector dose to each putamen via intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED).
  • iMRI intraoperative magnetic resonance image
  • CED convection enhanced delivery
  • local introduction further comprises introducing an MRI contrast agent at the same time or substantially the same time as the AAV vector.
  • MRI contrast agents include gadoterate; gadobutro; gadoteridol; gadopentetate; gadobenate; gadopentetic acid dimeglumine; gadoxentate; gadoversetamide; gadodiamide; gadofosveset; gadocoletic acid; gadomelitol and gadomer.
  • the MRI contrast agent is introduced to the subject in the same composition as the rAAV. In one embodiment, the MRI contrast agent is introduced to the subject in a different composition as the rAAV, but are administered at substantially the same time.
  • the rAAV is introduced via systemic introduction.
  • Another aspect provided herein relates to a method of slowing or inhibiting a progression of PD in a subject in need thereof, the method comprising locally introducing to the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line- derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF nucleic acid.
  • rAAV recombinant adeno-associated virus
  • transducing the putamen is transducing the putaminal neuron population.
  • at least 30% of the volume of the subject’s putaminal neuron population are transduced with the GDNF nucleic acid
  • Another aspect provided herein relates to a method of slowing or inhibiting a progression of PD in a subject in need thereof comprising transducing greater than or equal to about 30% of the volume of the subject’s putamen with a glial cell line-derived neurotrophic factor (GDNF) gene, wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months following the transducing.
  • transducing is performed by administering a rAAV comprising the GDNF gene to each putamen of the subject’s brain hemisphere.
  • transducing the putamen is transducing the putaminal neuron population.
  • at least 30% of the volume of the subject’s putaminal neuron population are transduced with the GDNF nucleic acid
  • Another aspect provided herein relates to a method of reducing or stabilizing an initial Movement Disorder Society-Unified PD Rating Scale Part III (MDS-UPDRS III) score in a subject having PD comprising administering to the subject’s putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the subject has a second MDS-UPDRS III score at 6 months following the administration is decreased or stabilized as compared to the initial MDS-UPDRS III score of the subject prior to administering.
  • MDS-UPDRS III Movement Disorder Society-Unified PD Rating Scale Part III
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • Another aspect provided herein relates to a method of treating a subject mildly affected by PD comprising administering to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with GDNF and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV, and wherein the subject has a second MDS-UPDRS III score at 6 months post-administering that is stabilized as compared to the initial MDS-UPDRS III score.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • Another aspect provided herein relates to a method of treating a subject moderately affected by PD comprising administering to each of the subject’s putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with the transgene and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV, and wherein the subject has a second MDS-UPDRS III score at 6 months post-administering that is at least about 20% lower than the initial MDS-UPDRS III score.
  • AAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • Another aspect provided herein relates to a method of slowing or inhibiting progression of PD in a subject in need thereof comprising locally introducing to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line- derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing an MRI contrast agent to each of the subject’s putamen at substantially the same time as the rAAV, wherein at least 30% of the volume of the subject’s putamen is transduced with the nucleic acid and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • Another aspect provided herein relates to a method of slowing or inhibiting progression of PD in a subject in need thereof comprising introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more of the volume of the subject’s putamen is transduced with the GDNF gene.
  • transduction of the subject’s putamen is assessed via non-invasive imaging, for example, via MRI.
  • One skilled in the art can assess the transduction of the rAAV by measuring the total volume of the putamen comprising the rAAV (e.g., as assessed by the infused MRI contrast agent) as compared to the total volume that does not comprise the rAAV.
  • transducing the putamen is transducing intrinsic medium spiny neurons (MSNs) of the putamen.
  • MSNs intrinsic medium spiny neurons
  • MSNs are transduced with the nucleic acid, e.g., GDNF.
  • transducing the putamen is transducing the putaminal neuron population.
  • at least 30% of the volume of the subject’s putaminal neuron population are transduced with the GDNF nucleic acid.
  • the coverage is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 6
  • the coverage is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least
  • coverage of the subject’s putamen is assessed via non-invasive imaging, for example, via MRI.
  • One skilled in the art can assess the coverage of the rAAV by measuring the total volume of the putamen comprising the rAAV (e.g., as assessed by the infused MRI contrast agent) as compared to the total volume of the putamen.
  • the MRI contrast agent is co-administered or co-introduced with any of the rAAVs described herein to provide enhanced real-time intraoperative MRI monitoring of the CED distribution and to assess the volume of transduction.
  • local administration is performed simultaneously with non-invasive imaging, e.g., to guide local delivery to a preferred or predetermined location or example, the putamen, and/or to visualize transduction following administration.
  • the non- invasive imaging is intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED).
  • iMRI intraoperative magnetic resonance image
  • CED convection enhanced delivery
  • intraoperative magnetic resonance image refers to an MRI image, for example, of the brain, acquired during a neurosurgical procedure. iMRI technology can be relied upon to create accurate, real time pictures of the brain for guidance during a neurosurgical procedure, e.g., removal of a tumor or placement of a therapeutic to a desired location (e.g., the putamen).
  • CED Convection-enhanced delivery
  • Direct intracerebral CED circumvents the blood-brain barrier and provides a wider, more homogenous distribution than bolus deposition (focal injection) or other diffusion-based direct delivery approaches.
  • CED is further described in, e.g., Rogawski MA, Neurotherapeutics. 2009 Apr; 6(2): 344-351 and Mehta AM, et al. Neurotherapeutics. 2017 Apr;14(2):358-371., the contents of each of which are incorporated herein by reference in their entireties.
  • iMRI will be used to monitor administration of the rAAV or composition thereof described herein using T1 -weighted sequences to visualize the MRI contrast agent, e.g., the gadolinium-based contrast agent, that is co-infused with the rAAV or composition thereof.
  • the MRI contrast agent e.g., the gadolinium-based contrast agent
  • local introduction further comprises introducing an MRI contrast agent at substantially the same time as the AAV vector.
  • MRI contrast agents are utilized to improve the visibility of internal brain structures captured in an MRI image.
  • paramagnetic contrast agents comprising gadolinium(III), known in the art as gadolinium-based MRI contrast agents (GBCAs), see “Gadolinium(III) Chelates as MRI Contrast Agents Structure, Dynamics, and Applications” by P. Caravan et al. Chem. Rev. 99, 2293-2352 (1999), incorporated herein in its entirety by reference.
  • contrast agents that may be used include gadoxetate disodium (e.g., EovistTM gadoxetate disodium; Schering AG); the contrast agents disclosed in U.S. Pat. Nos. 5,798,092 and 5,695,739; gadobenate dimeglumine (e.g., MultiHanceTM gadobenate dimeglumine, Bracco SpA); and the contrast agents disclosed in U.S. Pat. No. 5,733,528.
  • gadoxetate disodium e.g., EovistTM gadoxetate disodium; Schering AG
  • gadobenate dimeglumine e.g., MultiHanceTM gadobenate dimeglumine, Bracco SpA
  • contrast agents disclosed in U.S. Pat. No. 5,733,528 Particularly preferred are “blood pool” MRI contrast agents, see “Blood pool Contrast Agents for Cardiovascular MR Imaging” by L. J. M. Kroft et al.
  • blood pool agents include but are not limited to, ferucarbotran (e.g., ResovistTM ferucarbotran) or SHU 555 A and C (Schering).
  • ferucarbotran e.g., ResovistTM ferucarbotran
  • SHU 555 A and C Schering
  • Exemplary MRI contrast agents include gadoterate; gadobutro; gadoteridol; gadopentetate; gadobenate; gadopentetic acid dimeglumine; gadoxentate; gadoversetamide; gadodiamide; gadofosveset; gadocoletic acid; gadomelitol and gadomer.
  • the MRI contrast agent is gadoteridol (e.g., ProHance® gadoteridol). In one embodiment, gadoteridol (e.g., ProHance® gadoteridol) is administered in a 2mM solution.
  • MRI contrast agents may be administered by injection into the blood stream (intravenously) or orally, depending on the subject of interest. Oral administration is well suited to G.E tract scans, while intravascular administration proves more useful for most other scans. In one embodiment, the MRI contrast agent is administered in the same composition as the rAAV.
  • the MRI contrast agent is administered in a separate composition as the rAAV, but is administered concurrently with the separate rAAV composition.
  • the MRI contrast agent need not be administered in the same manner as the rAAV.
  • the MRI contrast agent can be administered intravenously or orally.
  • MRI brain scans can be performed pre-operatively as part of the screening process, as well as during the gene therapy infusion procedure and at 6- and 18-months after dosing. Scans may be obtained at other time points if deemed necessary by the investigators.
  • MRI brain scans can be obtained, for example, on a 1.5 or 3T scanner and sequences may include Tl, T2, turbo FLAIR, T2 gradient echo and diffusion.
  • Optional imaging at screening 18-months may also include expanded diffusion weighted sequences, resting state, and functional assessments with image acquisition while participants perform simple tasks (i.e. finger tapping or hand grasping). Total imaging time is 90 minutes per session, with the inclusion of functional and resting state imaging.
  • Exemplary non-invasive imaging techniques that can be utilized in the methods described herein include ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); iMRI; positron emission tomography (PET); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); DaTscan Dopamine Transporter Imaging; FDG imaging and combinations thereof.
  • CT computed tomography
  • fMRI functional magnetic resonance imaging
  • iMRI positron emission tomography
  • PET electroencephalography
  • EEG electroencephalography
  • MEG magnetoencephalography
  • fNIRS functional near-infrared spectroscopy
  • DaTscan Dopamine Transporter Imaging FDG imaging and combinations thereof.
  • loflupane 1-123 (e.g., DaTscanTM ioflupane 123) selectively binds to presynaptic dopamine transporters and provides a method for imaging nigrostriatal terminals in the striatum.
  • DaTscanTM ioflupane 123 is an FDA-approved radiopharmaceutical used in conjunction with single photon emission computed tomography (SPECT) scan for use in adults.
  • Iodine- 123 is a cyclotron-produced radionuclide that decays to 123 Te by electron capture and has a physical half-life of 13.2 hours.
  • the recommended dose is 111 to 185 MBq (3 to 5 mCi) administered intravenously in adults.
  • the Effective Dose resulting from a DaTscan administration with an administered activity of 185 MBq (5 mCi) is 3.94 mSv in an adult.
  • DaTscan injection may contain up to 6% of free iodide (iodine 123).
  • iodine 123 free iodide
  • a dose up to 100 mg of Potassium Iodide Oral Solution or Lugol's Solution will be administered.
  • Fluoro-2-Deoxyglucose is a common FDA-approved radiopharmaceutical tracer used with positron emission topography (PET) imaging to measure glucose metabolism in the brain and other organs. Brain metabolic patterns specific to PD, and not present in other parkinsonian-like diseases, have been characterized. FDG PET will be utilized to confirm PD diagnosis during screening. FDG is F 18 labeled with a half-life of 110 minutes. The recommended dose is 111 to 185 MBq (3 to 5 mCi) administered intravenously in adults. The Effective Dose resulting from an FDG scan with an administered activity of 185 MBq (5 mCi) is 3.51 mSv in an adult.
  • FDG Fluoro-2-Deoxyglucose
  • the coverage or transduction of the putamen is assessed via non-invasive imaging, for example, intraoperative MRI.
  • Volume of the putamen that is transduced with GDNF is indirectly determined by measuring the percent of the putamen showing CED-infiised MRI contrast within the putamen as compared to the total volume of the putamen.
  • the volume of the putamen transduced is assessed via F-DOPA PET imaging that is correlated to the intraoperative CED-infiised MRI contrast localization.
  • coverage refers to the volume of the putamen occupied by the infused rAAV relative to the total volume of the putamen. The coverage provides that putamen cells are exposed to the rAAV.
  • coverage of the putamen can be assessed via non-invasive imaging, for example, via co-infusion with a MRI contrast agent that can be visualized.
  • the coverage of the putamen following introduction/administration can be determined by measuring the area or volume of the putamen that displays the co-infused MRI agent and comparing it to the area or volume of the putamen that does not display the agent.
  • the rAAV can be further comprise a reporter gene, e.g., a fluorescent tag, that can be visualized postmortem using standard histological methods, e.g., microscopy.
  • a reporter gene e.g., a fluorescent tag
  • transduction refers to a cell within the putamen that comprises the administered rAAV or composition thereof.
  • the transduced cell comprises the genome of the rAAV, and has the potential to express the GDNF transgene.
  • the transduced cell comprises the genome of the rAAV and does not need to comprise the ability to express the GDNF transgene.
  • the transduced cell transiently expresses the GDNF transgene.
  • the transduced cell stably expresses the GDNF transgene.
  • transduction can be assessed via coinfusion with a MRI contrast agent that can be visualized.
  • the percentage of transduced cells of the putamen can be determined, e.g., by measuring the percent of the cells in the putamen that comprises the rAAV or composition, as compared to the total volume of the putamen.
  • the transduction can be accessed via non-invasive imaging, for example, via co-infusion with a MRI contrast agent that can be visualized.
  • the rAAV can be further comprise a reporter gene, e.g., a fluorescent tag, that can be visualized using standard methods, e.g., microscopy; such reporter gene can be utilized to determine the percent transduction.
  • Probes designed to target the rAAV e.g., a capsid protein
  • probes designed to target the GDNF nucleic action can be used to assess whether the cell expresses the GDNF transgene.
  • the volume of the putamen transduced is assessed via F-DOPA PET imaging.
  • the rAAV can be further comprise a reporter gene, e.g., a fluorescent tag) that can be visualized using standard methods, e.g., microscopy.
  • the progression and severity of PD is often measured using a various clinical surveys which assess various symptoms and the mental status of a subject having or thought to have PD. These clinical surveys can be completed by the subject, the subject’s caretaker, and/or a skilled physician. Often, these surveys are used by clinicians and researchers to assess the longitudinal course of PD during the course of treatment or in a clinical study. The results of such surveys can, e.g., aid the clinician or researcher in determining the best course of action for treating a subject, i.e., altering the type of a therapeutic, the dosage of a therapeutic, or frequency of administration for a therapeutic.
  • the methods described herein further comprise the step of determining an initial score of at least one diagnostic assay described herein for a subject prior to introducing an rAAV described herein.
  • the diagnostic assay can be Movement Disorder Society- Unified Parkinson Disease Rating Scale (MDS-UPDRS), Non-Motor Symptoms Scale (NMSS), Parkinson's Disease Questionnaire (PDQ-39) score; MDS-UPDRS Part III; Modified Hoehn and Yahr; Stand-Walk-Sit; 9-Hole Pegboard Dexterity Test; and Standing Balance Test; Global Impression (CGI & PGI); Brief Smell Identification Test (BSIT); Parkinson’s Disease Sleep Scale (PDSS-2); Scales for Outcomes in Parkinson's Disease-Autonomic (SCOPA-AUT); Global Cognitive Assessment via Montreal Cognitive Assessment (MoCA); 30-Item Boston Naming Test (BNT); Verbal Fluency Test; Cambridge Neuropsychological Test Automated Battery (CANTAB); Beck Depression
  • MDS-UPDRS Movement
  • the methods described herein further comprises the step of receiving an initial score of at least one diagnostic assay described herein for a subject prior to introducing any of the rAAVs described herein.
  • MDS-UPDRS Movement Disorder Society-Unified Parkinson Disease Rating Scale
  • the Unified Parkinson Disease Rating Scale is a rating scale to assess the short term (less than 1 year) and long term (greater than 1 year) progression of PD.
  • the UPDRS is a uniform and accepted assay utilized in a clinical setting that allows a clinician to follow the progression of patients' symptoms in an objective manner. This test is made up of six parts and is both self-administered and clinican/researcher-administered.
  • the six parts include — Part I: evaluation of mentation, behavior, and mood, including intellectual impairment, thought disorder, motivation/initative, depression; Part II: self-evaluation of the activities of daily life (ADUs) speech, salivation, swallowing, handwriting, cutting food, dressing, hygiene, turning in bed, falling, freezing, walking, tremor, sensory complaints; Part III: clinician-scored monitored motor evaluation, including speech, facial expression, tremor at rest, action tremor, rigidity, finger taps, hand movements, hand pronation and supination, leg agility, arising from chair, posture, gait, postural stability, body bradykinesia; Part IV : complications of therapy, including dyskinesia-duration, dyskinesia-disability, dyskinesia-pain, early morning dystonia, OFF-predictable, OFF -unpredictable, OFF-sudden, OFF- duration, anorexia-nausea- vomiting, sleep disturbance, symptomatic orthosta
  • the Movement Disorder Society-Unified Parkinson Disease Rating Scale is a rating scale (i.e., from 0-272) to assess the short term (less than 1 year) and long term (greater than 1 year) progression of PD.
  • MDS-UPDRS is an updated version of the UPDRS took aspects of nonmotor functioning out of each subcategory.
  • MDS-UPDRS Part I is titled “Non-Motor Experiences of Daily Living” and includes Part IA (concerning a number of behaviors that are assessed by an investigator with all pertinent information from patients and caregivers) and Part IB (completed by the patient with or without the aid of a caregiver, but independently of an investigator).
  • MDS-UPDRS Part II is identical to the second part of the original UPDRS, but has been renamed “Motor Experiences of Daily Living” to separate it from the new title of Part I.
  • MDS-UPDRS Part III is titled “Motor Examination.”
  • MDS-UPDRS Part IV has been condensed relative to UPDRS to include only "Motor Complications.”
  • a total MDS-UPDRS score is a sum of Parts I, II, III (in Off state), and IV, which provides a score of disease severity and progression as it provides both functional and rater-derived subscores.
  • an initial MDS-UPDRS score i.e., prior to administration of an rAAV described herein
  • a second MDS-UPDRS score i.e., a MDS- UPDRS score subsequent to the administration of rAAV, e.g., at least 6 months or at least 12 months immediately following administration of an rAAV described herein.
  • additional MDS-UPDRS scores e.g., third, fourth, fifth, and so on
  • the initial and second MDS-UPDRS scores can be any individual part of the MDS-UPDRS survey (e.g., Part I, Part II, Part III, Part IV) or a total MDS-UPDRS score.
  • the methods described herein further comprise the step of determining an initial MDS-UPDRS score for a subject prior to introducing any of the rAAVs described herein. In one embodiment, the methods described herein further comprise the step of receiving an initial MDS- UPDRS score for a subject prior to introducing any of the rAAVs described herein, i.e., receiving an in initial MDS-UPDRS score that was previously determined by a skilled practitioner that is not performing the administration of the rAAV.
  • the subject is mildly affected by PD.
  • mildly affected refers to a subject having an initial MDS-UPDRS score that is less 32.
  • the subject mildly affected by PD has an initial MDS-UPDRS score less than 32 prior to the introduction of rAAV and was diagnosed with PD less than 5 years prior to the introduction of rAAV.
  • the MDS-UPDRS refers to the MDS-UPDRS III score.
  • the subject is moderately affected by PD.
  • “moderately affected” refers to a subject having an initial MDS-UPDRS score that is equal to or greater than 32.
  • the subject moderately affected by PD has an initial MDS-UPDRS score (e.g., MDS-UPDRS III score) equal to or greater than 32 prior to the introduction of rAAV and was diagnosed with PD less than 4 years prior to the introduction of rAAV.
  • the methods described herein further include the step of determining a second MDS-UPDRS score at least 6 months or at least 12 months immediately following administration/introduction of the rAAV.
  • the methods described herein further include the step of determining a second MDS-UPDRS score at least 6 months immediately following administration/introduction of the rAAV to the subject who is moderately affected by PD (i.e., the subject having an initial MDS- UPDRS score that is equal to or greater than 32).
  • the methods described herein further include the step of determining a second MDS-UPDRS score at least 12 months immediately following administration/introduction of the rAAV to the subject who is mildly affected by PD (i.e., the subject having an initial MDS-UPDRS score that is less 32).
  • the subject who is mildly affected by PD does not exhibit an increase of their initial MDS-UPDRS score for at least 12 months immediately following introducing any of the rAAVs described herein. In one embodiment, the subject who is mildly affected by PD does not exhibit an increase of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the subject who is mildly affected by PD does not exhibit a substantial increase of their initial MDS-UPDRS score for at least 12 months immediately following introducing any of the rAAVs described herein.
  • substantial increase refers to an increase that is no more than 10% of the initial MDS-UPDRS score.
  • the subject who is mildly affected by PD does not exhibit a substantial increase of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the subject who is mildly affected by PD exhibit a stabilization of their initial MDS-UPDRS score for at least 6 months immediately following introducing any of the rAAVs described herein.
  • stabilization refers to an initial MDS-UPDRS score that does not increase or decrease by greater than 10%, i.e., the second MDS-UPDRS score is no more than +/-10% of the initial MDS-UPDRS score.
  • the subject who is mildly affected by PD exhibits a stabilization of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the subject who is mildly affected by PD exhibits a reduction of their initial MDS-UPDRS score for at least 12 months immediately following introducing any of the rAAVs described herein. In one embodiment, the subject who is mildly affected by PD exhibits a reduction of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months;
  • the reduction of the initial MDS-UPDRS score is by at least 1 point, 2 points; 3 points; 4 points; 5 points; 6 points; 7 points; 8 points; 9 points; 10 points; 11 points; 12 points; 13 points; 14 points; 15 points; 16 points; 17 points; 18 points; 19 points; 20 points; 21 points; 22 points; 23 points; 24 points; 25 points; 26 points; 27 points; 28 points; 29 points; 30 points; or 31 points; or by at least 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95%; or greater.
  • the subject who is moderately affected by PD exhibits a reduction of their initial MDS-UPDRS score for at least 6 months immediately following introducing any of the rAAVs described herein. In one embodiment, the subject who is moderately affected by PD exhibits a reduction of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months;
  • the reduction of the initial MDS-UPDRS score is by at least 20%. In one embodiment, the reduction of the initial MDS-UPDRS score is by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%; 23%; 24%; 25%; 26%; 27%;
  • the subject who is moderately affected by PD does not exhibit an increase of their initial MDS-UPDRS score for at least 6 months immediately following introducing any of the rAAVs described herein. In one embodiment, the subject who is moderately affected by PD does not exhibit an increase of their initial MDS-UPDRS score for at least Imonth; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the subject who is moderately affected by PD does not exhibit a substantial increase of their initial MDS-UPDRS score for at least 6 months immediately following introducing any of the rAAVs described herein.
  • substantially increase refers to an increase that is no more than 10% of the initial MDS-UPDRS score.
  • the subject who is moderately affected by PD does not exhibit a substantial increase of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months;
  • NMSS Non-Motor Symptoms Scale
  • Non-Motor Symptoms Scale is a 30-item self-administered survey scale to assess a wide range of non-motor symptoms in subjects with PD.
  • Non-motor symptoms in PD generally include neuropsychiatric symptoms, sleep disorders, autonomic dysfunction, gastrointestinal symptoms and sensory symptoms, and can significantly reduce quality of life.
  • the NMSS measures the severity and frequency of non-motor symptoms across nine dimensions: cardiovascular, sleep/fatigue, mood/cognition, perceptual problems, attention/memory, gastrointestinal, urinary, sexual function, and miscellaneous.
  • the scale can be used for patients at all stages of PD.
  • the scores for each item are based on a combination of severity (from 0 to 3) and frequency scores (from 1 to 4), to capture symptoms that are severe but relatively infrequent, or that are less severe but persistent.
  • the total NMSS score ranges from 0 to 360
  • the methods described herein further comprises the step of determining an initial NMSS score for a subject prior to introducing any of the rAAVs described herein. In one embodiment, the methods described herein further comprise the step of receiving an initial NMSS score for a subject prior to introducing any of the rAAVs described herein, i.e., receiving an in initial NMSS score that was previously determined by a clinican/research that is not performing the administration of the rAAV.
  • the methods described herein further include the step of determining a second NMSS score at least 6 months or at least 12 months immediately following administration/introduction of the rAAV.
  • the subject exhibits a decrease of their initial NMSS score for at least 6 months following introducing any of the rAAVs described herein. In one embodiment, the subject exhibits a decrease of their initial NMSS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer following introducing any of the rAAVs described herein. In one embodiment, the decrease of the initial NMSS score is by at least 20%.
  • the decrease of the initial NMSS score is by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%;
  • the subject does not exhibit an increase of their initial NMSS score for at least 6 months following introducing any of the rAAVs described herein.
  • a subject does not exhibit an increase of their initial NMSS score for at least Imonth; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer following introducing any of the rAAVs described herein.
  • the subject does not exhibit a substantial increase of their initial NMSS score for at least 6 months following introducing any of the rAAVs described herein.
  • substantially increase refers to an increase that is no more than 10% of the initial NMSS score.
  • a subject does not exhibit a substantial increase of their initial NMSS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer following introducing any of the rAAVs described herein.
  • the subject exhibits a stabilization of their initial NMSS score for at least 6 months immediately following introducing any of the rAAVs described herein.
  • stabilization refers to an initial NMSS score that does not increase or decrease by greater than 10%.
  • the subject exhibits a stabilization of their initial NMSS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the Parkinson's Disease Questionnaire (PDQ-39) is a 39-item self-administered questionnaire with eight subscales: mobility, activities of daily living (ADLs), emotional well-being, stigma, social support, cognitive impairment, communication, and physical discomfort.
  • the questionnaire is scored on a scale from 0 to 100 with lower scores indicating a better perception of health status, and higher scores indicating a more severe state of the disease.
  • the PDQ-39 can be used as a reliable tool for measuring quality of life for individuals with PD. Its inclusion in comprehensive assessment is especially important due to the tendency of treatment to focus on motor deficits and cardinal features rather than other clinical features including depression, cognitive impairment, and fall risk which can significantly impact quality of life.
  • the methods described herein further comprises the step of determining an initial PDQ-39 score for a subject prior to introducing any of the rAAVs described herein. In one embodiment, the methods described herein further comprise the step of receiving an initial PDQ- 39score for a subject prior to introducing any of the rAAVs described herein, i.e., receiving an in initial PDQ-39score that was previously determined by a clinican/research that is not performing the administration of the rAAV.
  • the methods described herein further include the step of determining a second PDQ-39 score at least 6 months or at least 12 months immediately following administration/introduction of the rAAV.
  • the subject exhibits a decrease of their initial PDQ-39 score for at least 6 months following introducing any of the rAAVs described herein.
  • the decrease of the initial PDQ-39 score is by at least 20%. In one embodiment, the decrease of the initial PDQ-39 score is by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%;
  • the subject does not exhibit an increase of their initial PDQ-39 score for at least 6 months following introducing any of the rAAVs described herein.
  • a subject does not exhibit an increase of their initial PDQ-39 score for at least Imonth; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer following introducing any of the rAAVs described herein.
  • the subject does not exhibit a substantial increase of their initial PDQ-39 score for at least 6 months following introducing any of the rAAVs described herein.
  • substantially increase refers to an increase that is no more than 10% of the initial PDQ-39 score.
  • a subject does not exhibit a substantial increase of their initial PDQ-39 score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months;
  • the subject exhibits a stabilization of their initial PDQ-39 score for at least 6 months immediately following introducing any of the rAAVs described herein.
  • stabilization refers to an initial PDQ-39 score that does not increase or decrease by greater than 10%.
  • the subject exhibits a stabilization of their initial PDQ-39 score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing any of the rAAVs described herein.
  • the subject is administered at least one standard clinical test to assess PD patients’ motor symptoms and function prior to and after administration/introduction of any of the rAAVs described herein.
  • assessments include MDS-UPDRS Part III; Modified Hoehn and Yahr; Stand-Walk-Sit; 9-Hole Pegboard Dexterity Test; and Standing Balance Test.
  • the Modified Hoehn & Yahr scale is a 5 -stage scale that measures the overall level of disability due to PD (Hoehn and Yahr, 1967).
  • Stand -Walk-Sit is a postural stability and gait assessment that involves standing up from a chair, walking 7 meters (23 feet) in a straight line, turning around and walking back to the chair and sitting down. Timer will be stopped when subjects back contacts back of chair. Subjects will perform this test in practically defined OFF and ON medication states. The SWS test will be video recorded and can be combined with the MDS-UPDRS assessment.
  • the 9-Hole Pegboard Dexterity Test is a simple test of manual dexterity; it records the time required for the participant to accurately place and remove nine plastic pegs into a plastic pegboard.
  • the Standing Balance Test assesses a person’s ability to orient their body in space, maintain an upright posture under both static and dynamic conditions, and move and walk without falling. It involves the participant assuming and maintaining up to five poses for 50 seconds each. The sequence of poses includes: eyes open on a solid surface, eyes closed on solid surface, eyes open on foam surface, eyes closed on foam surface, and eyes open in tandem stance on solid surface. Detailed stopping rules are in place to ensure participant safety with these progressively demanding poses. Postural sway is recorded for each pose using an accelerometer that the participant wears at waist level. This test takes approximately seven minutes to administer.
  • the subject is administered any of the standard clinical tests described herein prior to administration/introduction, and again at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the subject exhibits an improved score on any of the standard clinical test at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the improvement is at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%; 23%; 24%;
  • dyskinesia severity is measured with the Unified Dyskinesia Rating Scale (UDysRS).
  • UDSRS Unified Dyskinesia Rating Scale
  • This scale evaluates involuntary movements often associated with treated PD. It includes two primary sections: Historical [Part 1 (ON-Dyskinesia) and Part 2 (OFF -Dystonia)] and Objective [Part 3 (Impairment) and Part 4 (Disability)].
  • ON-Dyskinesia refers to the choreiform and dystonic movements described to the patient as jerking or twisting movements that occur when PD medication is working.
  • OFF-Dystonia refers to spasms or cramps that can be painful and occur when PD medications are not taken or are not working.
  • the focus is on these two forms of movements and a continual emphasis must be placed on excluding from the evaluation the impact of parkinsonism itself and tremor from the ratings (Goetz 2008).
  • the subject completes a subject-reported PD Motor Diary. Hauser and colleagues have developed a paper motor diary to assess PD motor symptoms over a 24-hour period (Hauser 2004). The diary captures the duration of time, in half-hour intervals, the participant is in the ON state without dyskinesia, ON with non-troublesome dyskinesia, ON with troublesome dyskinesia, in the OFF state, or asleep. Participants are required to record this information at half hour intervals throughout the day.
  • the subject is administered UDysRS and/or PD Motor diary prior to administration/introduction, and again at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the subject exhibits an improved score on the UDysRS and/or PD Motor at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the improvement is at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%; 23%; 24%;
  • the subject is provided a wearable activity monitor (e.g. Fitbit®) to assess daily activity for at least 18 months immediately following administration/introduction.
  • a wearable activity monitor e.g. Fitbit®
  • the subject undergoes a Global Disability and Quality of Life Assessment prior to and/or at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • Exemplary Global Disability and Quality of Life Assessments include Global Impression (CGI & PGI); Brief Smell Identification Test (BSIT); Parkinson’s Disease Sleep Scale (PDSS-2); and Scales for Outcomes in Parkinson's Disease-Autonomic (SCOPA-AUT) [00194]
  • the Clinical Global Impression (CGI) provides an overall clinician-determined summary measure that takes into account all available information, including a knowledge of the patient's history, psychosocial circumstances, symptoms, behavior, and the impact of the symptoms on the patient's ability to function.
  • the CGI actually comprises 2 companion l-item measures evaluating the following: (a) severity of illness from 1 to 7 and (b) change from the initiation of treatment on a similar 7-point scale.
  • the Patient Global Impression is the same as the CGI but is completed by the patient.
  • the PGI and CGI will be completed separately.
  • the PGI is a self-rating tool and will be completed independently by the participant at home with either paper assessment or by answering on a Sponsor provided tablet.
  • the Brief Smell Identification Test is a 12-item test of olfactory system function using “scratch and sniff’ strips. After each scent is released by scratching with a pencil, the participant smells the odor and then answers a four-option multiple choice question related to the scent. This is a self-directed assessment.
  • the Parkinson’s Disease Sleep Scale (PDSS-2) uses visual analogue scales to address 15 commonly reported symptoms associated with sleep disturbance in PD. Subject complete the 15 questions based on their experiences over the previous week. This is a self-directed measure.
  • the Scales for Outcomes in Parkinson's Disease-Autonomic is a 26 item self-report questionnaire of autonomic function. Questions cover upper and lower gastro-intestinal function, urinary function, cardio-circulatory function, sexuality, and other miscellaneous autonomic problems. This is a self-directed symptom scale.
  • the subject is administered any of the Global Disability and Quality of Life Assessments described herein prior to administration/introduction, and again at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the subject exhibits an improved score on the Global Disability and Quality of Life Assessment at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the improvement is at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; 70%; 71%; 72%; 73%; 74%; 75%; 76%; 77%; 78%; 79%; 80%; 81%; 82%; 83%; 84%; 85%; 86%
  • the subject undergoes a neuropsychological testing prior to and/or at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • exemplary neuropsychological tests include Global Cognitive Assessment via Montreal Cognitive Assessment (MoCA); 30-Item Boston Naming Test (BNT); Verbal Fluency Test; Cambridge Neuropsychological Test Automated Battery (CANTAB); Beck Depression Inventory-II (BDI-II); Beck Anxiety Inventory (BAI); and Questionnaire for Impulsive-Compulsive Disorders in Parkinson’s (QUIP -RS).
  • the Montreal Cognitive Assessment was designed as a rapid screening instrument for mild cognitive dysfunction. It assesses different cognitive domains: attention and concentration, executive functions, memory, language, visuoconstructional skills, conceptual thinking, calculations, and orientation.
  • the BNT evaluates confrontation naming and language deficits that can be present in PD. Participants are presented with stimuli of line drawings of objects with increasing naming difficulty and required to provide a response within 20 seconds. The score is based on number of spontaneously provided correct responses, number of cues given, and number of responses after cuing. This brief verbal assessment will be completed with remote guidance from the study team via a video call.
  • the Cambridge Neuropsychological Test Automated Battery (CANTAB) was developed to include sensitive and objective measures of cognitive function in the evaluation of neurologic disorders.
  • the cognitive assessments have been developed to detect changes in neuropsychological performance over time and as an effect of an intervention.
  • assessments have been validated in PD with a focus on domains of working memory, episodic memory, executive function, planning, and information processing. All tasks will be completed by subjects using a tablet that includes the collection of response times.
  • CANTAB allows for electronically captured outcome measures that have been validated in a variety of neurodegenerative diseases. CANTAB can be completed by selfdirection or guided remotely with the study coordinator or investigator on a Sponsor provided tablet.
  • Reaction Time (RTI) assesses mental response times as well as a measure of movement time, reaction time, response accuracy and impulsivity.
  • Motor Screening Task provides a general assessment of whether a sensorimotor deficit or lack of comprehension may limit the ability to collect valid data from a participant. This task measures the participant’s speed of response and accuracy of pointing to the center of an object on the screen.
  • One Touch Stockings of Cambridge is an assessment of executive function through evaluation of spatial planning and working memory subdomains. Participants are asked to create a 3- D arrangement on screen in a prescribed number of moves. This is measured by number of problems solved on first choice, mean choices correct, meant latency of response, and mean latency to correct.
  • Paired Associates Learning PAL
  • PAL Paired Associates Learning
  • Pattern Recognition Memory is a test of visual pattern recognition memory in a 2- choice forced discrimination paradigm. Unrelated words presented via audio recording and participant recalls as many as possible immediately or after a delay. This is measured by number and percentage of correct trials and latency of responses.
  • Multitasking Test is an assessment of an individual’s ability to interpret and manage conflicting information and to correctly ignore task-irrelevant information. Changing rules between trials places a higher cognitive demand on a participant to reveal underlying deficits of executive dysfunction, a domain often affected in PD patients. This is measured by latency of response and number of errors.
  • the Beck Depression Inventory-II (BDI-II) can be performed, e.g., at screening as part of the eligibility evaluation, and participants with a score >20 at screening will be excluded from the study and referred to their primary care physician for psychiatric evaluation and treatment.
  • Score guidelines for the BDI-II are provided with the recommendation that thresholds be adjusted based on the characteristics of the sample and the purpose for using the BDI-II. In general, total BDI-II scores of 0-13 indicate minimal depression, scores of 1419 indicate mild depression, scores of 20-28 indicate moderate depression, and scores of 29-63 indicate severe depression. If post-treatment BDI-II scores are greater than 28, then the participant will continue the study but will be referred to their primary care physician for psychiatric evaluation and treatment. This assessment is a self-reported measure.
  • Beck Anxiety Inventory is a 21 -question multiple-choice self-report inventory that is used for measuring the severity of anxiety in children and adults. This assessment is a self-reported measure.
  • Compulsive Disorders Questionnaire for PD Rating Scale is a rating scale designed to measure severity of symptoms and support a diagnosis of impulse control disorders and related disorders in PD. This rating scale covers impulse control behaviors on a 5 -point Likert scale to assess the frequency of the following behaviors: gambling, shopping, eating, hypersexuality, simple (punding) and/or complex (hobbyism) repetitive behaviors, and compulsive overuse of medication (dopamine dysregulation syndrome). This assessment is a self-reported measure.
  • the subject is administered any neuropsychological tests described herein prior to administration/introduction, and again at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the subject exhibits an improved score on the neuropsychological test at at least 3, 6, 9, 12 months or later immediately following administration/introduction.
  • the improvement is at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 21%; 22%;
  • the subject does not exhibit any serious adverse event for a least 6 months immediately following the introducing or administering. In one embodiment, the subject does not exhibit any serious adverse event for a least 12 months immediately following the introducing or administering. In one embodiment, the subject does not exhibit any serious adverse event for a for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately following introducing or administering.
  • Serious adverse events include, but are not limited to Blood and lymphatic system disorders (e.g., Anemia, Decreased lymphocyte count, and Leukocytosis); Gastrointestinal disorders (e.g., Dyspepsia, Dysphagia, and Constipation); Localized edema; Fatigue; Fall; Bruising ofthe neck; Aspartate aminotransferase increased; Platelet count decrease; Activated partial thromboplastin time prolonged; Weight loss; Blood lactate dehydrogenase; Metabolism and nutrition disorders (e.g., Hyperglycemia, Hypocalcemia, Hypoalbuminemiaa, Hypophosphatemia, Hypernatremia, Hypoglycemia, Hyperkalemia, and Hyponatremia); Musculoskeletal and connective tissue disorders (e.g., pain, neck pain, back pain, chest wall pain and extremity pain); Nervous system disorders (e.g., Headache, Involuntary Movements, Memory impairment, Sleep disorder - increased dreams, Sensory neuropathy and Hypers
  • nucleic acids that are useful for expressing the GDNF gene or GDNF gene product.
  • a "nucleic acid” sequence refers to a DNA or RNA sequence.
  • nucleic acids, and proteins translated therefrom, of the disclosure are isolated.
  • isolated means artificially produced.
  • isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known, or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not.
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides.
  • nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • isolated refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).
  • conservative amino acid substitutions may be made to provide functionally equivalent variants, or homologs of the capsid proteins.
  • the disclosure embraces sequence alterations that result in conservative amino acid substitutions.
  • a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one can make conservative amino acid substitutions to the amino acid sequences of the proteins and polypeptides disclosed herein.
  • the isolated nucleic acids described herein may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors).
  • AAV adeno-associated virus
  • an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof.
  • the isolated nucleic acid e.g., the recombinant AAV vector
  • Recombinant AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • the transgene may comprise a region encoding, for example, a protein and/or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.
  • ITR sequences are about 145 bp in length.
  • left and right ITRs are independently 145 bp or fewer or 130 bp or fewer.
  • the left and right ITRs can be the same length or different lengths.
  • the left and right ITRs can independently be 145 bp, 130 bp, 128 bp, 124 bp, or 119 bp.
  • substantially the entire sequences encoding the ITRs are used in the nucleic acid sequence, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art.
  • AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.
  • the isolated nucleic acid (e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof.
  • the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.
  • the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR.
  • the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof.
  • the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS).
  • lacking a terminal resolution site can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR).
  • TRS terminal resolution site
  • a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648-1656.
  • the vector in addition to the elements identified above for the recombinant AAV vector, the vector also includes conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the vector or infected with the virus produced by methods described herein.
  • "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest (i.e., GDNF) and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency ⁇ i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (polyA) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency ⁇ i.e., Kozak consensus sequence
  • sequences that enhance protein stability e.g., telomereon sequences that enhance protein.
  • a number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • nucleic acid sequence e.g., coding sequence
  • regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences.
  • nucleic acid sequences be translated into a functional protein
  • two DNA sequences are said to be operably linked if induction of a promoter in the regulatory sequence (e.g., a 5' regulatory sequences) results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region is operably linked to a nucleic acid sequence when the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame.
  • operably linked coding sequences yield a fusion protein.
  • the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., a promoter, etc.).
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • poly A splicing and polyadenylation
  • sequences that enhance translation efficiency i.e., Kozak consensus sequence
  • sequences that enhance protein stability i.e., Kozak consensus sequence
  • a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene (e.g., a coding sequence of a gene.
  • the phrases “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • a polyadenylation sequence generally is inserted following the transgene sequences, i.e., downstream of the transgene sequences or 3’ of the transgene sequences, and before the 3' AAV ITR sequence.
  • a rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • One exemplary intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • Another vector element that may be used is an internal ribosome entry site (IRES).
  • IRES sequence is used to produce more than one polypeptide from a single gene transcript.
  • An IRES sequence would be used to produce a protein that contain more than one polypeptide chains. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al., and references cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989], In some embodiments, a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.
  • 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 [3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF 1 a promoter [Invitrogen] .
  • a promoter is an enhanced chicken [3-actin promoter.
  • a promoter is a U6 promoter.
  • 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 metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline -repressible system (Gossen et al., Proc. Natl. Acad. Sci.
  • MT zinc-inducible sheep metallothionein
  • 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 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.
  • native promoter refers to the endogenous promoter of the transgene.
  • 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.
  • tissue-specific regulatory sequences are well known in the art.
  • tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin- 1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.
  • Beta-actin promoter hepatitis B virus core promoter, Sandig et al., Gene Ther., 3: 1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7: 1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24: 185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11 :654-64 (1996)), CD2 promoter (Hansal et al., J.
  • Immunol., 161: 1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, 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. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373- 84 (1995)), among others which will be apparent to the skilled artisan.
  • NSE neuron- specific enolase
  • Nervous system (NS)-specific promoters contemplated for use in the present methods and compositions also include those described in International Patent Application Numbers WO/2022/049385 and WO/2021/214443, which are incorporated by reference herein in their entireties.
  • the NS-specific promoter is a promoter of Table 1, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a promoter of Table 1.
  • the NS-specific promoter is a promoter of Table 1, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a promoter of Table 1 and retaining the NS-specific promoter activity of the promoter of Table 1.
  • CNS-specific promoters contemplated for use in the present methods and compositions also include those described in International Patent Application WO/2021/214443, the contents of which are incorporated by reference herein in their entireties.
  • the CNS-specific promoter is a promoter of Tables 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a promoter of Tables 2-4.
  • the CNS-specific promoter is a promoter of Tables 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a promoter of Tables 2-4 and retaining the CNS-specific promoter activity of the promoter of Tables 2-4.
  • the nucleic acid comprises one or more cis-regulatory elements (CREs). In some embodiments, the nucleic acid comprises one or more NS-specific CREs or CNS- specific CREs. In some embodiments, the nucleic acid comprises one or more CREs of Tables 4-6, or a CRE having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a CRE of Tables 4-6.
  • CREs cis-regulatory elements
  • the CRE is a CRE of Tables 4-6, or a CRE having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to a CRE of Tables 4-6 and retaining the activity of the CRE of Tables 4-6.
  • the CRE can comprise one or more CREs known in the art.
  • the one or more CREs may be selected from SEQ ID NOs: 19-24, 27, 28, 37, 38 in International Patent Application Number WO/2022/049385.
  • the one or more CREs may be selected from: SEQ ID NOs: 1-8 from WO 2019/199867A1, SEQ ID NOs: 1-7 from W02020/076614A1 and SEQ ID NOs: 25-51, 177-178, 188 from W02020/097121.
  • the foregoing references are incorporated by reference herein in their entireties.
  • Cis-regulatory elements comprised in the promoters of Table 2
  • the promoter is a synapsin (Synl) promoter (see e.g., SEQ ID NO: 61).
  • the promoter comprises a nucleic acid sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical, to SEQ ID NO: 61.
  • composition comprising a recombinant viral vector comprising a promoter comprising a nucleic acid sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical, to SEQ ID NO: 61.
  • the nucleic acid further includes an enhancer sequence helpful in driving expression to the CNS, for example, to specified CNS tissues or cell types.
  • enhancer sequences are described in, e.g., US Patent Application Nos 17/283,232; US 17/291 , 584; or International Patent Publication Nos WO2020168279A2; WO2021195591A2; WO2021248085A2; WO2021216778A2; the contents of each are incorporated herein by reference in their entireties.
  • the nucleic acid comprises a transgene that encodes a protein.
  • the protein can be a therapeutic protein (e.g., a peptide, protein, or polypeptide useful for the treatment or prevention of disease states in a mammalian subject) or a reporter protein.
  • the protein is GDNF.
  • the protein is human GDNF.
  • the GDNF gene encodes SEQ ID NO: 2 or a protein comprising SEQ ID NO: 2.
  • the GDNF gene encodes a protein with a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98% to SEQ ID NO: 2.
  • the therapeutic protein, and gene encoding such protein is useful for treatment or slowing of the progression of PD.
  • a nucleic acid described herein may further comprise a reporter sequence (e.g., nucleic acid sequences encoding a reporter protein).
  • Reporter sequences include, without limitation, DNA sequences encoding [3-lactamase, [3 - galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the reporter sequences When associated with regulatory elements which drive their expression, the reporter sequences, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for [3- galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
  • Such reporters can, for example, be useful in verifying the tissue- specific targeting capabilities and tissue specific promoter regulatory activity of a nucleic acid.
  • Glial cell line-derived neurotrophic factor (GDNF; NCBI Gene ID: 2668), also known as ATF, ATF1, ATF2, HSCR3, or HFB1-GDNF, is a neurotrophic factor that supports the development and survival of peripheral sympathetic, parasympathetic, enteric and sensory neurons as well as midbrain dopamine neurons and motoneurons.
  • GDNF Parkinson's disease
  • GDNF can prevent the neurotoxin-induced death of dopamine neurons and can promote axonal sprouting leading to functional recovery.
  • GDNF splice variants Two GDNF splice variants, called pre-(a)pro- GDNF (previously called GDNFa) and pre-(P)pro-GDNF (previously called GDNFJ3), have been described (Suter-Crazzolara and Unsicker, Neuroreport, 5:2486-2488 (1994)). These splice variants are produced by alternative splicing of the GDNF mRNA.
  • Many secreted proteins including neurotrophic factors, are synthesized in the forms of precursors, pre-pro-mature proteins.
  • the pre-region consisting of the ER signal peptide, is clipped off during translation by a signal peptidase, and the pro-mature protein is released into the lumen of the ER immediately after being synthesized.
  • the proteolytic cleavage of the mature protein can occur either inside the cell or in the extracellular matrix, or both.
  • the pro-mature protein can also remain uncleaved and have different function than the cleaved mature protein.
  • BDNF mature brain-derived neurotrophic factor
  • pro-BDNF pro-BDNF are secreted from neuronal cells.
  • Mature BDNF binds to TrkB receptor inducing neuronal survival, differentiation and synaptic modulation
  • pro-BDNF binds to p75 NTR and sortilin receptors inducing apoptosis (to review, see Thomas and Davies, Curr. Biol., 15:262-264 (2005); Teng et al., J. Neurosci., 25:5455-5463 (2005)).
  • GDNF mRNA and GDNF protein have been used for the full- length pre-(a)pro-GDNF mRNA and for the mature GDNF protein that is produced by proteolytic cleavage of the (a)pro-GDNF protein.
  • This mature GDNF protein has been extensively studied, and in PubMed more than 2500 citations are available for GDNF.
  • GDNF was identified based on its ability to increase neurite length, cell size, and the number of dopaminergic neurons as well as their high affinity dopamine uptake in culture (Lin et al., Science, 260: 1130-1132 (1993)).
  • GDNF is a potent factor for the protection of nigral dopaminergic neurons against their toxin-induced degeneration in animal models of PD and also in the treatment of patients with PD (reviewed in Airaksinen and Saarma, Nat. Rev. Neurosci. 3:383-394 (2002) and Bespalov and Saarma, Trends Pharmacol. Sci. 28:68-74 (2007)).
  • GDNF has a therapeutic role in the treatment of animal models of amyotrophic lateral sclerosis (ALS), addiction, alcoholism and depression (reviewed in Bohn, Exp. Neurol., 190:263-275 (2004); Messer et al., Neuron, 26:247-257 (2000); He et al., J.
  • GDNF has important roles also outside the nervous system. It acts as a morphogen in kidney development and regulates the differentiation of spermatogonia (reviewed in Sariola and Saarma, J. Cell Sci. 116:3855- 3862 (2003)).
  • a viral vector for slowing or inhibiting progression of PD wherein the vector comprises a GDNF encoding nucleic acid.
  • the viral vector is an Adeno-Associated Virus (AAV) vector (e.g., an rAAV).
  • AAV Adeno-Associated Virus
  • the viral vector comprises a nucleic acid sequence that encodes the amino acid sequence SEQ ID NO: 2. In some embodiments, the viral vector comprises a nucleic acid sequence that encodes an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 2.
  • the viral vector comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1. In some embodiments, the viral vector comprises the sequence of SEQ ID NO: 1.
  • gene refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed or translated.
  • coding sequence or "a sequence which encodes a particular protein”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • Genbank Access NM_000514.4 SEQ ID NO: 1
  • the amino acid sequence is shown in SEQ ID NO: 2.
  • Methods described herein makes use of a nucleic acid construct comprising sequence SEQ ID NO: 1 or a variant thereof for slowing or inhibiting the progression of PD.
  • the variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc.
  • the term variant also includes GDNF gene sequences from other sources or organisms.
  • Variants are preferably substantially homologous to SEQ ID NO: 1 and/or 2 , i.e., exhibit a nucleotide sequence identity of typically at least about 75%, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95% with SEQ ID NO: 1 or 2.
  • the nucleic acid construct comprises a sequence with at least 95% sequence identity to SEQ ID NO: 1 and which retains the activity of SEQ ID NO: 1 or 2.
  • Variants of a GDNF gene also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions.
  • Typical stringent hybridization conditions include temperatures above 30° C, preferably above 35 °C, more preferably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM. Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc.
  • variants at nucleotide 277, 633, and 1389 of GDNF For example, a C to T point mutation at nucleotide 277 (see, e.g., SEQ ID NO: 62), a C to G point mutation at nucleotide 633 (see, e.g., SEQ ID NO: 63), and a A to G point mutation at nucleotide 1389.
  • Other variants are possible including codon optimized sequences, and conservative changes. Conservative substitutions are well known in the art.
  • GDNF gene is codon optimized.
  • the GDNF nucleic acid sequence is codon optimized, for example, for any one or more of: (1) enhanced expression in vivo, (2) to reduce CpG islands or (3) reduce the innate immune response.
  • a skilled artisan can codon-optimize GDNF using standard techniques in the art.
  • the viral vector comprises a nucleic acid sequence that encodes the amino acid sequence SEQ ID NO: 2, or variant thereof. In some embodiments, the viral vector comprises a nucleic acid sequence that encodes an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 2.
  • the vector is adeno-associated virus (AAV) or recombinant AAV.
  • AAV adeno-associated virus
  • the disclosure provides isolated AAVs.
  • isolated refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as "recombinant AAVs".
  • Recombinant AAVs preferably have tissue- specific targeting capabilities, such that a nuclease and/or transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s).
  • the AAV capsid is an important element in determining these tissue-specific targeting capabilities.
  • an rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety).
  • the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.
  • ITRs AAV inverted terminal repeats
  • capsid proteins are structural proteins encoded by the cap gene of an AAV.
  • AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing.
  • the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa.
  • capsid proteins upon translation, form a spherical 60-mer protein shell around AAV genome.
  • the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
  • capsid proteins deliver the viral genome to a host in a tissue specific manner.
  • a recombinant AAV (rAAV) capsid protein is of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV 2G9, AAV 2.5G9, AAV9, and AAV10.
  • an AAV capsid protein is of a serotype derived from a non- human primate, for example AAVrh10 serotype.
  • an AAV capsid protein is of an AAV9 serotype.
  • the capsid protein is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV 12, or AAV 13 capsid protein or, a chimera thereof.
  • the rAAV comprises a capsid protein from serotype AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 2G9, AAV 2.5G9, AAV rh8, AAV rh10, AAV rh74, AAV10, or, AAV 11 or, a chimera thereof.
  • the AAV serotype and/or capsid described herein is selected from Table 7.
  • the rAAV comprises a chemically modified capsid as disclosed in WO 2017/212019 e.g., mannose ligand is chemically coupled to AAV2.
  • the rAAVs with chemically modified capsids disclosed in WO 2017/212019 is incorporated herein by reference in its entirety.
  • the AAV capsid proteins and virus capsids used herein can be polyploid (also referred to as rational haploid) in that they can comprise different combinations of VP1, VP2 and VP3 AAV serotypes in a single AAV capsid as described in PCT/US 18/22725, PCT/US2018/044632, or US 10,550,405 which are incorporated by reference.
  • the components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the required components ⁇ e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell will contain the required component(s) under the control of an inducible promoter.
  • the required component(s) may be under the control of a constitutive promoter.
  • a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
  • the instant disclosure relates to a host cell containing a nucleic acid that comprises a coding sequence encoding a protein (e.g., wild-type huntingtin protein, optionally "hardened” wildtype huntingtin protein).
  • a protein e.g., wild-type huntingtin protein, optionally "hardened” wildtype huntingtin protein.
  • the instant disclosure relates to a composition comprising the host cell described above.
  • the composition comprising the host cell above further comprises a cryopreservative.
  • the recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).
  • the selected genetic element may be delivered by any suitable method, including those described herein.
  • the methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure.
  • recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650).
  • the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector.
  • An AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation.
  • the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes).
  • vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein.
  • the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions").
  • the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. [00269]
  • the disclosure provides transfected host cells.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Uaboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197.
  • Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
  • a "host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
  • the term "vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term “vector” includes cloning and expression vehicles, as well as viral vectors.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments are ligated.
  • viral vector Another type of vector, wherein DNA segments are ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid” and “vector” is used interchangeably as the plasmid is the most commonly used form of vector.
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis.
  • replication can occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript.
  • Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., [3-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • the vectors used herein are capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • the recombinant AAV comprising a nucleic acid encoding GDNF is produced by the triple transfection method that uses close ended linear duplexed DNA molecules that lack bacterial backbone sequences, for example, as described in PCT/US2021/013689, published as WO/2021/146591, which is incorporated herein by reference in its entirety.
  • the recombinant AAV comprising a nucleic acid encoding GDNF is produced by the method as described in PCT/US2022/013279, published as WO2022159679, which is incorporated herein by reference in its entirety.
  • useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • the precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but in general can include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of described herein may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
  • expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically- active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.
  • one or more of the recombinantly expressed gene can be integrated into the genome of the cell.
  • a nucleic acid molecule described herein can be introduced into a cell or cells using methods and techniques that are standard in the art.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule described herein also may be accomplished by integrating the nucleic acid molecule into the genome.
  • the genome packaged within AAV2-GDNF comprises a sequence of SEQ ID NO: 64. In one embodiment, the genome packaged within AAV2-GDNF consists of or consists essentially of the sequence of SEQ ID NO: 64. In one embodiment, the genome packaged within AAV2-GDNF comprises, consists of, or consist essentially of a sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO: 64.
  • the AAV2-GDNF vector comprises the ITRto ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2,716 of SEQ ID NO: 64). In one embodiment, the AAV2-GDNF vector consists of or consists essentially of the ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2,716 of SEQ ID NO: 64).
  • the AAV2-GDNF vector comprises, consists of, or consists essentially of the ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2,716 of SEQ ID NO: 64) and is generated form a plasmid comprising, consisting of, or consisting essentially of SEQ ID NO: 64.
  • the AAV2-GDNF vector comprises, consists of, or consist essentially of a sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
  • rAAV is manufactured using plasmid DNA as set forth in SEQ ID NO: 64, which is depicted in Fig. 26.
  • rAAV is manufactured using close ended linear duplexed DNA.
  • AAV2-GDNF includes a plasmid comprising the ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2,716 of SEQ ID NO: 64).
  • AAV2-GDNF includes close ended linear duplexed DNA comprising the ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2,716 of SEQ ID NO: 64), a non-limiting example of which is Doggybone DNA (dbDNATM), as disclosed in US Application 2018/0037943 and Karbowniczek et al., Bioinsights, 2017, which is incorporated herein in its entirety by reference.
  • dbDNATM Doggybone DNA
  • the plasmid depicted in Fig. 26 is used to generate the AAV2-GDNF genome.
  • genome packaged within AAV2-GDNF is depicted in Fig. 26.
  • the AAV2-GDNF is manufactured using the plasmid depicted in Fig. 26. [00290] In one embodiment, AAV2-GDNF comprises at least one component listed in Table 8.
  • the capsid described herein is further modified to increase tropism for the CNS.
  • tropism of the capsid, and therefore the AAV is increased by at least
  • a capsid is modified such that its tropism for a non-CNS tissue is decreased.
  • a capsid having a liver-specific tropism can be modified such that it no longer has such tropism.
  • a capsid is modified such that its tropism for a non-CNS tissue is decreased by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%;
  • the modified capsid is modified such that its tropism for CNS tissue is increased and its tropism for a non-CNS tissue is decreased.
  • a capsid having liver-specific tropism can be modified such that it exhibits CNS-specific tropism and has decreased liver-specific tropism.
  • CNS-tropism of the capsid is increased by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 20%;
  • 750x, or l,000x or greater as compared to an unmodified capsid, and tropism for a non-CNS tissue is decreased by at least 1%; 2%; 3%; 4%; 5%; 6%; 7%; 8%; 9%; 10%; 11%; 12%; 13%; 14%; 15%; 16%; 17%; 18%; 19%; 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; 70%; 71%; 72%; 73%; 74%;
  • composition comprising a modified viral capsid comprising a payload, wherein the payload comprises a regulatory sequence and a nucleic sequence flanked by inverted terminal repeats (ITRs) that target a central nervous system disorder, and wherein the modification is a chemical, non-chemical or amino acid modification.
  • the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a transgene, e.g., GDNF.
  • the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a GDNF protein.
  • the modified viral capsid comprises modification that results in its preferential targeting of the CNS.
  • the modified viral capsid has increased tropism for the CNS, and/or decreased tropism for at least a second location, e.g., the liver.
  • Preferential targeting of the CNS does not exclude targeting to other sites, but rather indicates that it is more highly targeted to the CNS as compared to another site.
  • the modified viral capsid comprises modification that results in its targeting of the CNS.
  • a modification to a capsid that typically targets a non-CNS site e.g., the liver
  • the CNS-targeting does not need to be preferential.
  • the modification to the capsid is an amino acid modification, e.g., an amino acid deletion, insertion, or substitute.
  • the amino acid modification increases tropism for the CNS.
  • the amino acid modification targets the modified capsid to the CNS.
  • the modified viral capsid has or consists of, or consists essentially of a nucleic acid sequence that is 90% identical to SEQ ID NOs 1-4 of US Patent Application No.
  • the modified viral capsid is an AAV capsid protein comprising one or more amino acids substitutions, wherein the substitutions introduce a new glycan binding site into the AAV capsid protein.
  • the amino acid substitutions are in amino acid 266, amino acids 463-475 and amino acids 499-502 in AAV2 or the corresponding amino acid positions in AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV10.
  • AAV capsid protein is further described in, e.g., US Patent Application No. 16/110,773; the contents of which are incorporated herein by references in its entirety.
  • the modified viral capsid is an AAV capsid protein that comprises, consists of, or consists essentially of an AAV 2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT/US2020/029493; the contents of which are incorporated herein by references in its entirety) comprising one or more amino acid substitutions that introduce a new glycan binding site.
  • amino acid substitutions can target the capsid to neurons and glial cells, such as astrocytes.
  • the one or more amino acid substitutions comprise A267S, SQAGASDIRDQSR464-476SX1AGX2SX3X4X5X6QX7R (SEQ ID NOS 153 and 154, respectively), wherein X1-7 can be any amino acid, and EYSW 500-503 (SEQ ID NO: 155) EX 8 X 9 W, wherein X 8-9 can be any amino acid.
  • Xi is V
  • X2 is P
  • X3 is N
  • X4 is M
  • X5 is A
  • Xg is V
  • X7 is G
  • X x is F
  • X9 is A, wherein the new glycan binding site is a galactose binding site.
  • AAV capsid protein is further described in, e.g., International Patent Application No. WO/2020/219656; the contents of which are incorporated herein by references in its entirety.
  • the modified viral capsid is an AAV capsid protein particle comprising a surface-bound peptide, wherein the peptide bound to the surface of the AAV particle is Angiopep-2, GSH, HIV-1 TAT (48-60), ApoE (159-167)2, Leptin 30 (61-90), THR, PB5-3, PB5-5, PB5-14, or any combination thereof, as described in, e.g., US Patent Application No. 16/956,306; the contents of which are incorporated herein by references in its entirety.
  • AAV capsid permits delivery, e.g., of a payload, across the blood brain barrier.
  • the modified viral capsid comprises a AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of the capsid protein comprises modifications (e.g., replacement of a tyrosine residue with a non-tyrosine residue and/or a threonine residue with a non-threonine residue) at positions corresponding to: one or more of, or each of Y705, Y731, and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of US Patent Application No.
  • AAV capsid protein e.g., an AAV1, AAV5, or AAV6 capsid protein
  • modifications e.g., replacement of a tyrosine residue with a non-tyrosine residue and/or a threonine residue with a non-threonine residue
  • 16/565,191 the contents of which are incorporated herein by references in its entirety); one or more of, or each of Y436, Y693, and Y719 of a wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of US Patent Application No. 16/565,191); or one or more of, or each ofY705, Y731, and T492 of a wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of US Patent Application No. 16/565,191).
  • AAV capsids target neurons and astrocytes.
  • the modified viral capsid comprises a AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein) comprising Y to F (tyrosine to phenylalanine) modifications or T to V (threonine to valine) modifications in the VP3 region of the capsid at positions corresponding to: one or more of or each of Y705F, Y73 IF, and T492V of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of US Patent Application No.
  • AAV capsid protein e.g., an AAV1, AAV5, or AAV6 capsid protein
  • Y to F tyrosine to phenylalanine
  • T to V threonine to valine
  • AAV capsids target neurons and astrocytes.
  • the modified viral capsid comprises AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein), wherein a VP3 region of the capsid protein comprises modifications (e.g., replacement of a tyrosine residue with a non-tyrosine residue and/or a threonine residue with a non-threonine residue) at positions corresponding to: one or more of or each of Y705, Y731, and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of US Patent Application No.
  • AAV capsid protein e.g., an AAV1, AAV5, or AAV6 capsid protein
  • modifications e.g., replacement of a tyrosine residue with a non-tyrosine residue and/or a threonine residue with a non-threonine residue
  • AAV capsids target neurons and astrocytes.
  • the modified viral capsid is AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein) comprising Y to F (tyrosine to phenylalanine) modifications or T to V (threonine to valine) modifications in the VP3 region of the capsid protein at positions corresponding to: one or more of or each of Y705F, Y73 IF, and T492V of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of US Patent Application No.
  • AAV capsid protein e.g., an AAV1, AAV5, or AAV6 capsid protein
  • Y to F tyrosine to phenylalanine
  • T to V threonine to valine
  • AAV capsids target neurons and astrocytes.
  • the amino acid modification permits the modified capsid to evade neutralizing antibodies, for example, that are generated against a viral vector, e.g., of the same serotype.
  • the amino acid modification permits the modified capsid to be used for repeat administration, for example, the modification will enable the capsid to have a therapeutic effect upon re -administration.
  • the modified viral capsid is a chimeric capsid.
  • a “chimeric” capsid protein as used herein means an AAV capsid protein (e.g., any one or more of VP1, VP2 or VP3) that has been modified by substitutions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to wild type, as well as insertions and/or deletions of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to wild type.
  • complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wild type domain, functional region, epitope, etc. of a different AAV serotype, in any combination, to produce a chimeric capsid protein.
  • Production of a chimeric capsid protein can be carried out according to protocols well known in the art and a significant number of chimeric capsid proteins are described in the literature as well as herein that can be included in the capsid.
  • the modified viral capsid is a haploid capsid.
  • haploid AAV shall mean that AAV as described in International Application W02018/170310, or US Application US2018/037149, which are incorporated herein in their entirety by reference.
  • a population of virions is a haploid AAV population where a virion particle can be constructed wherein at least one viral protein from the group consisting of AAV capsid proteins, VP1, VP2 and VP3, is different from at least one of the other viral proteins, required to form the virion particle capable of encapsulating an AAV genome.
  • VP1 and VP2 are chimeric and only VP3 is non-chimeric.
  • VP1/VP2 the viral particle composed of VP1/VP2 from the chimeric AAV2/8 (the N-terminus of AAV2 and the C- terminus of AAV8) paired with only VP3 from AAV2; or only the chimeric VP1/VP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from AAV2 without mutation of VP3 start codon) paired with only VP3 from AAV2.
  • only VP3 is chimeric and VP1 and VP2 are non- chimeric.
  • at least one of the viral proteins is from a completely different serotype.
  • no chimeric protein is present.
  • a modified viral capsid comprises one or more modifications, e.g., a chemical modification, a non-chemical modification, or an amino acid modification to the capsid.
  • modifications can, for example, modify the tissue-type tropism or cell-type tropism of the modified capsid, among other things.
  • Modifications can alter the properties of the capsid, including biochemical properties such as receptor binding, directly, such that the modification itself alters the behavior of the capsid, or can permit further modification, such as the attachment of a ligand which in turn modifies behavior of the capsid in a desired manner.
  • cysteine residues which may be naturally present or introduced by genetic modification of a capsid polypeptide coding sequence, permits the covalent attachment of a ligand via disulfide bond formation (see, e.g., WO 2005/106046, the contents of which are incorporated herein by reference).
  • ligands are contemplated, including but not limited to antibodies or antigen-binding fragments thereof that, for example, target a cell-surface protein expressed by a target cell (see, e.g., WO 2000/002654, which is incorporated herein by reference).
  • WO2015/062516 describes the insertion of an amino acid comprising an azido group by genetic modification of the capsid gene, followed by chemical conjugation of a ligand via the azido group.
  • AAV capsid tropism by glycation, or chemical conjugation of sugar moieties, is described by Horowitz et al., Bioconjugate Chem. 22: 529-532 (2011). That approach, and similar approaches are contemplated for modification of capsids as described herein.
  • the coating of a viral capsid with a polymer such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl)methacrylamide (pHPMA) is specifically contemplated.
  • a polymer such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl)methacrylamide (pHPMA)
  • PEG polyethylene glycol
  • pHPMA poly-(N-hydroxypropyl)methacrylamide
  • carbodiimide coupling is specifically contemplated. See, e.g., Joo et al. ACS Nano 5, titled “Enhanced Real-time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates” (2011).
  • the viral capsid can be modified, e.g., as described in WO 2017/212019, see also U.S. National Phase USSN 16/308,740, the contents of which are each incorporated herein by reference.
  • the approach described therein couples a viral capsid to a ligand via bonds comprising -CSNH- and an aromatic moiety. While genetically modified viral capsids can be further modified by this approach, the modifications described therein do not require genetic modification of the viral capsid.
  • Ligands described therein include, for example, a targeting agent, a steric shielding agent for avoiding neutralizing antibody interactions, a labeling agent or a magnetic agent.
  • Targeting ligands described therein include, for example, a cell-type specific ligand, a protein, a mono- or polysaccharide, a steroid hormone, an RGD motif peptide (e.g., Arg-Gly-Asp, a cell adhesion motif which can mimic cell adhesion proteins and bind to integrins), a vitamin, and a small molecule.
  • a cell-type specific ligand e.g., a protein, a mono- or polysaccharide, a steroid hormone, an RGD motif peptide (e.g., Arg-Gly-Asp, a cell adhesion motif which can mimic cell adhesion proteins and bind to integrins), a vitamin, and a small molecule.
  • the chemical modification described herein is a modification described in International patent application WO/2017/212019, the content of which is incorporated herein by reference in its entirety.
  • the chemical modification described herein is a modification described in International patent application WO/2021/005210, the content of which is incorporated herein by reference in its entirety.
  • the capsid has at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I):
  • -XI is selected from the group consisting of:
  • -Ar is an aryl or a heteroaryl moiety optionally substituted.
  • the capsid has at least one chemically-modified tyrosine residue is of formula (la):
  • - Spacer is a group for linking the "Ar” group to the functional moiety "M” which preferably comprises up to 1000 carbon atoms and which is preferably in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties,
  • -M is a functional moiety comprising a steric agent, a labelling agent, cell-types specific ligand or a drug moiety.
  • Xi is of formula (a) and/or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl.
  • the capsid has at least one chemically-modified tyrosine is of formula
  • -X2 is at position para, meta or ortho, preferably at position para of the phenyl group
  • -Spacer, n and M are as defined herein above.
  • Spacer when present, is selected from the group consisting of saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains, optionally substituted, polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-
  • M comprises, or consists of, cell-type targeting ligand, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide (e.g., Arg- Gly-Asp, a cell adhesion motif which can mimic cell adhesion proteins and bind to integrins), a muscle targeting peptide (MTP) or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as antigen-binding fragment (Fab), Fab' (which is the antigen-binding fragment further comprising a free sulfhydryl group), and VHH, a single-chain fragment variable (ScF
  • "Spacer” (when present) is selected from the group consisting of linear or branched C2-C20 alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymer of alkyl diamine and combinations thereof, said polymers having from 2 to 20 monomers and/or "M” comprises, or consists of, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor 13FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N-acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, MTP selected from SEQ ID NO: 1 to SEQ ID NO:7, and vitamins such as folic acid.
  • EGF Epidermal Growth Factor
  • 13FGF basic Fibroblast Growth Factor 13FGF
  • the capsid further has at least one additional chemically modified amino acid residue in the capsid, which is different from a tyrosine residue, said amino acid residue preferably bearing an amino group chemically modified with a group of formula (V):
  • N* being the nitrogen of the amino group of an amino acid residue, e.g. of a lysine residue or arginine residue, and
  • the capsid is incubated a chemical reagent bearing a reactive group selected from an aryl diazonium, and a 4-phenyl-l, 2, 4-triazole-3, 5-dione (PTAD) moiety in conditions conducive for reacting said reactive group with a tyrosine residue present in the capsid so as to form a covalent bound.
  • a chemical reagent bearing a reactive group selected from an aryl diazonium, and a 4-phenyl-l, 2, 4-triazole-3, 5-dione (PTAD) moiety in conditions conducive for reacting said reactive group with a tyrosine residue present in the capsid so as to form a covalent bound.
  • PTAD 4-phenyl-l, 2, 4-triazole-3, 5-dione
  • the capsid is incubated with a chemical reagent of formula Vid to obtain the at least one chemically-modified tyrosine residue in the capsid of formula Ic.
  • the expression cassetes, vectors or virions of the present invention may be formulated in a pharmaceutical composition with a pharmaceutically acceptable excipient i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilizers, etc.
  • a pharmaceutically acceptable excipient i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilizers, etc.
  • the pharmaceutical composition may be provided in the form of a kit.
  • a further aspect of the present invention provides a pharmaceutical composition comprising an expression cassete, a vector or virion as described herein.
  • the pharmaceutical composition comprises a phosphate buffer.
  • the phosphate buffer comprises from about 1 mM to about 50 mM phosphate, such as phosphate at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.
  • the phosphate is prepared from a combination of dibasic phosphate (e.g., Na2HPC>4, K2HPO4) and monobasic phosphate (e.g., NaH2PO4, KH2PO4) at a dibasic phosphate: monobasic phosphate molar ratio of from about 1: 10 to about 10: 1.
  • dibasic phosphate e.g., Na2HPC>4, K2HPO4
  • monobasic phosphate e.g., NaH2PO4, KH2PO4
  • the 10 mM phosphate comprises 9.5 mM dibasic phosphate and 0.5 mM monobasic phosphate, 9 mM dibasic phosphate and 1 mM monobasic phosphate
  • the pH of the phosphate buffer is from about 6.5 to about 7.5, such as a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.
  • the pH of the phosphate buffer is 12-13. In one embodiment, the pH of the phosphate buffer is 7.22.
  • the phosphate buffer can also include NaCl at a concentration of from about 50 mM to about 200 mM, such as at a concentration of about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 135 mM, about 136 mM, about 137 mM, about 138 mM, about 139 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.
  • the phosphate buffer can also include KC1 at a concentration of from about 0.5 mM to about 10 mM, such as at a concentration of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 2.5 mM, about
  • the phosphate buffer can also include CaC’h at a concentration of from about 0.20 mM to about 10 mM, such as at a concentration of about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.81 mM, about 0.82 mM, about 0.83 mM, about 0.84 mM, about 0.85 mM, about 0.86 mM, about 0.87 mM, about 0.88 mM, about 0.89 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.
  • CaC’h at a concentration of from about 0.20 mM to about 10 mM, such as at a concentration of about 0.2
  • the phosphate buffer can also include MgCh at a concentration of from about 0.10 mM to about 1 mM, such as at a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM.
  • the phosphate buffer can also include Poloxamer 188 (e.g., PluronicTM F-68 non-ionic surfactant) at a concentration of from about 0.0001 wt.%to about 0.005 wt.%, such as at a concentration of about 0.0001 wt.%, about 0.0002 wt.%, about 0.0003 wt.%, about 0.0004 wt.%, about 0.0005 wt.%, about 0.0006 wt.%, about 0.0007 wt.%, about 0.0008 wt.%, about 0.0009 wt.%, about 0.001 wt.%, about 0.0015 wt.%, about 0.002 wt.%, about 0.0025 wt.%, about 0.003 wt.%, about 0.0035 wt.%, about 0.004 wt.%, about 0.0045 wt.%, or about 0.005 wt.%.
  • the phosphate buffer can also include sorbitol at a concentration of from about 0.005 wt.% to about 10 wt.%, such as at a concentration of about 0.005 wt.%, about 0.075 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 w
  • the rAAV comprising the nucleic acid (for example, AAV2 comprising the CMV promoter and the nucleic acid comprising a sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical, to SEQ ID NO: 1; AAV2-GDNF) can have a titer in the phosphate buffer of from about 1x10 12 vg/mL to about 4x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 1x10 12 vg/mL to about 3x10 12 vg/mL; 1x10 12 vg/mL to about 2x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 8x10 11 vg/mL to about 9x10 12 vg/mL; 9x10 11 vg/m
  • the pharmaceutical composition comprises, consists essentially of, or consists the composition described in Table 9.
  • the pharmaceutical composition comprises, consists essentially of, or consists of phosphate (monobasic and dibasic phosphate), NaCl, and poloxamer; pH 7.2-7.3.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 10 mM phosphate (monobasic and dibasic phosphate), about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM Na2HPO 4 , about 2 mM NaftPO 4 , about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and about 1x10 12 vg/mL to about 3.1x10 13 vg/mL AAV2- GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1x10 12 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3,' and at least 5x10 12 vg/mL vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1x10 13 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM dibasic phosphate, about 2 mM monobasic phosphate, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 3x10 13 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM K2HPO4, about 2 mM KH2PO4, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and about 1x10 12 vg/mL to about 3.1x10 13 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM K2HPO4, about 2 mM KH2PO4, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1x10 12 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM K2HPO4, about 2 mM KH2PO4, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 5x10 12 vg/mL vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM K2HPO4, about 2 mM KH2PO4, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1x10 13 vg/mL AAV2-GDNF.
  • the pharmaceutical composition comprises, consists essentially of, or consists of about 8 mM K2HPO4, about 2 mM KH2PO4, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 3x10 13 vg/mL AAV2-GDNF.
  • the rAAVs of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art.
  • an rAAV preferably suspended in a physiologically compatible carrier (i.e., in a composition) may be administered to a subject, i.e., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque).
  • a host animal does not include a human.
  • CNS all cells and tissue of the brain and spinal cord of a vertebrate.
  • the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like.
  • Recombinant AAVs may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum.
  • rAAV as described in the disclosure are administered by intravenous injection.
  • the rAAV are administered by intracerebral injection.
  • the rAAV are administered by intrathecal injection.
  • the rAAV are administered by intrastriatal injection.
  • the rAAV are delivered by intracranial injection.
  • the rAAV are delivered by cistema magna injection.
  • the rAAV are delivered by cerebral lateral ventricle injection.
  • the rAAVs or compositions thereof are delivered locally to the CNS, e.g., directly to the putamen, via a stepped cannula, e.g., as described in US Patent Nos 7,815,623; 8,337,458; and 9,302,070, the contents of each of which are incorporated herein by reference in their entireties.
  • the rAAVs or compositions thereof are delivered locally to the CNS, e.g., directly to the putamen, via SmartFlow cannula connected to MRI-compatible infusion pumps (e.g. Medfusion syringe pump, Smiths Medical Inc.
  • MRI-compatible infusion pumps e.g. Medfusion syringe pump, Smiths Medical Inc.
  • the compostions described herein are locally administered, e.g., to the putamen, at a flow rate of 1-30 pU/min via a cannula.
  • the flow rate is about 1-25 pU/min; 1-20 ⁇ L/min; 1-15 ⁇ L/min; 1-10 ⁇ L/min; 1-5 ⁇ L/min; 5-30 ⁇ L/min; 10-30 ⁇ L/min; 15-30 ⁇ L/min; 20-30 ⁇ L/min; 25-30 ⁇ L/min; 5-25 ⁇ L/min; 10-20 ⁇ L/min; 15-25 ⁇ L/min; 5-15 ⁇ L/min; 5-25 ⁇ L/min; or 10-15 ⁇ L/min.
  • the flow rate is about 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; 10 ⁇ L/min; 11 ⁇ L/min; 12 ⁇ L/min; 13 ⁇ L/min; 14 ⁇ L/min; 15 ⁇ L/min; 16 ⁇ L/min; 17 ⁇ L/min; 18 ⁇ L/min; 19 ⁇ L/min; 20 ⁇ L/min; 21 ⁇ L/min; 22 ⁇ L/min; 23 ⁇ L/min; 24 ⁇ L/min; 25 ⁇ L/min; 26 ⁇ L/min; 27 ⁇ L/min; 28 ⁇ L/min; 29 ⁇ L/min; or 30 ⁇ L/min.
  • the rAAVs may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit.
  • the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions.
  • isolated limb perfusion technique described in U.S. Pat. No.
  • rAAV or composition thereof is administered during the subject “off’ period.
  • the rAAV or composition thereof is administered during the subject “on” period.
  • compositions for slowing or inhibiting a progression of PD in a subject comprising any of the recombinant adeno-associated virus (rAAV) comprising a genome comprising a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter described herein and a pharmaceutically acceptable carrier.
  • rAAV adeno-associated virus
  • the composition comprises any of the viral vectors described herein, and optionally, a pharmaceutically acceptable carrier.
  • the compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes).
  • a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.
  • compositions of the disclosure may further comprise a second therapeutic, e.g., an antiParkinson’s therapeutic described herein.
  • the compositions of the disclosure may further comprise any immune modulator described herein.
  • the compositions of the disclosure may further comprise a second therapeutic, e.g., an anti -Parkinson’s therapeutic described herein and any immune modulator described herein.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
  • compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • the rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., delivery to the putamen), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.
  • all or, at least one of the nucleic acid sequences disclosed herein are delivered via non-viral DNA constructs comprising at least one DD-ITR.
  • the non viral DNA constructs as described in WO 2019/246554 can be utilized to deliver one or more of the nucleic acids described herein.
  • WO 2019/246554 is incorporated herein by reference in its entirety.
  • the dose of rAAV virions required to achieve a particular "therapeutic effect,” e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.
  • a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.
  • the rAAV is administered at a total dose within the range of 5x10 12 vg to about 1 ,5x10 13 vg. In another embodiment the rAAV is administered at a total dose within the range of 1x10 12 vg to about 6.5x10 13 vg; 2x10 12 vg to about 6.5x10 13 vg; 3x10 12 vg to about 6.5x10 13 vg; 4x10 12 vg to about 6.5x10 13 vg; 6x10 12 vg to about 6.5x10 13 vg; 7x10 12 vg to about 6.5x10 13 vg;
  • the rAAV is administered at atotal of at least 5.1x10 12 vg; 5.2x10 12 vg; 5.3x10 12 vg; 5.4x10 12 vg; 5.5x10 12 vg; 5.6x10 12 vg; 5.7x10 12 vg; 5.8x10 12 vg; 5.9x10 12 vg; 6x10 12 vg;
  • the rAAV is administered at a total of at least 1x10 12 vg; at least 2x10 12 vg; at least 3x10 12 vg; at least 4x10 12 vg; at least 5x10 12 vg; at least 6x10 12 vg; at least 7x10 12 vg; at least 8x10 12 vg; at least 9x10 12 vg; at least lx10 13 vg; at least 2x10 13 vg; at least 3x10 13 vg; at least 4x10 13 vg; at least 5x10 13 vg; at least 6x10 13 vg; and at least 7x10 13 vg or more.
  • one half of the total dose is administered to each of the subject’s putamen. Said another way, the total dose is divided substantially evenly between the subject’s left putamen and right putamen.
  • an effective amount of an rAAV is an amount sufficient to target an infection in an animal, or to target a desired tissue.
  • an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model.
  • the effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue.
  • an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about IO 9 to IO 16 genome copies. In some cases, a dosage between about 10 11 to 10 13 rAAV genome copies is appropriate. In certain embodiments, 10 12 or 10 13 rAAV genome copies is effective to target CNS tissue (i.e., the putamen). In some cases, stable transgenic animals are produced by multiple doses of an rAAV.
  • the rAAV is introduced or administered in a liquid composition.
  • the liquid composition has an rAAV concentration of from about 3x10 12 vg/mL to about 4x10I 2 vg/mL.
  • the liquid composition has an rAAV concentration of from about 1x10 12 vg/mL to about 4x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 1x10 12 vg/mL to about 3x10 12 vg/mL; 1x10 12 vg/mL to about 2x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 8x10 11 vg/mL to about 9x10 12 vg/mL; 9x10 11 vg/mL to about 9x10 12 vg/mL; 1x10 12 vg/mL to about 9x10 12 vg/mL; 2x10 12 vg/mL to about 9x10 12 vg/mL; 3x10 12 vg/mL to about 9x10 12 vg/mL; 4x10 12 vg//l
  • the liquid composition has an rAAV concentration of about 8x10 11 vg/mL; 9x10 11 vg/mL; 1x10 12 vg/mL; 2x10 12 vg/mL; 3x10 12 vg/mL; 3.1x10 12 vg/mL; 3.2x10 12 vg/mL; 3.3x10 12 vg/mL; 3.4x10 12 vg/mL; 3.5x10 12 vg/mL; 3.6x10 12 vg/mL; 3.7x10 12 vg/mL; 3.8x10 12 vg/mL; 3.9x10 12 vg/mL;4x10 12 vg/mL; 5x10 12 vg/mL; 6x10 12 vg/mL; 7x10 12 vg/mL; 8x10 12 vg/mL; and 9x10 12 vg/mL.
  • a dose of rAAV is administered to a subject no more than once, e.g., it is administered to each putamen no more than once.
  • a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period).
  • a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days.
  • a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days).
  • a dose of rAAV is administered to a subject no more than bi-weekly (e.g., once in a two calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per six calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).
  • the rAAV or composition thereof is administered as an infusion via a trans-frontal (e.g., bi-frontal”) trajectory.
  • the trans-frontal trajectory is at least one trajectory that is substantially perpendicular to the A-P axis of each putamen and accessed through the frontal bone of a skull.
  • the trans-frontal administration can include infusing the rAAV through more than one trajectory that are substantially perpendicular the A-P axis of each putamen.
  • Each trajectory may be accessed through a single burr hole through the frontal bone of the skull or through separate individual burr holes. Thus, at least one burr hole in the frontal bone is required for each of the left and right putamen.
  • the administration can be performed using a trans-frontal trajectory, such as a bi-frontal trajectory, in which a cannula is guided from the frontal bone at a first trajectory that is substantially perpendicular to the A-P axis of a first putamen and the rAAV is infused using the first trajectory.
  • the cannula is then positioned at a second trajectory that is substantially perpendicular to the A-P axis of the first putamen (the seoncond trajectory being different from the first trajectory) and the rAAV is infused using the second trajectory.
  • the process is then repeated at a second putamen. It is understood that each trajectory results in the cannula contacting the putamen at different locations.
  • the rAAV or composition thereof is administered as an infusion via a bi- occipital trajectory.
  • An occipital trajectory is a single posterior trajectory that is substantially parallel to the A-P axis of a putamen and accessed through the occipital bone of a skull. Accordingly, a single burr hole in the occipital bone is needed per putamen.
  • the administration can be performed using the bi-occiptial trajectory in which a cannula is guided from the occipital bone using a trajectory that is substantially parallel to the A-P axis of a first putamen and the rAAV is infused while the cannula is being advanced toward a rostral end of the first putamn. The process is then repeated at a second putamen.
  • the rAAV is infused into each putamen while the cannula is stantionary and not being advanced to the rostral ends of eachputamen.
  • Fig. 13B shows an exemplary occipital trajectory.
  • each putamen is infused using the same trajectory technique, i.e., each putamen is infused via a bi-frontal trajectory.
  • each putamen is infused using a different trajectory technique, i.e., one putamen is infused via a bi-frontal trajectory and the other putamen is infused via a bi-occipital trajectory.
  • the infusion volume into a single putamen is less than or equal to 2000 pl, and greater than 1,800 pll.
  • the single putamen can be infused with a total volume that is 1,800 ul; 1,810 pl; 1,820 pl; 1,830 pl; 1,840 pl; 1,850 pl; 1,860 pl; 1,870 pl; 1,880 pl; 1,890 pl; 1,900 pl; 1,910 pl; 1,920 pl; 1,930 pl; 1,940 pl; 1,950 pl; 1,960 pl; 1,970 pl; 1,980 pl; 1,990 pl; or 2,000 pl.
  • the infusion volume for each putamen is the same.
  • the infusion volume for each putamen is different (e.g., one putamen is infused with l,800ul volume and the other putamen is infused with 2,000um volume).
  • rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., -I0 13 GC/ml or more).
  • high rAAV concentrations e.g., -I0 13 GC/ml or more.
  • Methods for reducing aggregation of rAAVs include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
  • One aspect described herein is a formulation for slowing or inhibiting a progression of PD in a subject comprising any viral vector (e.g., an AAV) described herein at a concentration of 3x10 12 vg to 4x10 12 vg per mb of a pharmaceutically acceptable carrier.
  • any viral vector e.g., an AAV
  • the concentration of the viral vector is from about 1x10 12 vg/mL to about 4x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 1x10 12 vg/mL to about 3x10 12 vg/mL; 1x10 12 vg/mL to about 2x10 12 vg/mL; 2x10 12 vg/mL to about 4x10 12 vg/mL; 8x10 11 vg/mL to about 9x10 12 vg/mL; 9x10 11 vg/mL to about 9x10 12 vg/mL; 1x10 12 vg/mL to about 9x10 12 vg/mL; 2x10 12 vg/mL to about 9x10 12 vg/mL; 3x10 12 vg/mL to about 9x10 12 vg/mL; 4x10 12 vg/mL to about
  • the concentration of the viral vector is from about 8x10 11 vg/mL; 9x10 11 vg/mL; 1x10 12 vg/mL; 2x10 12 vg/mL; 3x10 12 vg/mL; 3.1x10 12 vg/mL; 3.2x10 12 vg/mL; 3.3x10 12 vg/mL; 3.4x10 12 vg/mL; 3.5x10 12 vg/mL; 3.6x10 12 vg/mL; 3.7x10 12 vg/mL; 3.8x10 12 vg/mL; 3.9x10 12 vg/mL;4x10 12 vg/mL; 5x10 12 vg/mL; 6x10 12 vg/mL; 7x10 12 vg/mL; 8x10 12 vg/mL; and 9x10 12 vg/mL.
  • these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1% or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of active compound in each therapeutically- useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelflife, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation.
  • the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 may be used to deliver rAAVs.
  • a preferred mode of administration is by portal vein injection.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
  • Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the rAAV compositions disclosed herein may also be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug -re lease capsules, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • dispersion media includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
  • the formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
  • Uiposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures.
  • liposomes are free of the DNA length constraints that are typical of viral -based delivery systems.
  • Uiposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals.
  • several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.
  • Uiposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 gm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Nanocapsule formulations of the rAAV may be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way.
  • ultrafine particles sized around 0.1 pm
  • Biodegradable polyalkyl -cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • Sonophoresis i.e., ultrasound
  • U.S. Pat. No. 5,656,016 has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system.
  • Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899).
  • the methods described herein relate to treating a subject having or diagnosed as having a PD with a nucleic acid described herein.
  • Subjects having a PD can be identified by a clinican using current methods of diagnosing such diseases and disorders, for example, as described herein above. Symptoms and/or complications of PD which characterize this conditions and will aid in diagnosis, as well as clinical test to do the same are well known in the art and are described herein above.
  • a family history of PD can also aid in determining if a subject is likely to have PD or in making a diagnosis of PD.
  • the subject has been diagnosed as having PD prior to receiving a treatment as described herein. In one embodiment, the subject was diagnosed as having PD at least 1 year; 2 years; 3 years; 4 years; 5 years; 6 years; 7 years; 8 years; 9 years; 10 years or more prior to receiving a treatment as described herein.
  • the subject has been diagnosed as being at risk of having PD prior to receiving a treatment as described herein.
  • the subject has not been diagnosed as having, or being at risk of having PD prior to receiving a treatment as described herein.
  • the subject is diagnosed as having PD prior to receiving a treatment as described herein. In one embodiment, the subject is diagnosed as being at risk of having PD prior to receiving a treatment as described herein.
  • the person administering the treatment receives results of a diagnostic assay(s) that diagnoses the subject as having PD. In one embodiment, prior to administering treatment, the person administering the treatment receives results of an assay(s) that diagnoses the subject as being at risk of having PD.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having PD.
  • the methods described herein comprise administering an effective amount of compositions described herein, e.g. a nucleic acid described herein to a subject in order to alleviate a symptom of a PD.
  • "alleviating a symptom" is ameliorating any condition or symptom associated with PD. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and/or maximal tolerated dose.
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for neuronal degradation or functionality among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the subject is administered at least one anti-PD therapeutic prior to instruction or administration of any of the rAAVs described herein.
  • the subject is administered at least one anti-PD therapeutic prior to instruction or administration of any of the rAAVs described herein.
  • the subject is administered at least one anti-PD therapeutic following to instruction or administration of any of the rAAVs described herein.
  • the at least one anti-PD therapeutic excludes Duopa.
  • the rAAV described herein is used as a monotherapy.
  • the rAAV described herein can be used in combination with other known agents and therapies for PD.
  • Administered "in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disease, e.g., the two or more treatments are delivered after the subject has been diagnosed with PD and before the disease has been cured or eliminated, or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration.
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disease is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • the rAAV described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially.
  • the agent described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • the agent and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease.
  • the agent can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.
  • Exemplary therapeutics used to treat PD are described herein above.
  • the additional therapeutic e.g., second or third anti-PD therapeutic
  • the additional therapeutic can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of each therapeutic used individually, e.g., as a monotherapy.
  • the administered amount or dosage of additional therapeutic is lower (e.g., at least 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95% or more lower) than the amount or dosage of each additional therapeutic used individually.
  • the amount or dosage of additional therapeutic that results in a desired effect is lower (e.g., at least 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95% or more lower) than the amount or dosage of each additional therapeutic individually required to achieve the same therapeutic effect.
  • the subject maintains the same amount or dosage of the at least one anti- PD therapeutic following introduction or administration of any of the rAAVs described herein.
  • the subject decreases the amount or dosage of the at least one anti-PD therapeutic following introduction or administration of any of the rAAVs described herein.
  • the amount or dosage of the at least one anti-PD therapeutic is decreased by at least 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95% or more as compared to the amount or dosage taken prior to introduction or administration of any of the rAAVs.
  • the subject is no longer administered an anti-PD therapeutic following introduction or administration of any of the rAAVs described herein.
  • Immune Modulators
  • the compositions described herein include an immune modulator, and the methods further comprise administering the immune modulator.
  • the immune modulator can be administered at the time of administration, before the administration or, after the administration.
  • the immune modulator can be administered prior to, with, or after the at least second administration.
  • the immune modulator is administered prior to administration of a recombinant viral vector.
  • the immune modulator is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more prior to administration of a recombinant viral vector.
  • the immune modulator is administered no more than 24 hours prior to administration of a recombinant viral vector.
  • the immune modulator is administered at substantially the same time as the recombinant viral vector, e.g., slightly before administration of the recombinant viral vector as disclosed herein (i.e., within 6-hours, or 5-hours, or 4-hours, or 3-hours, or 2-hours, or 1-hour). In some embodiments, the immune modulator is administered simultaneously, or within 6 hours after, administration of the viral vector, (i.e., within 1-hour, or within 2-hours, or within 3-hours, or within 4-hours, or within 5-hours or within 6-hours, or about 6-hours after administration of a viral vector composition as disclosed.
  • the immune modulator allows for the administration of a recombinant viral vector to a subject who would otherwise not be a good candidate to receive such vector.
  • a subject who would otherwise not be a good candidate to receive such a vector is, for example, a subject who has previously received administration of a recombinant viral vector and/or who was previously exposed to the recombinant viral vector and has subsequently developed an antibody response to the vector.
  • a subject is considered to be a candidate, i.e., a good candidate, for administration of a recombinant viral vector when they have a titer for viral vector binding antibodies that is less than 1:5 (e.g., 1: 1, 1:2, 1:3, or 1:4).
  • a subject is considered not to be a suitable candidate for administration of a recombinant viral vector when they have a titer for viral vector binding antibodies that is 1:5 or greater (e.g., 1:6, 1:6, 1:7, 1:8, 1:9, 1: 10, 1:20, 1:30, 1:50, 1: 100, 1 : 1,000 or more).
  • a titer for viral vector binding antibodies that is 1:5 or greater (e.g., 1:6, 1:6, 1:7, 1:8, 1:9, 1: 10, 1:20, 1:30, 1:50, 1: 100, 1 : 1,000 or more).
  • One skilled in the art can assess the antibody titer of a subject using standard techniques in the art, e.g., by taking a biological sample from a subject, e.g., the subject’s blood, challenging the biological sample with known antigens, and detecting the presence of the viral binding antibodies to the known antigens.
  • An antibody titer is a measure of how much a sample can be diluted before a 50% viral vector neutralization can be detected in the sample.
  • Antibody titers are usually expressed as ratios, such as 1: 100, meaning that one-part serum to 100 parts saline solution (i.e., dilutant) results in 50% antibody neutralization in the sample, i.e., a reciprocal dilution of serum required to inhibit viral infection by 50% can be designated as neutralizing antibody titer at 50% inhibition.
  • a titer of 1: 10 of viral vector antibody is, therefore, an indication of lower level of viral vector antibodies than a 1 : 100 titer.
  • the subject is assessed for the presence of anti-AAV antibodies to the AAV vector of a gene therapy prior to administration of the gene therapy.
  • the subject is assessed for the presence of neutralizing anti-AAV antibodies to the AAV vector of a gene therapy prior to administration of the gene therapy.
  • Methods for detecting neutralizing anti-AAV antibodies is further described in, e.g., Kasprzyk T., et al. Mol Therapy. Methods & Clinical Dev. Jan 6, 2022, the contents of which are incorporated herein in its entirety by reference.
  • the immune modulator is administered to a subject having a titer of viral vector binding antibodies present in the biological sample, e.g., a blood sample, from the subject that is less than about 1:5 (e.g., 1: 1, 1:2, 1:3, or 1:4), where 1 part of the biological sample diluted in 10,000 parts of buffer results in 50% viral vector neutralization.
  • a subject having a titer of viral vector binding antibodies present in the biological sample e.g., a blood sample
  • 1:5 e.g., 1: 1, 1:2, 1:3, or 1:4
  • the immune modulator is administered to a subject having a titer of viral vector binding antibodies present in the biological sample or blood product from the subject that is greater than or equal to 1 :5 and less than about 1: 10 (e.g., 1:6, 1:7, 1:8, or 1:9). where 1 part of the biological sample or blood product diluted in 10,000 parts of buffer results in 50% viral vector neutralization, to enlarge the pool of subjects that can effectively be treated with AAV gene therapy.
  • prospective patients with viral neutralizing antibody levels 1:5 or higher are excluded from such treatment, i.e., they are not good candidates.
  • Administration of the immune modulator to a subject having an antibody titer greater than or equal to 1 :5 but less than 1 : 10 is expected to decrease the antibody titer present in the subject to less that 1 :5, thereby qualifying the subject as a candidate for administration of the recombinant viral vector (e.g., a gene therapy vector).
  • the recombinant viral vector e.g., a gene therapy vector
  • the immune modulator is administered to a subject that was found to have a titer of viral vector binding antibodies present in the biological sample, e.g., a blood sample, from the subject that is greater than or equal to 1 :5 and less than about 1:25 (e.g., 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, 1: 18, 1: 19, 1:20, 1:21, 1:22, 1:23 andl:24), where 1 part of the biological sample or blood product diluted in 10,000 parts of buffer results in 50% viral vector neutralization.
  • a titer of viral vector binding antibodies present in the biological sample, e.g., a blood sample, from the subject that is greater than or equal to 1 :5 and less than about 1:25 (e.g., 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, 1: 18, 1:
  • administration of the immune modulator to a subject having a titer greater than or equal to 1:5 but less than 1 : 15 is expected to decrease the antibody titer present in the subject to less that 1:5. thereby qualifying the subject as a candidate for administration of the recombinant viral vector (e.g., a gene therapy vector).
  • the recombinant viral vector e.g., a gene therapy vector
  • the immune modulator is administered to a subject having an antibody titer of viral vector binding antibodies present in the biological sample from the subject that is greater than or equal to 1:5 and less than about 1: 100 (e.g., 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, 1: 18, 1: 19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, and 1:99), where 1 part of the biological sample or blood product diluted in 10,000 parts of buffer results in 50% viral vector neutralization.
  • Administration of the immune modulator to a subject having an antibody titer greater than or equal to 1:5 but less than 1 :25 is expected to decrease the antibody titer present in the subject to less that 1:5, thereby qualifying the subject as a candidate for administration of the recombinant viral vector (e.g.. a gene therapy vector).
  • the recombinant viral vector e.g.. a gene therapy vector
  • the immune modulator enables repeated dosages, or repeat administration of an AAV vector as disclosed herein.
  • administration of the viral vector, e.g., a AAV vector disclosed herein, with an immune modulator can be administered multiple times (i.e., greater than one time) over a defined time period.
  • the AAV vector can be administered several times, i.e., more than once, over a several weeks (e.g., 2-weeks) to several months (e.g., 2-months).
  • administration of the AAV vector with the immune modulator according to the methods as disclosed herein can be, as non-limiting examples, every month over a period of 6-months, 3-4 times over a period of 6-weeks, every week over a period of 1 -month (or about 4 weeks) or 2-months (or about 8-weeks).
  • the dose of the viral vector e.g., AAV vector is lower than typically used in a single-dose regimen, for example, at a dose lower than a single-dose regimen as described herein.
  • the dose of the AAV vector can be less than or equal to about 10 12 , or lower than about 10 12 , for example, the dose can be about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 , or any dose between 10 7 and 10 12 .
  • the immune modulator can be changed between the doses, i.e., the same or different immune modulators can be used in repeat doses.
  • the first dose of AAV is co-administered with an immune modulator A
  • a second immune modulator administered with the second or third dose of AAV is different than immune modulator A, e.g., the second immune modulator is immune modulator B.
  • a dosing regimen according to the methods as disclosed can be administration of a AAV vector at a concentration of 10 12 or less than 10 12 , where the immune modulator is administered as A-B-C-D, or A-A-B-C, or A-B-A-C, where A, B, C and D are different immune modulators as disclosed herein.
  • the dosing regimen can include a plurality of doses of immune modulator over the time period, wherein each dose of the plurality includes an immune modulator independently selected from immune modulator A, immune modulator B, immune modulator C, immune modulator D, and combinations thereof (i.e., each dose can include more than one immune modulator).
  • an immune modulator “A” can be such as IdeS
  • an immune modulator “B” can be ImmTORTM, as disclosed herein.
  • an AAV vector as disclosed herein? is administered at a first timepoint with an IdeS immune modulator
  • an AAV vector as disclosed herein is administered at a second timepoint with a different immune modulator, such as immunologlobulin degrading protein or a small molecule, e.g., ImmTORTM, or vice versa.
  • an AAV vector as disclosed here? can be administered at a first timepoint with an ImmTORTM immune modulator
  • an AAV vector as disclosed herein can be administered at a second timepoint with an IdeS.
  • One aspect herein provides a method for administering a recombinant viral vector (e.g., a gene therapy vector) to a subject who has previously received a recombinant viral vector, for example, the same recombinant viral vector or another viral vector having a similar serotype, the method comprising, prior to administering the recombinant viral vector, administering to the subject an immune modulator.
  • a recombinant viral vector e.g., a gene therapy vector
  • the previously received recombinant viral vector elicits an immune response resulting in anti-AAV antibodies that target (i.e., recognizes and binds) to the recombinant viral vector administered.
  • Another aspect herein provides a method for administering a recombinant viral vector (e.g., a gene therapy vector) to a subject who was previously exposed to a viral vector, wherein the exposure elicits an immune response resulting in anti-AAV antibodies that target the recombinant viral vector to be administered, and wherein the subject has anti-AAV antibody titer of at least 1 :5-l : 15, at least 1:5-1:25, at least 1:5-1:50, or at least 1:5-1: 100, the method comprising the steps of, prior to administering the recombinant viral vector, administering to the subject an immune modulator.
  • a recombinant viral vector e.g., a gene therapy vector
  • the immune modulator is administered systemically.
  • the immune modulator crosses the blood brain barrier. In alternative embodiments, the immune modulator does not cross the blood brain barrier.
  • the immune modulator is administered locally.
  • the recombinant viral vector is to be administer locally to the brain tissue and the immune modulator does not cross the blood brain barrier, it is preferred to administer the immune modulator locally to the brain tissue, e.g., via an appropriate catheter, either directly to the brain tissue or indirectly to the brain tissue through cerebrospinal fluid circulating about the spinal cord (i.e., a spinal tap).
  • the immune modulator is administered locally to central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF)).
  • CNS tissue also includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like.
  • Any composition described herein may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule.
  • the immune modulator is administered locally to any of the following: neural pathways, somatosensory systems, visual systems, auditory systems, nerves, neuro endocrine systems, neuro vascular systems, brain neurotransmitter systems, dural meningeal system, or combinations thereof.
  • the immune modulator is administered locally to the eye, e.g., the vitreous, the retina, or the sclera.
  • the immune modulator is administered systemically.
  • the immune modulator is an immunoglobulin degrading enzyme such as IdeS, IdeZ, IdeS/Z, Endo S, or, their functional variant.
  • immunoglobulin degrading enzymes and their uses are described in US 7,666,582, US 8,133,483, US 20180037962, US 20180023070, US 20170209550, US 8,889,128, WO 2010057626, US 9,707,279, US 8,323,908, US 20190345533, US 20190262434, US 20210246469 and WO 2020016318, each of which are incorporated in their entirety herein by reference.
  • an immune modulator disclosed herein can be administered to a subject at any suitable dose, such as a suitable dose determined by a medical professional.
  • a suitable dosage may be from about 0.05 mg/kg to about 5 mg/kg body weight of a subject, or from about 0. 1 mg/kg to about 4 mg/kg body weight of a subject.
  • an immune modulator disclosed herein e.g., IdeZ
  • a suitable dosage may be from about 0.05 mg/kg to about 5 mg/kg body weight of a subject, or from about 0. 1 mg/kg to about 4 mg/kg body weight of a subject.
  • an immune modulator disclosed herein e.g., IdeS
  • a suitable dosage may be from about 0.05 mg/kg to about 5 mg/kg body weight of a subject, or from about 0. 1 mg/kg to about 4mg/kg body weight of a subject.
  • an immune modulator disclosed herein e.g., EndoS
  • a suitable dosage may be from about 0.05 mg/kg to about 5 mg/kg body weight of a subject, or from about 0. 1 mg/kg to about 4mg/kg body weight of a subject.
  • a dosage of about “0.01 mg/kg to about 10 mg/kg” body weight of a subject includes 0.011 mg/kg, 0.012 mg/kg, 0.013 mg/kg, 0.014 mg/kg, 0.015 mg/kg etc., as well as 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg etc., and so forth.
  • administration of a recombinant viral vector to a subject is preceded by administration of a protease and/or glycosidase to inhibit, reduce, or prevent an immune response (e.g., a humoral immune response) against the recombinant viral vector or antibodies that bind to the heterologous polynucleotide or a protein or peptide encoded by the heterologous polynucleotide encapsidated by the viral vector.
  • an immune response e.g., a humoral immune response
  • administration of the viral vector can be preceded by administration of a protease and/or glycosidase by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours; or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days; or by at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months; or by at least 1 year, 2 years, 3 years, 4 years, 5 years, or more.
  • administration of a recombinant viral vector to a subject is performed concurrently with administration of a protease and/or glycosidase to inhibit, reduce, or prevent an immune response (e.g., a humoral immune response) against the recombinant viral vector or antibodies that bind to the heterologous polynucleotide or a protein or peptide encoded by the heterologous polynucleotide encapsidated by the viral vector.
  • an immune response e.g., a humoral immune response
  • a protease and/or glycosidase is administered to a subject before an immune response (e.g., a humoral immune response), such as before development of neutralizing antibodies or development of antibodies that bind to the heterologous polynucleotide, protein, or peptide encoded by the heterologous polynucleotide encapsidated by the viral vector.
  • an immune response e.g., a humoral immune response
  • an immune response occurs within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours; or within 1 day, 2 days, 3 days, 4 days, 5 days, or more following administration of a recombinant viral vector.
  • the immune modulator is a proteasome inhibitor.
  • the immune modulator is a protease or glycosidase.
  • the proteasome inhibitor is Bortezomib.
  • the immune modulator comprises bortezomib and an anti-CD20 antibody, such as Rituximab.
  • the immune modulator comprises bortezomib, Rituximab, methotrexate, and intravenous gamma globulin.
  • Non-limiting examples of proteasome inhibitors and their combinations with Rituximab, methotrexate and intravenous gamma globulin are described in US 10,028,993, US 9,592,247, and US 8,809,282, each of which is incorporated in its entirety herein by reference.
  • the immune modulator is an inhibitor of the NF-kB pathway.
  • the immune modulator is Rapamycin or a functional variant thereof. Non-limiting examples of uses of rapamycin are described in US 10,071,114, US 20160067228, US 20160074531, US 20160074532, US 20190076458, US 10,046,064, which are each incorporated herein by reference in their entirety.
  • the immune modulator is synthetic nanocarriers comprising an immunosuppressant.
  • Non limiting examples of immunosuppressants, immunosuppressants coupled to synthetic nanocarriers, synthetic nanocarriers comprising rapamycin, and/or, tolerogenic synthetic nanocarriers, their doses, administration and use are described in US20150320728, US 20180193482, US 20190142974, US 20150328333, US20160243253, US 10,039,822, US 20190076522, US 20160022650, US 10,441,651, US 10,420,835, US 20150320870, US 2014035636, US 10,434,088, US 10,335,395, US 20200069659, US 10,357,483, US 20140335186, US 10,668,053, US 10,357,482, US 20160128986, US 20160128987, US 20200038462, US 20200038463, each of which is incorporated in its entirety herein by reference.
  • the immune modulator comprises synthetic nanocarriers comprising rapamycin (i.e., ImmTORTM nanoparticles) as disclosed in Kishimoto, et al., 2016, Nat Nanotechnol, 11(10): 890-899; Maldonado, et al., 2015, PNAS, 112(2): E156-165) and in US20200038463 and US Patent 9,006,254, each of which is incorporated herein by reference in its entirety.
  • the immune modulator is an engineered cell, e.g., an immune cell that has been modified using SQZ technology as described in WO2017192786, which is incorporated herein in its entirety by reference.
  • the immune modulator is selected from the group consisting of poly- ICUC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSUIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvhnmune, UipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's Q
  • the immune modulator is a small molecule that inhibits the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and/or 2-aminopurine (a PKR inhibitor), which can also be administered in combination with the composition comprising at least one rAAV as disclosed herein.
  • chloroquine a TLR signaling inhibitor
  • a PKR inhibitor 2-aminopurine
  • TLR- signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available for purchase from INVIVOGEN).
  • inhibitors of pattern recognition receptors which are involved in innate immunity signaling
  • PRR pattern recognition receptors
  • 2-aminopurine, BX795, chloroquine, and H-89 can also be used in the compositions and methods comprising at least one rAAV vector as disclosed herein for in vivo protein expression as disclosed herein.
  • the immune modulator is photopheresis, also known as extracorporeal photochemotherapy, or ECP. Photopheresis treatment is performed on a subject’s blood. Using either an IV or a catheter, blood is routed from the subject through a device which separates out a portion of white blood cells (leukocytes).
  • Photopheresis can be performed at least once daily. In one embodiment, photopheresis is performed at least 1, 2, 3, 4, 5, 6, 7 times a week prior to administration of the recombinant viral vector. In one embodiment, photopheresis is performed at least 1, 2, 3, 4, 5, 6, 7 times a week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of the recombinant viral vector.
  • the administering the immune modulator to the subject can include performing photopheresis on the subject. It is understood that the photopheresis can be performed in conjunction with administration of a second immune modulator selected from the enzymes, nanoparticles, and chemical compositions described herein and/or as a portion of a multiple dosing regimen.
  • a rAAV vector having the modified viral capsid can also encode a negative regulator of innate immunity such as NLRX1. Accordingly, in some embodiments, a rAAV vector can also optionally encode one or more of NLRX1, NS 1, NS3/4A, or A46R. Additionally, in some embodiments, a composition comprising at least one rAAV vector as disclosed herein can also comprise a synthetic, modified-RNA encoding inhibitor of the innate immune system to avoid the innate immune response generated by the tissue or the subject.
  • an immune modulator for use in the administration methods as disclosed herein is an immunosuppressive drug or agent.
  • immunosuppressive drug or agent refers to pharmaceutical agents that inhibit or interfere with normal immune function.
  • immunosuppressive drugs or agents suitable for the methods disclosed herein include agents that inhibit T-cell/B- cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No 2002/0182211, which is incorporated herein by reference in its entirety.
  • an immunosuppressive agent is cyclosporine A.
  • immunosuppressive agents include myophenylate mofetil, rapamicin, and anti-thymocyte globulin.
  • the immunosuppressive drug is administered in a composition comprising at least one rAAV vector as disclosed herein, or in a separate composition but simultaneously with, or before or after administration of a composition comprising at least one rAAV vector according to the methods of administration as disclosed herein.
  • An immunosuppressive drug is administered in a formulation which is compatible with the route of administration and is administered to a subject at a dosage sufficient to achieve the desired therapeutic effect.
  • the immunosuppressive drug is administered transiently for a sufficient time to induce tolerance to the rAAV vector as disclosed herein.
  • a subject being administered a composition disclosed herein is also administered an immunosuppressive agent.
  • an immunosuppressive agent such as a proteasome inhibitor.
  • One such proteasome inhibitor known in the art for instance as disclosed in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15/796,137, both of which are incorporated herein by reference in their entireties, is bortezomib.
  • the immunosuppressive agent is an antibody, including polyclonal, monoclonal, scfv or other antibody-derived molecule that is capable of suppressing the immune response, for instance, through the elimination or suppression of antibody producing cells.
  • the immunosuppressive element is a short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the coding region of the shRNA is included in the rAAV cassette and is generally located downstream, i.e., 3’, of the poly-A tail.
  • the shRNA can be targeted to reduce, reduce, or eliminate expression of immunostimulatory agents, such as cytokines, growth factors (including transforming growth factors [31 and [32, TNF and others that are known in the art).
  • immune modulating agents facilitates the ability to use multiple doses (e.g., multiple administration) over a plurality of months and/or years. This permits using multiple agents as discussed below, e.g., a rAAV vector encoding multiple genes, or multiple administrations to the subject.
  • an "effective amount" of a substance is an amount sufficient to produce a desired effect.
  • an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect in the context of rAAV mediated delivery) a sufficient number of target cells of a target tissue of a subject.
  • a target tissue is central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.).
  • CNS central nervous system
  • an effective amount of an isolated nucleic acid may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to decrease or stabilize a subject’s MDS- UPDRS score, to extend the lifespan of a subject, to improve in the subject one or more symptoms of disease (e.g., a symptom of PD), etc.
  • the effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue as described elsewhere in the disclosure.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant
  • a "subject” means a human or non-human animal.
  • the non-human animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of PD.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. PD) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • a variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA, miRNA.
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • a variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
  • vector refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral.
  • vector encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
  • non-native e.g., heterologous
  • the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system.
  • the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism).
  • the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
  • sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • Non-limiting examples of a viral vector described herein include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector a baculovirus vector, and a chimeric virus vector.
  • the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies.
  • the vector is episomal.
  • the use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. PD.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition disease or disorder, e.g., as assessed by decreasing or stabilizing the subject’s initial (i.e., prior to administration) MDS-UPDRS score.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • “on” refers to a period of time in which the subject is administered an antiParkinson’s therapeutic (e.g., levodopa) and wherein the subject has a beneficial response to the therapeutic.
  • an antiParkinson e.g., levodopa
  • off refers to a period of time (e.g., up to 12 hours) in which the subject is not administered an anti -Parkinson’s therapeutic (e.g., levodopa), or a period of time in which the subject is administered an anti -Parkinson’s therapeutic (e.g., levodopa) but exhibits a low beneficial response to the therapeutic.
  • cDNA or a “cDNA molecule” refers to “complementary DNA” that is synthesized by RNA-dependent DNA polymerase- or reverse transcriptase -catalyzed extension of a primer that anneals to an RNA molecule of interest using at least a portion of the RNA molecule of interest as a template (which process is also called “reverse transcription”).
  • the cDNA molecules synthesized are “homologous to” or “base pair with” or “form a complex with” at least a portion of the template.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the technology described herein.
  • the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid.
  • Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising: introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the nucleic acid and/or wherein at least 30% of the subject’s putamen volume is covered by the rAAV, and wherein the subject does not exhibit an increase in PD-associated symptoms for a least 6 months following the introducing as compared to prior to introducing.
  • rAAV adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • non-invasive imaging is selected from the group consisting of intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED), ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); positron emission tomography (PET); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof.
  • iMRI intraoperative magnetic resonance image
  • CED computed tomography
  • fMRI functional magnetic resonance imaging
  • PET positron emission tomography
  • EEG electroencephalography
  • MEG magnetoencephalography
  • fNIRS functional near-infrared spectroscopy
  • the local introduction comprises introducing about half of the rAAV vector to each putamen via intraoperative magnetic resonance image (iMRI)-guided convection enhanced delivery (CED).
  • iMRI intraoperative magnetic resonance image
  • CED convection enhanced delivery
  • local introduction further comprises introducing an MRI contrast agent at substantially the same time as the AAV vector.
  • the MRI contrast agent is gadoteridol.
  • the MRI contrast agent is introduced to the subject in the same composition as the rAAV.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the promoter is a nervous system (NS) or central nervous system (CNS) specific promoter.
  • NS specific promoter is selected from the NS specific promoters in Table 1.
  • nucleic acid comprises a sequence of SEQ ID NO: 1, or a functional variant that is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO: 1.
  • rAAV is AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, or a rational haploid thereof.
  • rAAV comprises a modification that increases its brain-specific tropism.
  • brain-specific tropism is increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater as compared to an unmodified AAV.
  • the rAAV is introduced at a total dose within the range of 5xl0 12 vg to about 1.5xl0 13 vg.
  • rAAV is introduced as a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier.
  • liquid composition has an rAAV concentration of from about 3xl0 12 vg/mL to about 4xlO 12 vg/mL.
  • the at least one anti-PD therapeutic is selected from the group consisting of levodopa, Sinemet, Rytary, Stalevo, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, entacapone.
  • the dose of the at least one anti-PD therapeutic is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.
  • a method of slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject in need thereof comprising: locally introducing to the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the GDNF gene.
  • rAAV recombinant adeno-associated virus
  • a method of slowing or inhibiting a progression of PD in a subject in need thereof comprising: transducing greater than or equal to about 30% of the volume of the subject’s putamen with a glial cell line-derived neurotrophic factor (GDNF) gene, wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months following the transducing.
  • GDNF glial cell line-derived neurotrophic factor
  • a metohod of slowing or inhibiting a progression of PD in a subject in need thereof comprising: covering greater than or equal to about 30% of the volume of the subject’s putamen with a glial cell line-derived neurotrophic factor (GDNF) gene,
  • GDNF glial cell line-derived neurotrophic factor
  • the subject does not exhibit a substantial increase in PD-associated symptoms for at least 6 months following the covering.
  • transducing is performed by administering a rAAV comprising the GDNF gene to each of the subject’s putamen.
  • MDS-UPDRS Movement Disorder Society-Unified Parkinson’s Disease Rating Scale Part
  • a method of treating a subject mildly affected by Parkinson’s disease comprising: administering to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with GDNF, and wherein the subject has a second MDS-UPDRS score at 6 months post-administering that is stabilized as compared to the initial MDS-UPDRS score.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • a method of treating a subject moderately affected by Parkinson’s disease comprising: administering to each of the subject’s putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is transduced with the nucleic acid, and wherein the subject has a second MDS-UPDRS score at 6 months post-administering that is at least about 20% lower than the initial MDS-UPDRS score.
  • AAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • a method of treating a subject moderately affected by Parkinson’s disease comprising: administering to each of the subject’s putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject’s putamen is covered with the rAAV, and wherein the subject has a second MDS-UPDRS score at 6 months post-administering that is at least about 20% lower than the initial MDS-UPDRS score
  • AAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising: locally introducing to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing an MRI contrast agent to each of the subject’s putamen at substantially the same time as the rAAV, wherein at least 30% of the volume of the subject’s putamen is transduced with the nucleic acid, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising: locally introducing to each of the subject’s putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing an MRI contrast agent to each of the subject’s putamen at substantially the same time as the rAAV, wherein at least 30% of the volume of the subject’s putamen is covered with the rAAV, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising: introducing to the subject a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is transduced with the nucleic acid, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing.
  • rAAV recombinant adeno-associated virus
  • GDNF glial cell line-derived neurotrophic factor
  • a method of slowing or inhibiting progression of Parkinson’s disease (PD) in a subject in need thereof comprising: introducing to the subject a recombinant adeno-associated vims (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject’s putamen is covered with the rAAV, and wherein the subject does not exhibit a substantial increase in PD-associated symptoms for a least 6 months immediately following the introducing as compared to prior to introducing
  • rAAV recombinant adeno-associated vims
  • GDNF glial cell line-derived neurotrophic factor
  • a composition for slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject comprising: a recombinant adeno-associated vims (rAAV) comprising a genome comprising a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter; and a pharmaceutically acceptable carrier.
  • rAAV recombinant adeno-associated vims
  • GDNF glial cell line-derived neurotrophic factor
  • a formulation for slowing or inhibiting a progression of Parkinson’s disease (PD) in a subject comprising: an adeno-associated vims (AAV) at a concentration of 3xl0 12 vg to 4xl0 12 vg per mb of a pharmaceutically acceptable earner, wherein the rAAV comprises a genome comprising a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter.
  • AAV adeno-associated vims
  • rAAV a recombinant adeno-associated vims
  • GDNF glial cell line-derived neurotrophic factor
  • Example 1 Production of viral vectors comprising nucleic acid encoding GDNF polypeptide operatively linked to a CMV promoter.
  • MCB Master Cell Bank
  • FBS fetal bovine serum
  • the MCB cells are cultured and passaged over a 4 week period while the amount of FBS in the tissue culture media is gradually reduced from 10% to 2.5%.
  • the cells are then transferred from DMEM 2.5% FBS into serum free suspension media and grown in shaker flasks.
  • the cells are then cultured in the serum- free media for another 3 weeks while their growth rate and viability is monitored.
  • the adapted cells are then expanded and frozen down.
  • a number of vials from this cell bank are subsequently thawed and used during process development studies to create a scalable manufacturing process using shaker flasks and wave bioreactor systems to generate rAAV vectors.
  • Suspension HEK293 cells are grown in serum-free suspension media that supports both growth and high transfection efficiency in shaker flasks and wave bioreactor bags.
  • Multitron Shaker Incubators are used for maintenance of the cells and generation of rAAV vectors at specific rpm shaking speeds (based on cell culture volumes), 80% humidity, and 5% CO2.
  • the plasmid DNA has a sequence comprising a heterologous nucleic acid sequence of a GDNF gene (i.e., the nucleic acid sequence encoding GDNF (SEQ ID NO: 1)) operatively linked to CMV promoter.
  • the cocktail further comprises a Packaging plasmid encoding Rep2 and serotype-specific Cap2: AAV-Rep/Cap, and the Ad-Helper plasmid (XX680: encoding adenoviral helper sequences).
  • the cocktail is inverted to mix prior to being incubated at room temperature.
  • the transfection cocktail is then pipetted into the flasks and placed back in the shaker/incubator. All optimization studies are carried out at 30 mL culture volumes followed by validation at larger culture volumes. Cells are harvested 48 hours post-transfection.
  • Wave bags are seeded 2 days prior to transfection. Two days post-seeding the wave bag, cell culture counts are taken and the cell culture is then expanded/diluted before transfection. The wave bioreactor cell culture is then transfected. Cell culture is harvested from the wave bioreactor bag at least 48 hours post-induction.
  • plasmid DNA is spiked into a non-transfected cell lysate with and without the addition of DNase.
  • 50 ul of EDTA/Sarkosyl solution (6.3% sarkosyl, 62.5 mM EDTA pH 8.0) is then added to each tube and incubated at 70°C for 20 minutes.
  • 50 ul of Proteinase K (10 mg/mL) is then added and incubated at 55°C for at least 2 hours. Samples are then boiled for 15 minutes to inactivate the Proteinase K. An aliquot is removed from each sample to be analyzed by qPCR. Two qPCR reactions are carried out in order to effectively determine how much rAAV vector is generated per cell.
  • AAV vector purification Clarified AAV lysate is purified by column chromatography methods as one skilled in the art would be aware of and described in the following manuscripts (Allay et al., Davidoff et al., Kaludov et al., Zolotukhin et al., Zolotukin et al, etc).
  • 125 ul of NaOH buffer (80 mM NaOH, 4 mM EDTA pH 8.0) is added to each well.
  • a series of transgene specific standards are created through a dilution series.
  • NaOH buffer is then added and incubated.
  • Nylon membrane is incubated at RT in 0.4 M Tris-HCl, pH 7.5 and then set up on dot blot apparatus. After a 10-15 minute incubation in NaOH buffer, the samples and standards are loaded into the dot blot apparatus onto the Gene Screen PlusR hybridization transfer membrane (PerkinElmer). The sample is then applied to the membrane using a vacuum.
  • the nylon membrane is soaked in 0.4 M Tris-HCl, pH 7.5 and then cross linked using UV strata linker 1800 (Stratagene) at 600 ujouls x 100.
  • the membrane is then pre -hybridized in CHURCH buffer (1% BSA, 7% SDS, 1 mM EDTA, 0.5 M NasPO4, pH 7.5).
  • CHURCH buffer 1% BSA, 7% SDS, 1 mM EDTA, 0.5 M NasPO4, pH 7.5
  • the membrane is hybridized overnight with a 32 P-CTP labeled transgene probe (Roche Random Prime DNA labeling kit). The following day, the membrane is washed with low stringency SSC buffer (1xSSC, 0.1% SDS) and high stringency (0. 1xSSC, 0.1% SDS). It is then exposed on a phosphorimager screen and analyzed for densitometry using a STORM840 scanner (GE).
  • GE STORM840 scanner
  • the membrane is then neutralized using 0.5 M Tris pH 7.5 with 1 M NaCl, and is hybridized overnight with a 32 P-CTP labeled transgene probe. After washing the membrane as previously described, the membrane is exposed to a phosphorimager screen and analyzed using a STORM840 scanner.
  • HeLaRC-32 cells (Chadeuf et al., J Gene Med. 2:260 (2000)) are plated at 2x10 5 cells/well of a 24 well plate and incubated at 37°C overnight. The cells are observed for 90-100% confluence. 50 mb of DMEM with 2% FBS, 1% Pen/Strep is pre-warmed, and adenovirus (dl309) is added at a MOI of 10. The dl309 containing media is aliquoted in 900 ul fractions and used to dilute the rAAV in a series of ten-fold dilutions. The rAAV is then plated at 400 pl and allowed to incubate for 48 hours at 37°C.
  • TEM Transmission electron microscopy
  • Purified dialyzed rAAV vectors are placed on a 400-mesh glow-discharged carbon grid by inversion of the grid on a 20 ul drop of virus. The grid is then washed 2 times by inversion on a 20 ul drop of ddH2O followed by inversion of the grid onto a 20 ul drop of 2% uranyl acetate for 30 seconds. The grids are blotted dry by gently touching Whatman paper to the edges of the grids. Each vector is visualized using a Zeiss EM 910 electron microscope.
  • Example 2 Local administration of AA V2- GDNF for treatment of Parkinson ’s disease.
  • GDNF is known to support the survival and promote differentiation of dopaminergic neurons and has long been evaluated as a putative therapeutic agent for Parkinson’s disease.
  • Nonclinical studies have demonstrated that local delivery of GDNF into the brain can both protect dopaminergic neurons against neurotoxic insults and stimulate anatomical and functional recovery in rodent and non-human primate models of PD (Kordower 2013).
  • MRI visualization of gadoteridol coadministered with AAV2 vectors provides an accurate anatomical representation of the parenchymal volume histologically showing neuronal gene transfer, and that the delivered contrast agent provides no intrinsic local toxicity (Su 2010, Richardson 2011).
  • Intraoperative MRI monitoring therefore, provides real-time feedback to the surgical team as to the drug distribution, and provides an opportunity to tailor the CED based on the individual patient’s anatomy, maximizing delivery to the target while limiting exposure of non-targeted regions from visualized reflux or leakage of infusate.
  • AAV2-GDNF the study drug, is an AAV serotype 2 vector containing a DNA expression cassette encoding the complementary sequence for human GDNF under the control of the cytomegalovirus immediate early promoter (CMV).
  • CMV cytomegalovirus immediate early promoter
  • the current study described herein features infusion of the study drug in study participants via intraoperative MRI-guided CED.
  • a maximum of 1.8mL of study drug was infused into each putamen with the anticipated distribution to 50-80% of the putaminal volume.
  • the one-time delivery of the study drug is intended to result in the continuous expression of GDNF protein within >50% of the putaminal volume, primarily transducing intrinsic medium spiny neurons (MSNs). Additional anterograde transport of the study drug from the MSNs to the SN (via direct and indirect pathways) will provide GDNF expression and trophic support directly to dopaminergic neuronal somata within the SN.
  • MSNs intrinsic medium spiny neurons
  • a total of 12 study participants have been administered the investigational product (i.e., AAV2-GDNF) in this study. Participants are enrolled into one of two parallel cohorts, based upon the duration and stage of their PD. Cohort A include subject that are mildly affected by PD (Fig. 2) and Cohort B include subject that are moderately affected by PD (Fig. 3).
  • PD related genetic factors e.g. PRKN, PINK1, or LRKK2 mutations
  • Presence of untreated or suboptimally treated depression (BDI-II score >20) or a history of a serious mood disorder (i.e., requiring psychiatric hospitalization or a prior suicide attempt)
  • Presence of substance (drug, alcohol) abuse as defined by DSM-5 criteria and in the judgement of the Investigator. Note: Use of tetrahydrocannabinol or cannabidiol would not be exclusionary
  • Clinically active infection including acute or chronic scalp infection
  • Chronic immunosuppressive therapy e.g., chronic steroids, tumor necrosis factor, antagonists, chemotherapy
  • AAV2-GDNF comprises an adeno-associated virus, serotype 2 (AAV2) containing human GDNF complementary DNA (cDNA), human cytomegalovirus (CMV) promoter and 3’ UTR sequences.
  • AAV2-GDNF is supplied in ImU aliquots at a concentration of 7.9 x 10 12 vg/mU.
  • the current study described herein is an open-label safety study of AAV2-GDNF (see, e.g., Fig. 15) delivered by CED bilaterally into the putamen of patients with PD.
  • the primary study objective was to confirm the safety of the delivered viral vector and subsequent expression of the GDNF transgene in both mild and moderately advanced PD patients.
  • Primary safety and clinical outcome assessments are performed 18 months after administration of the study drug. Schematics of the current trial is presented in Figs 1 and 16.
  • Neuroimaging o Brain MRI (ON or OFF state, with and without contrast for pre-surgical planning) o FDG PET (OFF state, evaluate for PD-related pattern) o DaT SPECT (OFF state, evaluate presynaptic terminal density)
  • Subjects can be rescreened up to 2 times at the discretion of the study investigator.
  • Study drug was delivered bilaterally into the putamen in a single surgical setting using SmartFlow cannula connected to MRI-compatible infusion pumps (e.g. Medfusion syringe pump, Smiths Medical Inc.). CED infusions with increasing rates of infusion (1-30 ⁇ L/min) was used to deliver the drug volume into each putamen.
  • MRI-compatible infusion pumps e.g. Medfusion syringe pump, Smiths Medical Inc.
  • Adjustments of the cannula depth and infusion rates was made by the surgical team to control distribution within the targeted volume, with a goal of covering 50-80% of each putaminal volume. Infusions were terminated when either target coverage is achieved, additional infusions were meaningfully increase target coverage or increases in infusion volume extends coverage beyond the target volume, or a maximum infusion volume of 1.8mL per putamen is reached.
  • the objective of the MRI-monitored dosing procedure was to maximize coverage of each putamen while minimizing off-target delivery.
  • Admixing a small quantity of gadoteridol with the study drug enabled the surgical team to visually monitor the distribution in real-time via iMRI, documenting coverage and allowing modification of cannula position, infusion rates, or infusion stoppage, as required.
  • Given individual variability in the size, shape and anatomical characteristics of the brain, and in particular the putamen of PD patients, the actual volume of infusion and resulting volume of distribution, and the percentage of putaminal volume coverage was expected to vary between subjects. Monitoring the drug infusate distribution throughout the administration procedure optimally allowed for a 50 to 80% putaminal coverage.
  • UDYSRS objective sub score was stable up to at least 18 months post administration in the mild cohort (Fig. 22A), and slightly decreased in the moderate cohort (Fig. 22B).
  • the moderate cohort the UDYSRS overall and historical sub score is decreased at 18 months post administration (Fig. 22B).
  • the levadopa equivalent daily dose average values score is stabilized as compared to baseline at at least 18 months post administration in the mild cohort (Fig. 23 A), and is decreased at at least 18 months post administration in the moderate cohort (Fig. 23B).
  • MDS-UPDRS III score was observed at 12 months post administration (Fig. 11) and 18 months post administration (Fig. 21).
  • Previous clinical trials included administrations of a lower total dose than described herein (i.e., 9.Ox10 10 vg and 3.0x10 11 vg) and resulted in a lower total coverage of the putamen (i.e., 26%). This administration resulted in only a slight decrease in the subjects’ initial MDS-UPDRS III score at 12 months post administration.
  • TEAEs TEAEs
  • Fig. 17 A total of fifty-six (56) TEAEs were observed in this study in 11 participants. The majority of these events were transient and expected perioperative events. These events are summarized in Fig. 17. No AAV2-GDNF-associated adverse events were reported. No subjects have discontinued the study for any reason (including TEAEs). No life-threatening TEAEs or deaths have occurred to date.
  • Example 3 Highly-Reproducible and Safe Intraputaminal Delivery of AAV2-GDNF via Bilateral Single Posterior Trajectories in Early and Moderate Stage Parkinson ’s Disease
  • This infusion method allows consistent volumes (averaging 1500 microliters) to be delivered within the putamen, providing >50% coverage in the current PD clinical trials, while maintaining clinical safety.
  • Perivascular leakage can be commonly noted with infuse-as-you-go CED in the putamen, however, such leakage is difficult to predict preoperative ly, in location and scope.
  • Primary mitigation strategies for neurosurgeons include a) advancement of the delivery cannula and b) minor adjustments in the infusion flow rate. It is potentially advantageous perivascular extension into the caudate nucleus following perivascular channels.
  • Results' Infusions were safely performed and well-tolerated by all participants. Evidence of a single, unilateral, asymptomatic cerebrovascular event was incidentally detected adjacent to the putamen on a scheduled 6-month MRI. Average putaminal volumetric coverage from 20 infusions was 62.5%, a marked improvement from the 26% average coverage in the initial AAV2-GDNF study and exceeds the >50% coverage goal (see, e.g., Figs 14A and 14B). Preliminary clinical findings indicate greater motor benefits and reductions in PD medications when compared to a previous Phase 1 study (Heiss 2019).
  • Example 4 AAV2-GDNF expression localized to putamen infusion sites
  • AAV2-GDNF will be administered to subjects having Parkinson’s disease.
  • the AAV2- GDNF will include close ended linear duplexed DNA encapsidated by AAV2.
  • the close ended linear duplexed DNA will comprise the ITRto ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12- 2,716 of SEQ ID NO: 64).
  • the close ended linear duplexed DNA can be manufactured from the plasmid DNA illustrated in Fig. 26.
  • the AAV2-GDNF will be infused into the putamen of the subjects by MRI-guided CED using bi-occipital trajectories. The results are expected to be substantially similar to those described above.

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KR1020247032559A KR20250006358A (ko) 2022-03-25 2023-03-24 파킨슨병의 치료를 위한 방법 및 조성물
CA3255025A CA3255025A1 (en) 2022-03-25 2023-03-24 METHODS AND COMPOSITIONS INTENDED FOR THE TREATMENT OF PARKINSON'S DISEASE
JP2024556470A JP2025510150A (ja) 2022-03-25 2023-03-24 パーキンソン病の処置のための方法および組成物
AU2023240256A AU2023240256A1 (en) 2022-03-25 2023-03-24 Methods and compositions for the treatment of parkinson's disease
CN202380042769.7A CN119256090A (zh) 2022-03-25 2023-03-24 用于治疗帕金森病的方法和组合物
EP23775736.4A EP4499155A4 (en) 2022-03-25 2023-03-24 METHODS AND COMPOSITIONS INTENDED FOR THE TREATMENT OF PARKINSON'S DISEASE
MX2024011436A MX2024011436A (es) 2022-03-25 2024-09-18 Metodos y composiciones para el tratamiento de enfermedad de parkinson

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