Methods and compositions for treating Parkinson's disease

Abstract

Aspects of the present disclosure relate to compositions and methods useful for treating Parkinson's disease. In some embodiments, the present disclosure provides a method for treating Parkinson's disease comprising administering a viral vector comprising a GDNF nucleic acid sequence. In some embodiments, a subject's shell core is topically administered. In some embodiments, systemic administration, e.g., by a viral vector comprising a modified viral capsid, e.g., to preferentially target cells in the CNS or PNS.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 USC §119(e), this application claims the benefit of U.S. Provisional Application No. 63 / 323,830, filed on March 25, 2022; U.S. Provisional Application No. 63 / 326,236, filed on March 31, 2022; U.S. Provisional Application No. 63 / 341,841, filed on May 13, 2022; U.S. Provisional Application No. 63 / 393,196, filed on July 28, 2022; and U.S. Provisional Application No. 63 / 438,164, filed on January 10, 2023, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0002] The technology described herein relates to methods for treating neurological diseases or disorders, such as Parkinson's disease. Government support

[0003] This invention was made in performance of a cooperative research and development agreement with the Department of Veterans Affairs, an agency of the United States Government, which has certain rights in the invention. Background Art

[0004] Parkinson's disease (PD) is a progressive neurodegenerative disorder that inevitably progresses to disability and death over 10 to 30 years. Drugs that are generally designed to alleviate known striatal dopamine deficiency can provide substantial clinical benefit for the cardinal motor signs of PD (i.e., resting tremor, rigidity, bradykinesia, and postural instability). However, disease progression continues because dopamine replacement and other medical therapies do not affect the underlying neurodegenerative process. The clinical response to antiparkinsonian drugs diminishes over time, and a variety of drug-related complications ensue, including motor fluctuations, dyskinesias, and neuropsychiatric manifestations.

[0005] Deep brain stimulation (DBS) is a reasonable and effective symptomatic treatment option for specific primary motor signs. However, the use of DBS is limited, in part due to the risks and complexity of surgical implantation and device programming, as well as hardware-related complications and maintenance. Recently, Duopa has been approved for use in advanced patients with severe motor fluctuations. Duopa is a levodopa / carbidopa enteral gel that is administered through a gastrostomy tube connected to an external portable pump, thereby providing a steady dose. While this circumvents intracranial surgery, Duopa requires maintenance of a stoma site, and carrying an external component is inconvenient. Due to oxidation of Duopa, this therapy is approved for 16hr / day, so some patients are undertreated at night.

[0006] Research efforts have pointed to a number of potential mechanisms that may underlie the neurodegenerative process in PD. Oxidative stress, mitochondrial dysfunction, and abnormal intracellular protein processing are common postulated mechanisms. Experimental therapeutic studies are designed to correct such pathobiological disturbances, thereby aiming to slow, prevent, or reverse the neurodegenerative process. Neurotrophic factors such as GDNF have the potential to modify the course of PD rather than simply treat specific clinical features.

[0007] Parkinson's disease is a progressive, multicentric neurodegenerative disorder characterized by resting tremor, rigidity, bradykinesia, and postural instability. Most cases of PD are idiopathic and it is the second most common neurodegenerative disorder after Alzheimer's disease. Patients experience mood symptoms, including depression and anxiety, as well as characteristic motor features and movement disturbances. There is currently no cure for PD; treatment options are limited to relieving disease symptoms. Summary of the invention

[0008] One aspect provided herein describes a method for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising introducing into the subject a recombinant adeno-associated virus (rAAV), the recombinant 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, for at least 6 months after the introduction, the subject does not exhibit an increase in PD-related symptoms compared to before the introduction.

[0009] In one embodiment of any of the aspects herein, the rAAV is introduced by systemic introduction.

[0010] In one embodiment of any of the aspects herein, the rAAV is introduced by topical introduction.

[0011] In one embodiment of any of the aspects herein, the local introduction is directly into the putamen of the subject.

[0012] In one embodiment of any of the aspects herein, the local introduction comprises introducing the rAAV directly into each of the putamen of the subject.

[0013] In one embodiment of any aspect of the present invention, local introduction is performed simultaneously with non-invasive imaging. Exemplary non-invasive imaging techniques include intraoperative magnetic resonance imaging (iMRI)-guided convection enhanced delivery (CED), ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); positron emission tomography (PET); electroencephalogram (EEG); magnetoencephalogram (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof.

[0014] In one embodiment of any of the aspects herein, the local introduction comprises introducing about half of the total delivered dose of the rAAV vector into each putamen by convection enhanced delivery (CED) guided by intraoperative magnetic resonance imaging (iMRI).

[0015] In one embodiment of any of the aspects herein, the local introduction further comprises introducing an MRI contrast agent substantially simultaneously with the AAV vector.

[0016] In one embodiment of any of the aspects herein, the MRI contrast agent is gadoteridol.

[0017] In one embodiment of any aspect herein, the MRI contrast agent and the rAAV are introduced into the subject in the same composition. In one embodiment of any aspect herein, the MRI contrast agent and the rAAV are introduced into the subject in different compositions.

[0018] In one embodiment of any of the aspects herein, the rAAV is introduced by systemic (eg, intravenous) introduction.

[0019] In one embodiment of any aspect herein, transduction and / or coverage of the putamen is assessed by magnetic resonance imaging.In one embodiment of any aspect herein, at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the putamen volume of the subject is transduced with the GDNF gene.

[0020] In one embodiment of any aspect herein, the subject does not show a significant increase in PD-related symptoms for at least 12 months immediately following introduction compared to before introduction.

[0021] In one embodiment of any of the aspects herein, the subject exhibits a reduction in PD-related symptoms for at least 6 months or longer immediately following introduction compared to before introduction.

[0022] In one embodiment of any of the aspects herein, the subject exhibits a reduction in PD-related symptoms for at least 12 months or longer immediately following introduction compared to before introduction.

[0023] In one embodiment of any of the aspects herein, the subject has an initial Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) score of less than 32 prior to introduction.

[0024] In one embodiment of any of the aspects herein, slowing or inhibiting progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score 6 months following introduction being not substantially higher than the initial MDS-UPDSS score.

[0025] In one embodiment of any of the aspects herein, slowing or inhibiting progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score about 12 months following introduction being not substantially higher than the initial MDS-UPDRS score.

[0026] In one embodiment of any of the aspects herein, the subject's initial MDS-UPDRS score is greater than or equal to 32 prior to introduction.

[0027] In one embodiment of any of the aspects herein, the subject exhibits a decrease in initial MDS-UPDRS score at least 6 months immediately following introduction compared to before introduction.

[0028] In one embodiment of any of the aspects herein, slowing or inhibiting progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score that is at least about 20% lower than the initial MDS-UPDRS score at about 6 months following introduction.

[0029] In one embodiment of any of the aspects herein, slowing or inhibiting progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score that is at least about 30% lower than the initial MDS-UPDRS score at about 12 months following introduction.

[0030] In one embodiment of any of the aspects herein, the method further comprises determining the subject's initial MDS-UPDRS score prior to introduction.

[0031] In one embodiment of any of the aspects herein, the method further comprises, prior to the introduction, receiving an assay result providing information about the subject's initial MDS-UPDRS score.

[0032] In one embodiment of any of the aspects herein, slowing or inhibiting progression of PD in the subject is characterized by a decrease in the initial MDS-UPDRS score following introduction.

[0033] In one embodiment of any of the aspects herein, the reduction is at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more reduction in the initial MDS-UPDRS score 6 months after introduction.

[0034] In one embodiment of any of the aspects herein, the reduction is at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more reduction in the initial MDS-UPDRS score 12 months after introduction.

[0035] In one embodiment of any of the aspects herein, slowing or inhibiting progression of PD in the subject is characterized by stabilization of the initial MDS-UPDRS score following introduction.

[0036] In one embodiment of any aspect herein, stabilization is characterized by an increase or decrease of no more than 10% in the initial MDS-UPDRS score. In one embodiment of any aspect herein, stabilization occurs for at least 6 months or more.

[0037] In one embodiment of any aspect herein, the subject is mildly affected by PD. In one embodiment of any aspect herein, the subject mildly affected by PD has an initial MDS-UPDRS score of less than 32 prior to the introduction of rAAV and was diagnosed with PD less than 5 years prior to said introduction.

[0038] In one embodiment of any of the aspects herein, the method comprises, prior to introducing, diagnosing the subject as being mildly affected by PD.

[0039] In one embodiment of any of the aspects herein, the method further comprises, prior to the introducing, receiving an assay result diagnosing the subject as mildly affected by PD.

[0040] In one embodiment of any aspect herein, the subject is moderately affected by PD. In one embodiment of any aspect herein, 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.

[0041] In one embodiment of any of the aspects herein, the method further comprises, prior to the introducing, diagnosing the subject as moderately affected by PD.

[0042] In one embodiment of any of the aspects herein, the method further comprises, prior to the introducing, receiving an assay result diagnosing the subject as moderately affected by PD.

[0043] In one embodiment of any aspect herein, the promoter is a cytomegalovirus (CMV) promoter.

[0044] In one embodiment of any aspect of the present invention, the promoter is a nervous system (NS) or central nervous system (CNS) specific promoter. In one embodiment of any aspect of the present invention, the NS specific promoter is selected from the NS specific promoters in Table 1. In one embodiment of any aspect of the present invention, the CNS specific promoter is selected from the CNS specific promoters in Table 2.

[0045] In one embodiment of any aspect herein, the nucleic acid comprises the 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.

[0046] In one embodiment of any aspect herein, 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.

[0047] In one embodiment of any aspect herein, 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, the brain-specific tropism is increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to unmodified AAV.

[0048] In one embodiment of any aspect herein, 5×10 12 vg is about 1.5×10 13 The total dose introduced into rAAV was within the range of vg.

[0049] In one embodiment of any of the aspects herein, about half of the total dose is administered to each putamen of the subject.

[0050] In one embodiment of any of the aspects herein, the introducing is performed at a flow rate of about 1 μL / min to about 30 μL / min.

[0051] In one embodiment of any aspect herein, the rAAV is introduced in a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier.

[0052] In one embodiment of any aspect herein, the rAAV concentration of the liquid composition is about 3×10 12 vg / mL to about 4×10 12 vg / mL.

[0053] In one embodiment of any of the aspects herein, at least one anti-PD therapeutic agent is administered to the subject prior to the introduction of the rAAV.

[0054] In one embodiment of any aspect of the present invention, at least one anti-PD therapeutic agent is administered to the subject before and after the introduction of rAAV. In one embodiment of any aspect of the present invention, the at least one anti-PD therapeutic agent is selected from the group consisting of: levodopa, Sinemet, Rytary, Stalevo, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, and entacapone.

[0055] In one embodiment of any aspect herein, the subject maintains or reduces the dose of at least one anti-PD therapeutic agent after introduction. In one embodiment of any aspect herein, the dose of the at least one anti-PD therapeutic agent is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.

[0056] Another aspect provided herein describes a method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising locally introducing into the putamen of the subject a recombinant adeno-associated viral (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 putamen volume of the subject is transduced with the GDNF gene.

[0057] Another aspect provided herein describes a method of slowing or inhibiting the progression of PD in a subject in need thereof, the method 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 significant increase in PD-related symptoms for at least 6 months after transduction. In one embodiment of any aspect herein, transduction is performed by administering a rAAV comprising a GDNF gene to each putamen of the subject.

[0058] Another aspect provided herein describes a method of reducing or stabilizing an initial Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) score in a subject with Parkinson's disease (PD), the method comprising administering to the putamen of the subject a recombinant adeno-associated virus (rAAV), the recombinant rAAV comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein, 6 months after the administration, the subject's second MDS-UPDRS score is reduced or stabilized compared to the subject's initial MDS-UPDRS score before the administration.

[0059] In one embodiment of any aspect herein, the method further comprises a step of obtaining or receiving an initial MDS-UPDRS score from a subject prior to administration. In one embodiment of any aspect herein, a second MDS-UPDRS score 12 months after administration is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the initial MDS-UPDRS score.

[0060] In one embodiment of any of the aspects herein, stabilization is an increase or decrease in the initial MDS-UPDRS score of no more than 10%.

[0061] Another aspect provided herein describes a method of treating a subject mildly affected by Parkinson's disease (PD), the method comprising administering to each putamen of the subject a recombinant adeno-associated virus (rAAV), the recombinant rAAV comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the putamen of the subject is transduced with GDNF, and wherein at 6 months after administration, the subject has a second MDS-UPDRS score that is stable compared to the initial MDS-UPDRS score.

[0062] In one embodiment of any of the aspects herein, 12 months after the administration, the subject has a stable MDS-UPDRS score compared to the initial MDS-UPDSS score before the administration.

[0063] Another aspect provided herein describes a method for treating a subject moderately affected by Parkinson's disease (PD), the method comprising administering a recombinant adeno-associated virus (AAV) to each putamen of the subject, the recombinant AAV comprising a nucleic acid encoding glial cell-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, 6 months after administration, the subject's second MDS-UPDRS score is at least about 20% lower than the initial MDS-UPDRS score. In one embodiment of any aspect herein, the reduction is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the initial MDS-UPDRS score.

[0064] Another aspect provided herein describes a method for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising locally introducing a recombinant adeno-associated viral (rAAV) vector into each putamen of the subject, the recombinant adeno-associated viral 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 into each putamen of the subject substantially simultaneously with the rAAV, wherein at least 30% of the volume of the subject's putamen is transduced with the nucleic acid, and wherein, for at least 6 months immediately following the introduction, the subject does not exhibit a significant increase in PD-related symptoms compared to before the introduction.

[0065] Another aspect provided herein describes a method for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising introducing into 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 subject's putamen volume is transduced with the GDNF gene, and wherein, for at least 6 months immediately following the introduction, the subject does not exhibit a significant increase in PD-related symptoms compared to before the introduction.

[0066] Another aspect provided herein describes a composition for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, the composition 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.

[0067] In one embodiment of any aspect herein, the rAAV concentration of the composition is 3×10 12 vg / mL to 4×10 12 vg / mL.

[0068] In one embodiment of any aspect herein, the composition comprises a concentration of 3.3×10 12 vg / mL of rAAV.

[0069] Another aspect provided herein describes a formulation for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, the formulation comprising a concentration of 3×10 12 vg / mL pharmaceutically acceptable carrier to 4×10 12 vg / mL of an adeno-associated virus (AAV) containing 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 A schematic diagram of the clinical study schedule is shown. Subjects will cycle "ON" and "OFF" their prescribed anti-Parkinson's disease therapeutic agents as indicated by the "OFF" and "ON" arrows. MRI, FDG, and DaT scans are administered as indicated by the crosses. Blood tests are performed as indicated by the droplets. Subjects' activities are monitored as indicated by the hexagons. For visits that require evaluation in a defined off-drug state, participants are required to stop all PD medications (such as carbidopa / levodopa, Sinemet, Rytary, Darlingfort, Amantadine, Pramipexole, Rotigotine, Ropinirole, Apomorphine, Entacapone) the night before the visit and should remain on for at least 12 hours.

[0071] Figure 2 A summary of the subjects included in Cohort A (mildly affected by PD) is shown.

[0072] Figure 3 A summary of the subjects included in Cohort B (moderately affected by PD) is shown.

[0073] Figure 4 A summary of the percentage of putamen transduced with GDNF in subjects following topical administration of AAV2-GDNF is shown. The average putamen volume transduced with GDNF was 63%.

[0074] Figure 5 A bar graph is presented showing the percentage of putamen volume transduced with GDNF in subjects following topical administration of AAV2-GDNF operably linked to a CMV promoter. The average putamen volume transduced with GDNF was -63%.

[0075] Fig. 6A and Figure 6B Representative postoperative MRI of subjects in Cohort A is shown. Fig. 6A MRI T1 (pre-contrast) images are shown at a time after administration of the therapeutic agent. Figure 6B MRI T2 images at a time after the administration of the therapeutic agent are shown. Different manifestations of putaminal hyperintensities are observed.

[0076] Fig. 7A and Figure 7B Representative postoperative MRI of subjects in Cohort B is shown. Fig. 7A MRI T1 (pre-contrast) images are shown some time after administration. Figure 7B MRI T2 images at a certain time after administration are shown. Different manifestations of putamen hyperintensity are observed.

[0077] Figure 8A-8H Data evaluating PD progression at different time points after administration are shown. Figure 8A-8D Presents a method showing the use of anti-Parkinson's disease therapeutic agents ( Fig. 8A ) or discontinuation of anti-Parkinson's disease therapy ( Figure 8B ) in the MDS-UPDRS combined motor skills. Stable MDS-UPDRS scores were observed in cohort A (represented by the "mild PD cohort") for 12 months. A significant reduction in MDS-UPDRS scores was observed in cohort B (represented by the "moderate PD cohort"). MDS-UPDRS scores were assessed at pre-administration screening to establish a baseline and then at 3, 6, 9, and 12 months after surgery. Total UPDRS score ( Figure 8C ) and MDS-UPDRS II score ( Fig.8D ) followed the same trend within the specified time after surgery. Figure 8E-Figure 8H showed that motor measures were stable over 18 months after AAV2-GDNF administration in the mild PD cohort ( Fig. 8E and Figure 8F ), and motor improvement after 18 months of AAV2-GDNF administration in the moderate PD cohort ( Figure 8G and Figure 8H ).exist Fig. 8E and Figure 8F In the figure, (A) shows that mild PD shows stability over 18 months. (B) shows that the window for measuring large functional improvements in mild PD is limited. (C) shows an outlier identified as having a TH mutation.

[0078] Figure 9A-9D Data showing PD exercise log data are presented. ( Figure 9A-9B ) The bar chart shows the queue A ( Fig. 9A ) and queue B( Fig. 9B ) PD exercise log data. The “on / off” times in the logs were normalized to 16 hours of wakefulness. ( Fig. 9C ) The pie chart illustrates the significant improvements in subjects in cohort B at 12 and 18 months post-administration. Good ON time improved by 27% from baseline and OFF time improved by 52% from baseline. ( Fig.9D ) Pie charts illustrate the significant improvements in subjects in Cohort A at 12 and 18 months post-administration. Good ON time was reduced by 12% from baseline, and OFF time was increased by 46% from baseline.

[0079] Fig. 10A and Fig. 10B Cohort A (mildly affected; Fig. 10A ) and cohort B (moderate effect; Fig. 10B ) are shown in Figure 3. NMSS (circles) and PDQ-39 (squares) scores were assessed at screening before administration, 6 months, 12 months, 18 months, and 24 months after surgery.

[0080] Fig.11 A line graph showing the dose response of AAV2-GDNF in Cohort B (moderate effects) versus Phase 1 of the trial is presented. The data demonstrate that dose and putamen coverage (>50%) correlate with clinical response in moderate stage PD. The magnitude of functional motor improvement in moderate stage PD exceeded the expected placebo effect (-5pt).

[0081] Fig.12 Graphs are presented showing that the volume distribution of putamen infusions, including gene therapy, is highly dependent on the delivered infusion volume, as predicted in animal models. For clinical PD cases, the average volume of the unilateral putamen is approximately 4200 cubic millimeters. As shown below the horizontal dashed line in this figure, early gene therapy products infused into the putamen provided limited distribution volume, well below 50% of the total putamen volume. The initial limitation in distribution volume was primarily due to the small infusion volumes delivered and the use of a standard bifrontal trajectory to reach the putamen. The standard bifrontal approach provides a trajectory that is nearly perpendicular to the long axis of the putamen; volume coverage of the putamen with this trajectory is limited by the small dorsoventral putamen size, requiring multiple trajectories to expand volume coverage. The development of gene therapy infusions parallel to the long axis of the putamen has provided larger infusion volumes (up to 1800 μL / putamen) and achieved >50% putamen coverage.

[0082] Fig.13A and Fig. 13B Schematic diagram of the dual frontal and dual occipital trajectory technique is shown. Fig.13A ) Bifrontal trajectories - one or more frontal burr holes are made on both sides. At least 2 trajectories per putamen to cover both the prefascicular and postfascicular putamen. Trajectories are nearly perpendicular to the long axis of the putamen, and volume coverage is limited primarily by the short dorsoventral dimension of the putamen and the number of trajectories used. Typically less than 50% putamen volume coverage is achieved. Fig. 13B ) Double occipital trajectory - an occipital burr hole is made in each putamen. This technique requires a single trajectory per putamen to cover both the prefascicular and postfascicular putamen. Trajectories parallel to the long axis of the putamen and volume coverage is limited primarily by perivascular leakage from within the putamen. Putaminal volume coverage of >50% is typically achieved.

[0083] Fig.14A and Fig. 14B A summary of previously completed trials and currently ongoing trials is shown. Fig.14A A chart showing clinical experience with AAV2-GDNF gene delivery to the putamen in Parkinson's disease using bifrontal and bioccipital delivery approaches is presented. Fig.14ADetails of previously completed Phase 1 and ongoing clinical trials are provided that test the safety and tolerability of different vector doses and putamen coverage in late, intermediate, and early stages of PD. The current clinical trial (described in Examples 1-3 below) is the first human gene therapy trial approved to test the safety of a gene therapy product in participants with early PD. As of the end of March 2022, the clinical study has enrolled and treated 11 of the 12 planned participants. The Phase 1 study delivered an infusion volume of 450 microliters (9×10 10 vg to 9×10 11 vg), resulting in an average putamen coverage of 26%. In the clinical trials described herein, dual pillow delivery has provided up to 1800 microliters of infusate in each putamen and provided an average putamen distribution of 62.5% in 11 participants to date. Fig. 14B A summary of putamen coverage achieved by the indicated cohorts in the previously completed Phase 1 trial and the current clinical trial (Phase 1b; described in Examples 1-3 below) is shown.

[0084] Fig.15 A schematic diagram of AAV-GDNF is shown. CMV, cytomegalovirus; hGDNF, human glial cell-derived neurotrophic factor; hGH, human growth hormone; ITR, inverted terminal repeat.

[0085] Fig.16 A schematic diagram of the study design is presented. AAV2-GDNF was administered bilaterally to the putamen (up to 1.8 mL per putamen, with a maximum dose of 1.2 × 10 13 vg) and the distribution was observed by contrast agent (2 mM gadoteridol).

[0086] Fig.17 A summary of postoperative adverse reactions observed for more than 1 month after surgery, i.e., treatment-emergent adverse events (TEAEs), is presented.

[0087] Fig.18A and Fig.18B A mild queue ( Fig.18A ) and moderate cohort ( Fig.18B ) Summary of individual post-treatment changes in motor and non-motor assessments.

[0088] Figures 19A-19F Data showing AAV2-GDNF expression 3.5 years after administration are presented. Fig.19A )MRI images of participants to whom AAV2-GDNF was administered intradermally. Figure 19B-Figure 19D )Tyrosine hydroxylase staining of putamen biopsy samples showed enrichment of dopaminergic neurons in the putamen. Fig.19B Shows Fig.19A The area indicated by the arrow. Fig.19C The arrows show Fig.19B Enhanced and enlarged image of the area in . Fig.19D The arrows show Fig.19C An enhanced, magnified image of the area in . Fig.19E ) Locations of the 6 biopsies performed in the sample. Biopsy sites #1 and 5 were the infusion sites used during surgery. Biopsy site #6 was located outside the putamen of the white matter tract. Fig.19F ) GDNF transgene levels (pg GDNF / mg protein) at the indicated biopsy sites. The highest levels of GDNF were seen at sites #1 and 5. No GDNF expression was identified at biopsy site #6.

[0089] Fig. 20 Longitudinal MRI monitoring data showing safety readouts are presented. The left column of T1 (top row) and T2 (bottom row) weighted MRI brain scans show the distribution of gadoteridol (bright white signal in T1 images) after bilateral infusion into the putamen (outline). Matched MRI scans acquired at the 6-month and 18-month time points show no residual gadoteridol signal or tissue abnormalities in the putamen or other brain structures.

[0090] Fig.21 A graph depicting the response of the moderate PD cohort is presented. Stronger and more progressive recovery and motor function were found compared with previous CGT. The 18-month clinical data showed: (1) stronger improvements than previous neurotrophic CGT, (2) the effects of AAV2-GDNF were more progressive than previous neurotrophic factor GTx, with sustained improvements after six months, unlike the transient improvements seen with other CGTs, and (3) clinically meaningful improvements over six months were consistent with the expected mechanisms of action, such as progressive recovery of terminal sprouting and dopamine function.

[0091] Fig.22A and Fig. 22B Shows mild ( Fig.22A ) and moderate ( Fig. 22B ) Charts of the Unified Movement Disorder Rating Scale history, target, and total scores for the cohort up to 18 months after treatment.

[0092] Fig.23A and Fig. 23B Presented with mild ( Fig.23A ) and moderate ( Fig. 23B ) cohort.

[0093] Figure 24A-Figure 24CPreliminary analysis data showing functional imaging with DaT scanning in mild and moderate cohorts are presented. Fig.24A Numerical bar graphs are presented. Fig. 24B and Fig.24C Shown Fig.24A About mild ( Fig. 24B ) and moderate ( Fig.24C ) cohorts. A preliminary analysis of DAT binding over time is shown. In the mild and moderate cohorts, decreased binding was observed in the caudate nucleus. Put Him showing relatively stable or increased DaT signal in both cohorts.

[0094] Figure 25A-Figure 25D Data showing changes in infusion site F-dopa (L-dopa) uptake 6 and 18 months after gene therapy administration are presented. Figure 25A-Figure 25C ) MRI images showing the distribution of gadoteridol in the axial (left column) and coronal (right column) planes after bilateral infusion into the internal (anterior) and posterior (posterior) putamen. Fig.25A One patient had a baseline ( Fig.25B ) and 18 months after surgery ( Fig.25C ) shows that the region corresponding to the infusion site has increased Ki, which is visualized as gadoteridol signal in MRI.

[0095] Fig.26 A plasmid schematic for generating the AAV2-GDNF vector is presented, for example, SEQ ID NO: 64. DETAILED DESCRIPTION

[0096] Aspects of the technology disclosed herein relate to administration (e.g., local or systemic administration) of a glial cell-derived neurotrophic factor (GDNF) gene such that at least 30% of the subject's putamen is covered and / or transduced by the gene. Such coverage and / or transduction levels have been shown to be effective in reducing, slowing, or inhibiting the progression of symptoms associated with PD. Therefore, in some embodiments, the methods and compositions described herein are useful for treating PD. Parkinson's disease (PD)

[0097] Methods provided herein relate to slowing or inhibiting the progression of Parkinson's disease. As used herein, "Parkinson's disease" or "PD" refers to a neurodegenerative disease characterized by gradually worsening tremors and rigidity, as well as increasingly severe problems with balance, walking, and coordination. Although most PDs are considered idiopathic in nature, a combination of environmental influences and genetic susceptibility, often acting on epigenetic control, as well as mutations in a number of genes, can cause or increase the risk of PD, including those in synuclein alpha (SNCA; NCBI Gene ID: 6622), leucine-rich repeat kinase 2 (LRRK2 / PARK8; NCBI GeneID 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 55737), and mitochondrial isosterol kinase 2 (MIT; NCBI Gene ID 55710). ID 1981), DnaJ heat shock protein family member C13 (DNAJC13; NCBI Gene ID 23317), coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2; NCBI Gene ID 51142), and / or ubiquitin C-terminal hydrolase L1 (UCHL1; NCBI Gene ID 7345) genes. The sequences of such PD-related genes are known in many species, for example, human mRNA and protein sequences can be obtained in the NCBI database using the provided Gene ID numbers.These PD-associated genes and other genes, as well as their PD-associated alleles (e.g., mutations, duplications, SNPs, etc.) are known in the art and are further described, for example, in: 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., Neurologica 2017 135:273-84; Jain et al., Clinical Science 2005 109:355-64; Fagan et al., European Journal of Neurology 2017 24:561-e20; Campelo et al., Parkinson's Disease 2017 4318416; and Porter et al., Neurodegeneration and Alzheimer's Disease 2019, Chapter 15; each of which is incorporated herein by reference in its entirety.

[0098] Risk factors for developing PD include, but are not limited to, age, genetics, exposure to certain toxins, and gender. It is rare for adolescents and young adults to be diagnosed with PD. Starting in middle and old age, the risk of developing Parkinson's increases with age; subjects usually develop the disease around age 60 or older. Having a close relative (such as an immediate family member, uncle, aunt, or grandparent) with PD increases the chance that a subject will develop the disease. However, unless multiple relatives have been diagnosed with PD, the risk is still considered low. Continued exposure to certain herbicides and pesticides has been shown to slightly increase a subject's risk of PD. Finally, males are more likely to develop PD than females.

[0099] Symptoms of PD are well documented and known to those skilled in the art. Early symptoms of PD include, but are not limited to, tremors (e.g., shaking when the body is at rest, usually starting in the limbs, often in the hands or fingers); pilling-rolling tremors (e.g., rubbing the thumb and index finger back and forth when the body is at rest); bradykinesia (slow movements); muscle rigidity (i.e., stiffness of muscles anywhere in the body that may be painful and limit a person's range of motion); impaired posture and balance (e.g., posture may become hunched, or a person may have balance problems); loss of automatic movements (e.g., reduced ability to perform involuntary movements, including blinking, smiling, or swinging the arms while walking); changes in speech (e.g., a person may speak softer and faster, slur or hesitate before speaking; or have changes in intonation and speech patterns); and changes in handwriting (e.g., handwriting may appear smaller and more crowded).

[0100] Complications of PD include, but are not limited to, cognitive problems (dementia) and difficulty thinking in late PD; depression and mood changes (i.e., fear, anxiety, or loss of motivation) in early and late PD; swallowing problems (e.g., dysphagia, saliva accumulation, and drooling) as the disease progresses; chewing and eating problems in late PD, which can lead to choking and malnutrition; sleep problems and sleep disturbances (i.e., frequent waking, early rising, and daytime sleeping); rapid eye movement sleep behavior disorder; bladder problems (i.e., inability to control urine or difficulty urinating); constipation; orthostatic hypotension (i.e., a sudden drop in blood pressure); olfactory dysfunction (e.g., loss of smell or difficulty identifying certain smells or the difference between smells); fatigue; pain, i.e., pain in a specific part of the body or throughout the body; and sexual dysfunction.

[0101] There is no one specific test for diagnosing Parkinson's disease, instead, a skilled clinician will diagnose a subject based on the subject's medical history, family history, signs and / or symptoms present, and a neurological and physical examination. A specific single photon emission computed tomography (SPECT) scan called a dopamine transporter scan (DaTscan) may be performed to support the diagnosis, but this is unlikely to be a key determinant of the diagnosis. Most patients do not require a DaTscan. Non-invasive imaging such as MRI, brain ultrasound, and PET scans may be performed to rule out other neurological diseases but are not helpful in diagnosing PD. In addition, an adequate (i.e., high) dose of an anti-Parkinson's disease therapeutic (such as carbidopa-levodopa) may be administered to a subject suspected of having Parkinson's disease and monitored for improvement in symptoms; improvement following administration would indicate / confirm a diagnosis of PD.

[0102] Treatments for PD include, but are not limited to, therapeutic agents designed to treat persistent symptoms of the disease. These therapeutic agents include, but are not limited to, carbidopa-levodopa; inhaled carbidopa-levodopa; carbidopa-levodopa infusion; dopamine agonists; MAO B inhibitors; catechol O-methyltransferase (COMT) inhibitors; anticholinergic drugs; amantadine.

[0103] Levodopa, the most effective PD medication, is a natural chemical that enters the brain and is converted into dopamine. Levodopa is often combined with carbidopa (eg The combination of levodopa and carbidopa can prevent premature conversion of levodopa to dopamine outside the brain, preventing or alleviating side effects such as nausea. As the disease progresses to later stages, the benefits of levodopa may become less stable and tend to wax and wane (i.e., "wearing off"). Involuntary movements (dyskinesias) are associated with higher doses of levodopa. Levodopa inhalation powder is a therapeutic drug that delivers levodopa in an inhaled form. TM Carbidopa / levodopa suspension is a brand-name medication consisting of carbidopa and levodopa that is administered through a feeding tube, allowing the medication to be delivered directly to the small intestine in a gel form. TM Carbidopa / levodopa suspension is indicated for patients with more advanced Parkinson's disease who still respond to carbidopa-levodopa but whose responses fluctuate significantly. TM With continuous infusion, blood levels of both drugs (carbidopa and levodopa) remain constant.

[0104] Unlike levodopa, dopamine agonists do not convert to dopamine, but rather mimic the effects of dopamine in the patient's brain. Dopamine agonists are not as effective as levodopa in treating PD symptoms; however, they last longer and can be used with levodopa to support the off-and-on effects of levodopa. Exemplary dopamine agonists include pramipexole (e.g., Pramipexole), ropinirole (e.g. Ropinirole), rotigotine (e.g. Rotigotine transdermal system, available in patch form) and apomorphine (e.g. Apomorphine), which is a short-acting injectable dopamine agonist.

[0105] MAO B inhibitors help prevent the breakdown of brain dopamine by inhibiting the brain enzyme monoamine oxidase B (MAO B), which metabolizes brain dopamine. Exemplary MAO B inhibitors include selegiline (e.g., Selegiline hydrochloride), rasagiline (eg Rasagiline) and safinamide (such as Taking selegiline with levodopa can help prevent the effects of the drug from wearing off.

[0106] Catechol O-methyltransferase (COMT) inhibitors modestly prolong the effects of levodopa treatment by blocking the enzyme that breaks down dopamine. Exemplary COMT inhibitors include entacapone (e.g. Entacapone), opicapone (such as opicapone) and tolcapone (eg Tolcapone). Tolcapone is rarely prescribed because of the risk of severe liver damage and liver failure.

[0107] Anticholinergic drugs have been used previously to help control tremors associated with PD. Exemplary anticholinergic drugs include antipsychotics (clozapine, quetiapine), atropine, benztropine (eg Benztropine Mesylate), Biperiden, Chlorpheniramine, Certain SSRIs (Paroxetine), Dicyclomine, Dimenhydrinate, Diphenhydramine, Doxepi, Doxylamine, Flavoxate, Glycopyrrolate, Glycopyrronium, Scopolamine, Ipratropium, Orphenadrine, Oxitropium, Oxybutynin, Promethazine, Propantheline Bromide, Scopolamine, Solifenacin, Tolterodine, Tiotropium, Tricyclic Antidepressants, Trihexyphenidyl, Tropicamide, and Umeclidinium.

[0108] Amantadine (eg Amantadine) is an antidyskinesia drug prescribed as monotherapy, providing short-term relief of symptoms in mild, early PD. In more advanced stages of Parkinson's disease, it is also prescribed along with carbidopa-levodopa therapy to control carbidopa-levodopa-induced involuntary movements (dyskinesias).

[0109] Parkinson's disease patients may further undergo surgery to have deep brain stimulation (DBS) implanted to reduce symptoms associated with the disease. DBS involves implanting electrodes in specific parts of the patient's brain; the electrodes are connected to a generator implanted in the patient's chest near the collarbone, which sends electrical pulses to the patient's brain. DBS is effective in controlling erratic and fluctuating responses to levodopa, or controlling movement disorders that do not improve after medication adjustments. DBS is more often used in more advanced patients who have an erratic response to medications such as levopoda. Treatment

[0110] The present disclosure provides methods for delivering nucleic acids and / or transgenes (e.g., nucleic acids encoding GDNF) to a subject. These methods generally involve administering to a subject an effective amount of a nucleic acid encoding GDNF. In some embodiments, administration is systemic administration. In some embodiments, administration is topical administration. In some embodiments, the nucleic acid is provided in the form of a viral vector and / or viral particle (e.g., rAAV).

[0111] One aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising introducing into 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 subject's putamen volume is transduced with the GDNF gene, and wherein, for at least 6 months immediately following the introduction, the subject does not exhibit an increase in PD-related symptoms compared to before the introduction.

[0112] One aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising introducing into 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 introduction of the rAAV results in at least 30% of the subject's putamen being covered by the rAAV, and wherein the subject does not exhibit an increase in PD-related symptoms for at least 6 months immediately following the introduction compared to before the introduction.

[0113] The putamen includes two bilaterally called oval-shaped subcortical lobes, which extend longitudinally around the anterior-posterior (AP) axis. As used herein, and as can be determined by context, the term "putamen" may refer to a single putamen (i.e., left putamen or right putamen) or a general term for two putamen. The putamen is located in the paraventricular deep white matter of the forebrain of each cerebral hemisphere (telencephalon), comprising a plurality of nerve cell (neuron) bodies. The putamen forms the striatum together with the adjacent caudate nucleus. The striatum is one of the many components that form the basal ganglia of each cerebral hemisphere. In addition to many areas of the cerebral cortex, the putamen is also connected to the substantia nigra (including the pars compacta and pars reticularis), the globus pallidus, the claustrum, and the thalamus through various pathways. The main function of the putamen is to regulate the preparation and execution of body movements, and to play a role in various types of learning. The putamen also plays an important role in the development of degenerative neurological disorders (such as PD). Retrograde axonal transport of GDNF protein and / or AAV2 vector from the putamen to the substantia nigra is possible; however, in the PD state, anterograde axonal transport of GDNF protein and / or AAV2 vector to the pars reticulata is more likely. The direction of axonal transport can be determined by the vector used to deliver the GDNF transgene.

[0114] One aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising introducing into 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 volume of the subject's putamen is covered by the rAAV.

[0115] In one embodiment, the rAAV is introduced by topical introduction. In one embodiment, the AAV capsid is reasonably haploid, for example, the capsid is AAV8, AAV9, and contains at least one capsid protein from a rhesus monkey AAV strain.

[0116] In various embodiments, the local introduction is directly into the putamen of the subject. For example, the local introduction can include introducing the rAAV directly into one or both of the putamen of the subject.

[0117] In various embodiments, local introduction is performed simultaneously with non-invasive imaging. For example, local introduction includes introducing about half of the total dose of rAAV vector into each putamen by convection-enhanced delivery (CED) guided by intraoperative magnetic resonance imaging (iMRI).

[0118] In one embodiment, local introduction further comprises introducing an MRI contrast agent simultaneously or substantially simultaneously with the AAV vector. Exemplary MRI contrast agents include gadoterate, gadobutro, gadoteridol, gadopentatate, gadobenate, gadopentetic acid dimeglumine, gadoxentate, gadooversetamide, gadodiamide, gadofosveset, gadocolic acid, gadomelitol, and gadomer.

[0119] In one embodiment, the MRI contrast agent is introduced into the subject in the same composition as the rAAV. In one embodiment, the MRI contrast agent is introduced into the subject in different compositions than the rAAV, but is administered substantially simultaneously.

[0120] In one embodiment, rAAV is introduced by systemic introduction.

[0121] Another aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising locally introducing a recombinant adeno-associated viral (rAAV) vector into the putamen of a subject, the recombinant adeno-associated viral vector comprising a nucleic acid encoding glial cell-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the putamen of the subject is transduced with the GDNF nucleic acid. In one embodiment, transducing the putamen is transducing a putamen neuronal population. In one embodiment, at least 30% of the volume of the putamen neuronal population of a subject is transduced with the GDNF nucleic acid.

[0122] Another aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising transducing a subject's putamen greater than or equal to about 30% by volume with a glial cell-derived neurotrophic factor (GDNF) gene, wherein the subject does not show a significant increase in PD-related symptoms for at least 6 months after transduction. In one embodiment, transduction is performed by administering an rAAV comprising a GDNF gene to each putamen of the subject's cerebral hemisphere. In one embodiment, transduction of the putamen is transduction of a putamen neuron population. In one embodiment, at least 30% of the volume of the subject's putamen neuron population is transduced with a GDNF nucleic acid.

[0123] 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 with PD, the method comprising administering to the putamen of the subject a recombinant adeno-associated virus (rAAV), the recombinant rAAV comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the subject's second MDS-UPDRS III score at 6 months after the administration is reduced or stabilized compared to the subject's initial MDS-UPDRS III score before the administration.

[0124] Another aspect provided herein relates to a method of treating a subject mildly affected by PD, the method comprising administering a recombinant adeno-associated virus (rAAV) to each of the subject's putamen, the recombinant rAAV comprising a nucleic acid encoding glial cell-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 volume of the subject's putamen is covered by the rAAV, and wherein the subject has a second MDS-UPDRS III score that is stable compared to the initial MDS-UPDRS III score at 6 months after administration.

[0125] Another aspect provided herein relates to a method of treating a subject moderately affected by PD, the method comprising administering a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter to each of the subject's putamen, 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 that is at least about 20% lower than the initial MDS-UPDRS III score at 6 months after administration.

[0126] Another aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising locally introducing a recombinant adeno-associated viral (rAAV) vector into each of the subject's putamen, the recombinant adeno-associated viral 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 into 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 volume of the subject's putamen is covered by the rAAV, and wherein the subject does not exhibit a significant increase in PD-related symptoms for at least 6 months immediately following the introduction compared to before the introduction.

[0127] Another aspect provided herein relates to a method for slowing or inhibiting the progression of PD in a subject in need thereof, the method comprising introducing into the subject a recombinant adeno-associated virus (rAAV), the recombinant rAAV comprising a nucleic acid encoding glial cell-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 volume of the subject's putamen is covered by the rAAV, and wherein the subject does not exhibit a significant increase in PD-related symptoms for at least 6 months immediately following the introduction compared to before the introduction.

[0128] In various embodiments, 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 putamen of the subject is transduced with the GDNF gene. In one embodiment, transduction of the putamen of the subject is assessed by non-invasive imaging (e.g., by MRI). One skilled in the art can assess transduction of rAAV by measuring the total volume of the putamen containing rAAV (e.g., assessed by injected MRI contrast agent) compared to the total volume not including rAAV.

[0129] In one embodiment, transducing the putamen is transducing the intrinsic medium spiny neurons (MSNs) of the putamen. In one embodiment, 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more are transduced with a nucleic acid, such as GDNF.

[0130] In one embodiment, transducing the putamen is transducing the putamen neuron population. In one embodiment, at least 30% of the volume of the putamen neuron population of the subject is transduced with GDNF nucleic acid. In various embodiments, 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 9 %, 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more are transduced with GDNF nucleic acid.

[0132] In various embodiments, 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least In some embodiments, 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 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more are covered by rAAV. In some embodiments, the coverage of the subject's putamen is assessed by non-invasive imaging (e.g., by MRI). One skilled in the art can assess the coverage of rAAV by measuring the total volume of the putamen containing rAAV (e.g., assessed by an injected MRI contrast agent) compared to the total volume of the putamen.

[0132] In one embodiment, an 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 CED distribution and to assess transduction volume. Non-invasive imaging

[0133] In one embodiment, local administration is carried out simultaneously with non-invasive imaging, for example, guiding local delivery to a preferred or predetermined position or for example, the putamen, and / or visualizing transduction after administration. In one embodiment, non-invasive imaging is convection enhanced delivery (CED) guided by intraoperative magnetic resonance image (iMRI). As used herein, "intraoperative magnetic resonance image (iMRI)" refers to an MRI image of, for example, the brain obtained during neurosurgery. Accurate, real-time brain pictures can be created by relying on iMRI technology to provide guidance during neurosurgery, such as tumor removal or placement of therapeutic drugs in a desired position (such as putamen). As used herein, "convection enhanced delivery (CED)" refers to a drug delivery technology that uses positive hydrostatic pressure to deliver a fluid containing a therapeutic substance directly to the interstitial space in a local area of ​​brain parenchyma by a bulk flow. Direct intracerebral CED bypasses the blood-brain barrier and provides a wider and more uniform distribution than bolus deposition (focal injection) or other direct delivery methods based on diffusion. 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 which are incorporated herein by reference in their entirety.

[0134] In one embodiment, iMRI will be used to monitor administration of the rAAV or compositions thereof described herein using T1-weighted sequences to visualize MRI contrast agents, such as gadolinium-based contrast agents, co-infused with the rAAV or compositions thereof.

[0135] In one embodiment, local introduction further includes introducing an MRI contrast agent substantially simultaneously with the AAV vector. In this case, the MRI contrast agent is used to increase the visibility of internal brain structures captured in MRI images. Preferred are paramagnetic contrast agents containing gadolinium (III), referred to in the art as gadolinium-based MRI contrast agents (GBCAs), see P. Caravan et al. "Gadolinium (III) Chelates as MRI Contrast Agents Structure, Dynamics, and Applications", Chem. Rev. 99, 2293-2352 (1999), the contents of which are incorporated herein by reference in their entirety. Other contrast agents that can be used include gadoxetate disodium (e.g., Eovist TMGadoxetate disodium; Schering AG); contrast agents disclosed in U.S. Pat. Nos. 5,798,092 and 5,695,739; gadobenatedimeglumine (e.g., MultiHance TM Gadobenate dimeglumine, Bracco SpA); and contrast agents disclosed in U.S. Pat. No. 5,733,528. Particularly preferred are "blood pool" MRI contrast agents, see LJM Croft et al., "Blood pool Contrast Agents for Cardiovascular MR Imaging", JMRI 10, 395-403 (1999), incorporated herein by reference, and A. Muhler Invest, "The Future of Contrast-Enhanced Magnetic Resonance Angiography: Are Blood Pool Agents Needed?" Radiol. 33, 709-714 (1998), also incorporated herein by reference. Examples of blood pool contrast agents include MP-2269 (Mallinckrodt, Inc.); the contrast agents disclosed in U.S. Pat. No. 5,888,576; MS-325 (EPIX Medical, Inc.); the contrast agents disclosed in PCT Publication No. WO 96 / 23526; P760 (Geurbet); gadolinium diethylenetriaminepentaacetate (GD-DTPA; e.g., Gadomer-17 TM , Schering AG); contrast agents disclosed in U.S. Pat. Nos. 5,876,698, 5,820,849, 5,681,543, 5,650,136, and 5,364,614; gadoterate meglumine (e.g., Clariscan TM gadoterate dimeglumine, Nycomed Amersham); contrast agents disclosed in PCT publications WO 96 / 09840 and WO 9725073; B22956 / 1 (Bracco SpA); and contrast agents disclosed in PCT publications WO 00 / 30688, WO 98 / 05625, WO 98 / 05626, WO 95 / 32741, WO 98 / 38738, WO 95 / 32441, and U.S. Pat. No. 5,649,537. Other examples of such blood pool agents include, but are not limited to, ferucarbotran (e.g., Resovist TMferucarbotran) or SHU 555A and C (Schering). The contents of all patents and patent applications mentioned above are expressly incorporated herein by reference in their entirety.

[0136] Exemplary MRI contrast agents include gadoterate, gadobutro, gadoteridol, gadopentatate, gadobenate, gadopentetic acid dimeglumine, gadoxentate, gadooversetamide, gadodiamide, gadofosveset, gadocoletic acid, gadomelitol, and gadomer.

[0137] In one embodiment, the MRI contrast agent is gadoteridol (eg In one embodiment, gadoteridol (e.g. Gadoteridol) was administered as a 2 mM solution.

[0138] MRI contrast agents can be administered by injection into the bloodstream (intravenously) or orally, depending on the subject of interest. Oral administration is well suited for GI tract scanning, while intravascular administration proves to be more useful for most other scans. In one embodiment, the MRI contrast agent is administered in the same composition as rAAV. In one embodiment, the MRI contrast agent is administered in a separate composition from rAAV, but is administered simultaneously with the separate rAAV composition. When administered in a separate composition, the MRI contrast agent does not need to be administered in the same manner as rAAV. For example, if rAAV is administered locally, for example to the putamen, the MRI contrast agent can be administered intravenously or orally.

[0139] MRI brain scans may be performed preoperatively as part of the screening process, during gene therapy infusion, and at 6 and 18 months after administration. Scans may be performed at other time points if the investigator deems it necessary. MRI brain scans may be obtained, for example, on a 1.5 or 3T scanner, and sequences may include T1, T2, turbo FLAIR, T2 gradient echo, and diffusion. Optional imaging at 18 months screening may also include extended diffusion-weighted sequences, resting state and functional assessments, and image acquisition while the participant performs simple tasks (i.e., finger tapping or grasping). Total imaging time for each procedure is 90 minutes, including functional imaging and resting state imaging.

[0140] Exemplary non-invasive imaging techniques that can be used 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.

[0141] Ioflupant I-123 (e.g. DaTscan TM Iodoflupant (I23) selectively binds to presynaptic dopamine transporters and provides a method for imaging nigrostriatal terminals in the striatum. DaTscan TM Iodine-123 is an FDA-approved radiopharmaceutical for use in adults in conjunction with single-photon emission computed tomography (SPECT). Iodine-123 is a cyclotron-produced radionuclide that decays by electron capture to 123 Te, with a physical half-life of 13.2 hours. The recommended dose for intravenous administration in adults is 111 to 185 MBq (3 to 5 mCi). The effective dose produced in an adult when administering 185 MBq (5 mCi) of DaTscan activity is 3.94 mSv. DaTscan injections may contain up to 6% free iodide (iodine-123). To reduce the accumulation of iodine-123 in the thyroid gland, potassium iodide oral solution or Lugol's Solution up to 100 mg will be taken.

[0142] Fluoro-2-deoxyglucose (FDG) is a common FDA-approved radiopharmaceutical tracer used with positron emission tomography (PET) imaging to measure glucose metabolism in the brain and other organs. Brain metabolic patterns unique to PD that are not present in other Parkinson's-like disorders have been identified. During screening, FDGPET will be used to confirm a PD diagnosis. FDG is a F 18 Labeled with a half-life of 110 minutes. The recommended dose for intravenous administration in adults is 111 to 185 MBq (3 to 5 mCi). The effective dose produced when an FDG scan is administered in adults with an activity of 185 MBq (5 mCi) is 3.51 mSv.

[0143] In one embodiment, the coverage or transduction of the putamen is assessed by non-invasive imaging (e.g., intraoperative MRI). The putamen volume transduced by GDNF is determined indirectly by measuring the percentage of the putamen that shows CED perfusion MRI contrast in the putamen to the total putamen volume. In one embodiment, the transduced putamen volume is assessed by F-DOPA PET imaging associated with intraoperative CED perfusion MRI contrast localization.

[0144] As used herein, "coverage" refers to the volume of the putamen occupied by the infused rAAV relative to the total volume of the putamen. The coverage provides exposure of the putamen cells to the rAAV. There are many methods to determine the coverage of the therapeutic agent delivered after administration. For example, in one embodiment, the coverage of the putamen can be assessed by non-invasive imaging, for example, by co-infusion with a visualized MRI contrast agent. For example, the coverage of the putamen after introduction / administration can be determined by measuring the area or volume of the putamen showing the co-infused MRI agent and comparing it with the area or volume of the putamen that does not show the agent. Similarly, coverage is assessed by F-DOPA PET imaging. In addition, in one embodiment, rAAV may further comprise a reporter gene, such as a fluorescent tag, which can be visualized using standard histological methods (e.g., microscopic examination) at autopsy.

[0145] As used herein, "transduction" or "transduced" refers to cells within the nucleus that contain the administered rAAV or a composition thereof. In one embodiment, the transduced cells contain the genome of the rAAV and have the potential to express the GDNF transgene. In one embodiment, the transduced cells contain the genome of the rAAV and do not need to contain the ability to express the GDNF transgene. In one embodiment, the transduced cells transiently express the GDNF transgene. In one embodiment, the transduced cells stably express the GDNF transgene.

[0146] There are many ways to determine transduction after administration; these are usually secondary, inferred (or indirect) techniques. For example, transduction can be assessed by co-infusion with an MRI contrast agent that can be visualized. For example, by measuring the percentage of cells containing rAAV or a composition in the putamen that accounts for the total volume of the putamen, the percentage of cells transduced in the putamen can be determined. Transduction can be approached by non-invasive imaging, for example, by co-infusion with an MRI contrast agent that can be visualized. In one embodiment, rAAV may also include a reporter gene, such as a fluorescent tag, which can be visualized using standard methods (e.g., microscopy); such a reporter gene can be used to determine the percentage of transduction. Probes designed to target rAAV (e.g., capsid protein) can be used to assess whether cells are transduced by rAAV. In addition, probes designed to target the action of GDNF nucleic acid can be used to assess whether cells express the GDNF transgene. In one embodiment, the volume of the putamen transduced is assessed by F-DOPA PET imaging. In addition, in one embodiment, rAAV may also include a reporter gene, such as a fluorescent tag, which can be visualized using standard methods (e.g., microscopy). Diagnostic Assays for Parkinson's Disease and Disease Progression

[0147] The progression and severity of PD are often measured using a variety of clinical surveys that assess various symptoms and mental states of subjects who have PD or are thought to have PD. These clinical surveys can be completed by the subject, the subject's caregiver, and / or a skilled physician. Typically, clinicians and researchers use these surveys to assess the longitudinal course of PD during treatment or in clinical studies. For example, the results of such surveys can help clinicians or researchers determine the best course of action for treating the subject, i.e., changing the type of therapeutic agent, the dose of the therapeutic agent, or the frequency of administration of the therapeutic agent.

[0148] In one embodiment, the methods described herein further comprise the step of determining an initial score for at least one diagnostic assay described herein for the subject prior to introducing the rAAV described herein. In one embodiment, the diagnostic assay can be the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), the Non-Motor Symptom Scale (NMSS), the Parkinson's Disease Questionnaire (PDQ-39) score, the MDS-UPDRS Part III, the Modified Hoehn and Yahr, Up-Walk-Sit, 9-hole Pegboard Dexterity Test, Standing Balance Test, Global Impression (CGI and PGI), Brief Smell Identification Test (BSIT), Parkinson's Disease Sleep Scale (PDSS-2), Parkinson's Disease Autonomic Outcome Scale (SCOPA-AUT), Global Cognitive Assessment by Montreal Cognitive Assessment (MoCA), the 30-Item Boston Naming Test (BNT), Verbal Fluency Test, Cambridge Automated Neuropsychological Test Battery (CANTAB), Beck Depression Inventory II (BDI-II), Beck Anxiety Inventory (BAI), and Questionnaire for Parkinson's Disease-Impulsive-Compulsive Disorder (QUIP-RS), or any combination thereof.

[0149] In one embodiment, the methods described herein further comprise the step of receiving an initial score of the subject with at least one diagnostic assay described herein prior to introducing any of the rAAV described herein.

[0150] Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS)

[0151] The Unified Parkinson's Disease Rating Scale (UPDRS) is a rating scale that assesses the short-term (less than 1 year) and long-term (greater than 1 year) progression of PD. The UPDRS is a uniform and accepted measurement used in clinical settings that allows clinicians to objectively track the progression of a patient's symptoms. The test consists of six parts and can be either self-administered or administered by a clinician / researcher. The six parts include - Part I: Assessment of psychology, behavior and emotions, including intellectual disability, thinking disorder, motivation / initiative, depression; Part II: Self-evaluation of activities of daily living (ADL) - speech, drooling, swallowing, writing, cutting vegetables, dressing, hygiene, turning over in bed, falls, freezing, walking, tremor, feeling unwell; Part III: Clinician scoring of monitored movement assessment, including speech, facial expression, tremor at rest, action tremor, rigidity, finger tapping, hand movement, hand pronation and supination, leg flexibility, getting up from a chair, posture, gait, postural stability, body movement slowness; Part IV: Complications of treatment, including duration of movement disorder, disability of movement disorder, pain of movement disorder, morning dystonia, OFF-predictable, OFF-unpredictable, OFF-sudden, OFF-duration, anorexia-nausea-vomiting, sleep disturbance, symptomatic orthostatic; Part V: Hoehn and Yahr staging of PD severity; Part VI: Schwab and British ADL scales.

[0152] The Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) is a rating scale (i.e., from 0-272) used to assess short-term (less than 1 year) and long-term (greater than 1 year) progression of PD. Historically, researchers have used the UPDRS to measure the treatment effect of a given therapy in a uniform and accepted rating system. The MDS-UPDRS is an updated version of the UPDRS that extracts aspects of non-motor function from each subcategory. MDS-UPDRS Part I, entitled "Non-motor Experiences of Daily Life," includes Part IA (involving some behaviors assessed by the researcher based on all relevant information from the patient and caregiver) and Part IB (completed by the patient with or without the assistance of a caregiver, but independent of the researcher). MDS-UPDRS Part II is the same as Part II of the original UPDRS, but has been renamed "Motor Experiences of Daily Life," separating 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 the UPDRS and includes only "Motor Complications." The total score of MDS-UPDRS is the sum of Part I, Part II, Part III (in the Off state) and Part IV, which provides a score for disease severity and progression because it provides a sub-score derived from a function and a scorer. Those of ordinary skill in the art will be able to correctly conduct an MDS-UPDRS survey and instruct the subject to conduct the self-management portion of the MDS-UPDRS survey to determine, for example, an initial MDS-UPDRS score (i.e., before applying rAAV as described herein) and a second MDS-UPDRS score (i.e., the MDS-UPDRS score after applying rAAV, for example, at least 6 months or at least 12 months immediately following rAAV as described herein). It is understood that clinicians can determine additional MDS-UPDRS scores (e.g., the third, fourth, fifth, etc.) as needed. It is further understood that the initial and second MDS-UPDRS scores can be any separate part (e.g., Part I, Part II, Part III, Part IV) or a total score of MDS-UPDRS in an MDS-UPDRS survey.

[0153] In one embodiment, the methods described herein further include the step of determining the subject's initial MDS-UPDRS score prior to the introduction of any rAAV described herein. In one embodiment, the methods described herein further include the step of receiving the subject's initial MDS-UPDRS score prior to the introduction of any rAAV described herein, i.e., receiving an initial MDS-UPDRS score previously determined by a technician without rAAV administration.

[0154] In one embodiment, the subject is mildly affected by PD. As used herein, "mildly affected" refers to a subject with an initial MDS-UPDRS score of less than 32.

[0155] In one embodiment, the subject mildly affected by PD had an initial MDS-UPDRS score of less than 32 prior to the introduction of rAAV and was diagnosed with PD less than 5 years prior to the introduction of rAAV.

[0156] In one embodiment, the MDS-UPDRS refers to the MDS-UPDRS III score.

[0157] In one embodiment, the subject is moderately affected by PD. As used herein, "moderately affected" refers to a subject with an initial MDS-UPDRS score equal to or greater than 32.

[0158] In one embodiment, the subject moderately affected by PD has an initial MDS-UPDRS score (eg, 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.

[0159] In one embodiment, the methods described herein further comprise the step of determining a second MDS-UPDRS score at least 6 months or at least 12 months following administration / introduction of rAAV.

[0160] In one embodiment, the methods described herein further comprise the step of determining a second MDS-UPDRS score at least 6 months immediately following administration / introduction of rAAV to a subject moderately affected by PD (ie, a subject having an initial MDS-UPDRS score equal to or greater than 32).

[0161] In one embodiment, the methods described herein further comprise the step of determining a second MDS-UPDRS score at least 12 months following administration / introduction of rAAV to a subject mildly affected by PD (ie, a subject having an initial MDS-UPDRS score of less than 32).

[0162] In one embodiment, a subject mildly affected by PD does not show an increase in its initial MDS-UPDRS score within at least 12 months immediately following the introduction of any rAAV described herein. In one embodiment, a subject mildly affected by PD does not show an increase in its initial MDS-UPDRS score within 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 more immediately following the introduction of any rAAV described herein.

[0163] In one embodiment, at least 12 months immediately following the introduction of any of the rAAVs described herein, a subject who is mildly affected by PD does not show a significant increase in his initial MDS-UPDRS score. As used herein, "significant increase" refers to an increase of no more than 10% of the initial MDS-UPDRS score. In one embodiment, 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 the introduction of any of the rAAVs described herein, a subject who is mildly affected by PD does not show a significant increase in his initial MDS-UPDRS score.

[0164] In one embodiment, the initial MDS-UPDRS score of a subject mildly affected by PD is stable within at least 6 months immediately following the introduction of any rAAV described herein. As used herein, "stable" refers to an increase or decrease in the initial MDS-UPDRS score of no more than 10%, i.e., a second MDS-UPDRS score of no more than + / - 10% of its initial MDS-UPDRS score. In one embodiment, 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 the introduction of any rAAV described herein, a subject mildly affected by PD shows stability in its initial MDS-UPDRS score.

[0165] In one embodiment, at least 12 months immediately following the introduction of any rAAV described herein, a subject mildly affected by PD exhibits a decrease in its initial MDS-UPDRS score. In one embodiment, 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, 23 months, 24 months or longer immediately following the introduction of any rAAV described herein, a subject mildly affected by PD exhibits a decrease in its initial MDS-UPDRS score. In one embodiment, the initial MDS-UPDRS score 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, 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 more.

[0166] In one embodiment, at least 6 months immediately following the introduction of any rAAV described herein, a subject moderately affected by PD exhibits a reduction in its initial MDS-UPDRS score. In one embodiment, 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 the introduction of any rAAV described herein, a subject moderately affected by PD exhibits a reduction in its initial MDS-UPDRS score. In one embodiment, the reduction in the initial MDS-UPDRS score is at least 20%. In one embodiment, the initial MDS-UPDRS score is reduced 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%, 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; or decreased 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, 31 points, 32 points, 33 points, 34 points, 35 points, 36 points, 37 points, 38 points, 39 points, 40 points, 41 points, 42 points, 43 points, 44 points, 45 points, 46 points, 47 points, 48 ​​points, 49 points, 50 points, 51 points, 52 points, 53 points, 54 points, 55 points, 56 points, 57 points, 58 points, 59 points, 60 points, 61 points, 62 points, 63 points, 64 points, 65 points, 66 points, 67 points, 68 points, 69 points, 70 points, 71 points, 72 points, 73 points, 74 points, 75 points, 76 points, 77 points, 78 points, 79 points, 80 points, 81 points, 82 points,83 points, 84 points, 85 points, 86 points, 87 points, 88 points, 89 points, 90 points, 91 points, 92 points, 93 points, 94 points, 95 points, 96 points, 97 points, 98 points, 99 points, 100 points, 101 points, 102 points, 103 points, 104 points, 105 points, 106 points, 107 points, 108 points, 109 points, 110 points, 111 points, 112 points, 113 points, 114 points, 115 points, 116 points, 117 points, 118 points, 119 points, 120 points, 1 21 points, 122 points, 123 points, 124 points, 125 points, 126 points, 127 points, 128 points, 129 points, 130 points, 131 points, 132 points, 133 points, 134 points, 135 points, 136 points, 137 points, 138 points, 139 points, 140 points, 141 points, 142 points, 143 points, 144 points, 145 points, 146 points, 147 points, 148 points, 149 points, 150 points, 151 points, 152 points, 153 points, 154 points, 155 points, 1 56 points, 157 points, 158 points, 159 points, 160 points, 161 points, 162 points, 163 points, 164 points, 165 points, 166 points, 167 points , 168 points, 169 points, 170 points, 171 points, 172 points, 173 points, 174 points, 175 points, 176 points, 177 points, 178 points, 179 points, 180 points, 181 points, 182 points, 183 points, 184 points, 185 points, 186 points, 187 points, 188 points, 189 points, 190 points, 191 points, 192 points, 193 points, 194 points, 195 points, 196 points, 197 points, 198 points, 199 points, 200 points, 201 points, 202 points , 203 points, 204 points, 205 points, 206 points, 207 points, 208 points, 209 points, 210 points, 211 points, 212 points, 213 points, 21 4 points, 215 points, 216 points, 217 points, 218 points, 219 points, 220 points, 221 points, 222 points, 223 points, 224 points, 225 points, 2 26 points, 227 points, 228 points, 229 points, 230 points, 231 points, 232 points, 233 points, 234 points, 235 points, 236 points, 237 points, 238 points, 239 points, 240 points, 241 points, 242 points, 243 points, 244 points, 245 points, 246 points, 247 points, 248 points, 249 points, 250 points, 251 points, 252 points, 253 points, 254 points, 255 points, 256 points, 257 points, 258 points, 259 points, 260 points, 261 points, 262 points, 263 points, 264 points, 265 points, 266 points, 267 points, 268 points, 269 points, 270 points, 271 points or 272 points.

[0167] In one embodiment, a subject moderately affected by PD does not show an increase in its initial MDS-UPDRS score for at least 6 months immediately following the introduction of any rAAV described herein. In one embodiment, a subject moderately affected by PD does not show an increase in its 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 more immediately following the introduction of any rAAV described herein.

[0168] In one embodiment, at least 6 months after the introduction of any of the rAAVs described herein, the subject with moderate PD does not show a significant increase in its initial MDS-UPDRS score. As used herein, "significant increase" refers to an increase of no more than 10% of the initial MDS-UPDRS score. In one embodiment, 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 after the introduction of any of the rAAVs described herein, the subject with moderate PD does not show a significant increase in its initial MDS-UPDRS score. Non-Motor Symptom Scale (NMSS)

[0169] The Non-Motor Symptom Scale (NMSS) is a 30-item self-administered questionnaire that assesses various non-motor symptoms in subjects with PD. Non-motor symptoms of PD typically include neuropsychiatric symptoms, sleep disturbances, 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 along nine dimensions: cardiovascular, sleep / fatigue, mood / cognition, perceptual problems, attention / memory, gastrointestinal, urinary, sexual function, and other. The scale can be used for patients at all stages of PD. The score for each item is based on a combination of severity (0 to 3) and frequency score (1 to 4) to capture symptoms that are severe but relatively infrequent, or symptoms that are less severe but persistent. The total NMSS score ranges from 0 to 360.

[0170] In one embodiment, the methods described herein further include the step of determining an initial NMSS score of the subject prior to the introduction of any of the rAAV described herein. In one embodiment, the methods described herein further include the step of receiving an initial NMSS score of the subject prior to the introduction of any of the rAAV described herein, i.e., receiving an initial NMSS score previously determined by a clinician / researcher without rAAV administration.

[0171] In one embodiment, the methods described herein further comprise the step of determining a second NMSS score at least 6 months or at least 12 months following administration / introduction of rAAV.

[0172] In one embodiment, at least 6 months after the introduction of any rAAV described herein, the subject shows a reduction in the initial NMSS score. In one embodiment, 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 after the introduction of any rAAV described herein, the subject shows a reduction in its initial NMSS score. In one embodiment, the initial NMSS score is reduced by at least 20%. In one embodiment, an initial NMSS score is reduced 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%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 5%, 96%, 97%, 98%, 99% or more; or decreased 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, 31 points, 32 points, 33 points, 34 points, 35 points, 36 points, 37 points, 38 points, 39 points, 40 points, 41 points, 42 points, 43 points, 44 points , 45 points, 46 points, 47 points, 48 ​​points, 49 points, 50 points, 51 points, 52 points, 53 points, 54 points, 55 points, 56 points, 57 points, 58 points, 59 points, 60 points, 61 points, 62 points, 63 points, 64 points, 65 points, 66 points, 67 points, 68 points , 69 points, 70 points, 71 points, 72 points, 73 points, 74 points, 75 points, 76 points, 77 points, 78 points, 79 points, 80 points, 81 points, 82 points, 83 points, 84 points, 85 points, 86 points, 87 points, 88 points, 89 points, 90 points, 91 points, 92 points,93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 102 minutes, 103 minutes, 104 minutes, 105 minutes, 106 minutes, 107 minutes, 108 minutes, 109 minutes, 110 minutes, 111 minutes, 112 minutes, 113 minutes, 114 minutes, 115 minutes, 116 minutes, 117 minutes, 118 minutes, 1 19 minutes, 120 minutes, 121 minutes, 122 minutes, 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes, 143 minutes, 1 44 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes, 150 minutes, 151 minutes, 152 minutes, 153 minutes, 154 minutes, 155 minutes, 156 minutes, 157 minutes, 158 minutes, 159 minutes, 160 minutes, 161 minutes, 162 minutes, 163 minutes, 164 minutes, 165 minutes, 166 minutes, 167 minutes, 168 minutes, 1 69 minutes, 170 minutes, 171 minutes, 172 minutes, 173 minutes, 174 minutes, 175 minutes, 176 minutes, 177 minutes, 178 minutes, 179 minutes, 180 minutes, 181 minutes, 182 minutes, 183 minutes, 184 minutes, 185 minutes, 186 minutes, 187 minutes, 188 minutes, 189 minutes, 190 minutes, 191 minutes, 192 minutes, 193 minutes, 1 94 minutes, 195 minutes, 196 minutes, 197 minutes, 198 minutes, 199 minutes, 200 minutes, 201 minutes, 202 minutes, 203 minutes, 204 minutes, 205 minutes, 206 minutes, 207 minutes, 208 minutes, 209 minutes, 210 minutes, 211 minutes, 212 minutes, 213 minutes, 214 minutes, 215 minutes, 216 minutes, 217 minutes, 218 minutes, 219 minutes, 220 minutes, 221 minutes, 222 minutes, 223 minutes, 224 minutes, 225 minutes, 226 minutes, 227 minutes, 228 minutes, 229 minutes, 230 minutes, 231 minutes, 232 minutes, 233 minutes, 234 minutes, 235 minutes, 236 minutes, 237 minutes, 238 minutes, 239 minutes, 240 minutes, 241 minutes, 242 minutes, 243 minutes, 244 minutes, 245 minutes, 246 minutes, 247 minutes, 248 minutes, 249 minutes, 250 minutes, 251 minutes, 252 minutes, 253 minutes, 254 minutes, 255 minutes, 256 minutes, 257 minutes, 258 minutes, 259 minutes, 260 minutes, 261 minutes, 262 minutes, 263 minutes, 264 minutes, 265 minutes, 266 minutes, 267 minutes, 268 minutes, 269 ​​minutes, 270 minutes, 271 minutes, 272 minutes, 273 minutes, 274 minutes, 275 minutes, 276 minutes, 277 minutes, 278 minutes, 279 minutes, 280 minutes, 281 minutes, 282 minutes, 283 minutes, 284 minutes, 285 minutes, 286 minutes, 287 minutes, 288 minutes, 289 minutes, 290 minutes, 291 minutes, 292 minutes, 293 minutes,294 points, 295 points, 296 points, 297 points, 298 points, 299 points, 300 points, 301 points, 302 points, 303 points, 304 points, 305 points, 306 points, 307 points, 308 points, 309 points, 310 points, 311 points, 312 points, 313 points, 314 points, 315 points, 316 points, 317 points, 318 points, 319 points, 320 points, 321 points, 322 points, 323 points, 324 points, 325 points, 326 points, 32 7 points, 328 points, 329 points, 330 points, 331 points, 332 points, 333 points, 334 points, 335 points, 336 points, 337 points, 338 points, 339 points, 340 points, 341 points, 342 points, 343 points, 344 points, 345 points, 346 points, 347 points, 348 points, 349 points, 350 points, 351 points, 352 points, 353 points, 354 points, 355 points, 356 points, 357 points, 358 points, 359 points or 360 points.

[0173] In one embodiment, the subject does not show an increase in its initial NMSS score for at least 6 months after the introduction of any rAAV described herein. In one embodiment, the subject does not show an increase in its 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 more after the introduction of any rAAV described herein.

[0174] In one embodiment, the subject does not show a significant increase in his / her initial NMSS score for at least 6 months after the introduction of any of the rAAVs described herein. As used herein, "significant increase" refers to an increase of no more than 10% of the initial NMSS score. In one embodiment, the subject does not show a significant increase in his / her 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 more after the introduction of any of the rAAVs described herein.

[0175] In one embodiment, the subject exhibits stability of its initial NMSS score for at least 6 months immediately following the introduction of any rAAV described herein. As used herein, "stabilization" refers to an increase or decrease of no more than 10% in the initial NMSS score. In one embodiment, the subject exhibits stability of its 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 the introduction of any rAAV described herein. Parkinson's Disease Questionnaire (PDQ-39) score

[0176] The Parkinson's Disease Questionnaire (PDQ-39) is a 39-item self-administered questionnaire with eight subscales: mobility, activities of daily living (ADL), emotional well-being, stigma, social support, cognitive impairment, communication, and somatic complaints. Scores on the questionnaire range from 0 to 100, with lower scores indicating a better perception of health status and higher scores indicating a more severe disease state. The PDQ-39 can be used as a reliable tool to measure quality of life in individuals with PD. Its inclusion in a comprehensive assessment is particularly important because treatment tends to focus on motor deficits and basic features rather than other clinical features, including depression, cognitive impairment, and fall risk, which can significantly affect quality of life.

[0177] In one embodiment, the methods described herein further include the step of determining the subject's initial PDQ-39 score prior to the introduction of any of the rAAV described herein. In one embodiment, the methods described herein further include the step of receiving the subject's initial PDQ-39 score prior to the introduction of any of the rAAV described herein, i.e., receiving an initial PDQ-39 score previously determined by a clinician / researcher without rAAV administration.

[0178] In one embodiment, the methods described herein further comprise the step of determining a second PDQ-39 score at least 6 months or at least 12 months after the administration / introduction of rAAV.

[0179] In one embodiment, the subject exhibits a decrease in their initial PDQ-39 score at least 6 months after the introduction of any rAAV described herein. In one embodiment, the subject's initial PDQ-39 score is reduced 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 more after the introduction of any rAAV described herein. In one embodiment, the initial PDQ-39 score is reduced by at least 20%.In one embodiment, the initial PDQ-39 score is reduced 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%, 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%, 100% 9% or more; or a decrease of 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, 31 points, 32 points, 33 points, 34 points, 35 points, 36 points, 37 points, 38 points, 39 points, 40 points, 41 points, 42 points, 43 points, 44 points, 45 points, 46 points, 47 points, 48 ​​points, 49 points, 50 points , 51 points, 52 points, 53 points, 54 points, 55 points, 56 points, 57 points, 58 points, 59 points, 60 points, 61 points, 62 points, 63 points, 64 points, 65 points, 66 points, 67 points, 68 points, 69 points, 70 points, 71 points, 72 points, 73 points, 74 points, 75 points, 76 points, 77 points, 78 points, 79 points, 80 points, 81 points, 82 points, 83 points, 84 points, 85 points, 86 points, 87 points, 88 points, 89 points, 90 points, 91 points, 92 points, 93 points, 94 points, 95 points, 96 points, 97 points, 98 points, 99 points or 100 points.

[0180] In one embodiment, the subject does not show an increase in its initial PDQ-39 score for at least 6 months after the introduction of any rAAV described herein. In one embodiment, the subject does not show an increase in its 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 more after the introduction of any rAAV described herein.

[0181] In one embodiment, the subject does not show a significant increase in their initial PDQ-39 score for at least 6 months after the introduction of any of the rAAVs described herein. As used herein, "significant increase" refers to an increase of no more than 10% of the initial PDQ-39 score. In one embodiment, the subject does not show a significant increase in 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 more after the introduction of any of the rAAVs described herein.

[0182] In one embodiment, the subject exhibits stability of their initial PDQ-39 score for at least 6 months immediately following the introduction of any rAAV described herein. As used herein, "stable" refers to an increase or decrease of no more than 10% in the initial PDQ-39 score. In one embodiment, the subject exhibits stability 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 more immediately following the introduction of any rAAV described herein.

[0183] In one embodiment, before and after administration / introduction of any of the rAAVs described herein, the subject is subjected to at least one standard clinical test to assess motor symptoms and function in PD patients. Such assessments include MDS-UPDRS part 3; modified Hoehn and Yahr; stand-walk-sit; 9-hole pegboard dexterity test; and standing balance test.

[0184] The modified Hoehn & Yahr scale is a 5-point scale used to measure the overall level of impairment caused by PD ( Hoehn and Yahr, 1967 ).

[0185] The Stand-Walk-Sit (SWS) is a postural stability and gait assessment that involves standing up from a chair, walking in a straight line for 7 meters (23 feet), turning around and walking back to a chair to sit. The timer will stop when the subject's back touches the back of the chair. Subjects will perform this test in both the OFF and ON medication states as practically defined. The SWS test will be video recorded and can be combined with the MDS-UPDRS assessment.

[0186] The 9-hole pegboard dexterity test is a simple test of manual dexterity; it records the time required for participants to accurately place and remove nine plastic pegs on a plastic pegboard.

[0187] The standing balance test assesses a person's ability to orient the body in space, maintain an upright posture under static and dynamic conditions, and move and walk without falling. It involves the participant assuming and maintaining up to five postures for 50 seconds each. The sequence of postures includes: eyes open on a solid surface, eyes closed on a solid surface, eyes open on a foam surface, eyes closed on a foam surface, and a tandem posture with eyes open on a solid surface. Detailed stopping rules are in place to ensure that participants safely perform these progressively demanding postures. Postural sway is recorded for each posture using an accelerometer worn by the participant at the waist. This test takes approximately 7 minutes to perform.

[0188] In one embodiment, prior to administration / introduction, the subject is subjected to any of the standard clinical tests described herein, and again at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction. In one embodiment, at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction, the subject shows an improved score in any of the standard clinical tests. In one embodiment, 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%, 87%, 88%, 89%, 90%, , 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0189] In one embodiment, the severity of dyskinesias is measured using the Unified Dyskinesia Rating Scale (UDysRS). This scale evaluates involuntary movements commonly associated with treated PD. It consists of two main parts: historical [Part 1 (ON-dyskinesias) and Part 2 (OFF-dystonias)] and objective [Part 3 (Impairments) and Part 4 (Disabilities)]. ON-dyskinesias refer to choreic, dystonic movements that describe patients jerking or twisting when PD medications are effective. OFF-dystonias refer to muscle spasms or muscle twitches that may be painful or occur when PD medications are not taken or are not effective. Throughout the assessment, emphasis is placed on these two forms of movement, and it must be constantly emphasized that Parkinson's disease itself and tremor must be excluded from the assessment to influence the rating (Goetz 2008).

[0190] In one embodiment, the subject completes a subject-reported PD motor log. Hauser and colleagues developed a paper motor log for assessing PD motor symptoms over a 24-hour period (Hauser 2004). The log records the duration of the participant's ON state without dyskinesia, ON without bothersome dyskinesia, ON with bothersome dyskinesia, OFF state, or sleep at half-hour intervals. Participants are required to record this information at half-hour intervals throughout the day.

[0191] In one embodiment, the subject performs a UDysRS and / or PD movement log before administration / introduction and again at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction. In one embodiment, the subject shows an improvement in UDysRS and / or PD movement score at least 3 months, 6 months, 9 months, 12 months or later immediately after administration / introduction. In one embodiment, 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%, 87%, 88%, 89%, 90%, , 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0192] In one embodiment, a wearable activity monitor (e.g. ) to assess daily activities for at least 18 months immediately following administration / introduction.

[0193] In one embodiment, the subject receives global disability and quality of life assessment (Global Disability and Quality of Life Assessment) before administration / introduction or immediately after administration / introduction for at least 3 months, 6 months, 9 months, 12 months or later. Exemplary global disability and quality of life assessments include global impressions (CGI and PGI), brief odor identification test (BSIT), Parkinson's disease sleep scale (PDSS-2), Parkinson's disease autonomic outcome scale (SCOPA-AUT).

[0194] The Clinical Global Impression (CGI) provides an overall summary measure determined by the clinician that takes into account all available information, including knowledge of the patient's medical history, psychosocial environment, symptoms, behaviors, and the impact of symptoms on the patient's functional ability. The CGI actually consists of 2 companion 1-item measures that assess the following: (a) severity of illness, on a scale of 1 to 7; and (b) change from the start of treatment, measured on a similar 7-point scale. The Patient Global Impression (PGI) 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 instrument that will be completed independently by the participant at home, either on a paper assessment or by answering on a tablet provided by the sponsor.

[0195] The Brief Smell Identification Test (BSIT) is a 12-item test of olfactory system function using a "scratch and sniff" strip. After releasing each odor by scratching with a pen-like object, participants take a sniff and then answer multiple-choice questions with four options related to the odor. This is a self-directed assessment.

[0196] The Parkinson's Disease Sleep Scale (PDSS-2) uses a visual analog scale to address 15 common symptoms associated with sleep disturbances in PD. Subjects complete 15 questions based on their experiences during the previous week. This is a self-directed measure.

[0197] The Scale for Autonomic Outcomes in Parkinson's Disease-AUT (SCOPA-AUT) is a 26-item self-report questionnaire on autonomic function. Questions cover upper and lower gastrointestinal function, urinary function, cardiovascular circulatory function, sexual behavior, and various other autonomic problems. This is a self-directed symptom inventory.

[0198] In one embodiment, before administration / introduction, any one of global disability and quality of life assessment described herein is performed to the subject, and at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction are performed again. In one embodiment, at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction, the subject shows improved scores in global disability and quality of life assessment. In one embodiment, 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%, 87%, 88%, 89%, 90%, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0199] In one embodiment, the subject undergoes neuropsychological testing prior to or immediately after administration / introduction for at least 3 months, 6 months, 9 months, 12 months or later. Exemplary neuropsychological testing includes a global cognitive assessment by the Montreal Cognitive Assessment (MoCA); 30 Boston Naming Tests (BNT); Verbal Fluency Test; Cambridge Automated Battery of Neuropsychological Tests (CANTAB); Beck Depression Inventory II (BDI-II); Beck Anxiety Inventory (BAI); and the Questionnaire for Impulsive-Compulsive Disorder in Parkinson's (QUIP-RS).

[0200] The Montreal Cognitive Assessment (MoCA) was designed as a rapid screening tool for mild cognitive impairment. It assesses different cognitive domains: attention and concentration, executive function, memory, language, visual-constructive skills, conceptual thinking, calculation, and orientation. The BNT evaluates possible oppositional naming and language deficits in PD. Participants are presented with stimuli of line drawings of objects that are increasingly difficult to name and are asked to respond within 20 seconds. Scoring is based on the number of correct responses provided spontaneously, the number of clues given, and the number of responses following clues. This brief verbal assessment will be completed with remote guidance from the research team via video call.

[0201] In the verbal fluency test, subjects are asked to record as many words as possible from a pre-selected alphabet and category within 60 seconds, with instructions not to use proper nouns or words that have changed only by suffixes. The number of correct answers in each category is scored.

[0202] The Cambridge Neuropsychological Automated Test Battery (CANTAB) was developed to include sensitive and objective measures of cognitive function in the evaluation of neurodegenerative disorders. Cognitive assessments were developed to detect changes in neuropsychological performance over time and as an effect of intervention. Multiple assessments have been validated in PD, focusing on the domains of working memory, episodic memory, executive function, planning, and information processing. All tasks will be completed by the subject using a tablet that includes collection of response times. The CANTAB allows for the electronic capture of outcome measures that have been validated in a variety of neurodegenerative diseases. The CANTAB can be completed either self-directed or remotely by the study coordinator or investigator on a tablet provided by the sponsor.

[0203] Reaction time (RTI) assesses mental response time as well as motor timing, reaction time, response accuracy, and impulsivity.

[0204] The Motor Screening Task (MOT) provides an overall assessment of whether sensorimotor deficits or lack of understanding limit the ability to collect valid data from participants. This task measures the participant's response speed and accuracy in pointing to the center of an object on the screen.

[0205] One Touch Stockings of Cambridge (OTS) assesses executive function by evaluating spatial planning and working memory subdomains. Participants are asked to create a 3D arrangement on the screen with a prescribed number of actions. This is measured by the number of problems solved with the first choice, mean correct choice, mean response latency, and mean correction latency.

[0206] Paired Associate Learning (PAL) is a type of visual memory in which new learning abilities are assessed through this task. Participants are asked to select boxes of increasing difficulty levels in a pre-specified pattern. Results are measured by the number of errors made, the number of trials required to correctly identify the pattern, the stages completed, and the memory score. Estimated time to complete: 8 minutes.

[0207] Pattern recognition memory (PRM) is a test of visual pattern recognition memory in a 2-choice forced discrimination paradigm. Unrelated words are presented via an audio recording and participants recall as many words as possible either immediately or after a delay. This is measured by the number and percentage of correct trials and the latency of responding.

[0208] The Multi-Task Test (MTT) is an assessment of an individual's ability to interpret and manage conflicting information and correctly ignore task-irrelevant information. The rule changes between trials place a higher cognitive demand on the participants to reveal underlying deficits in executive function, an area often affected in PD patients. This is measured by response latency and number of errors.

[0209] The Beck Depression Inventory II (BDI-II) may be administered as part of the eligibility assessment, such as at screening, and participants with a screening score >20 will be excluded from the study and referred to their primary care physician for psychiatric evaluation and treatment. Guidelines for BDI-II scoring are provided, suggesting that the threshold be adjusted based on the characteristics of the sample and the purpose for which the BDI-I is being used. In general, a total BDI-II score of 0-13 indicates minimal depression, 1419 indicates mild depression, 20-28 indicates moderate depression, and 29-63 indicates severe depression. If the BDI-II score after treatment is greater than 28, the participant will continue in the study but will be referred to their primary care physician for psychiatric evaluation and treatment. This assessment is a self-report measure.

[0210] The Beck Anxiety Inventory (BAI) is a 21-question multiple-choice self-report scale used to measure the severity of anxiety in children and adults. This assessment is a self-report measure.

[0211] The PD Questionnaire-Obsessive-Compulsive Rating Scale (QUIP-RS) is a rating scale designed to measure symptom severity and support the diagnosis of impulse control disorders and related disorders in PD. The 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 (compulsive meaningless activities) and / or complex (hobbyism) repetitive behaviors, and compulsive excessive drug use (dopamine dysregulation syndrome). This assessment is a self-report measure.

[0212] In one embodiment, prior to administration / introduction, the subject is subjected to any of the neuropsychological tests described herein and again at least 3 months, 6 months, 9 months, 12 months or later immediately after administration or introduction. In one embodiment, at least 3 months, 6 months, 9 months, 12 months or later immediately after administration / introduction, the subject exhibits improved scores on the neuropsychological tests. In one embodiment, 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%, 87%, 88%, 89%, 90%, , 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0213] In one embodiment, at least 6 months immediately after introduction or application, there are no serious adverse events in the subject. In one embodiment, at least 12 months immediately after introduction or application, there are no serious adverse events in the subject. In one embodiment, 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 after introduction or application, there are no serious adverse events in the subject. Serious adverse events include, but are not limited to, blood and lymphatic system disorders (such as anemia, decreased lymphocyte count, and leukocytosis); gastrointestinal disorders (such as dyspepsia, dysphagia, and constipation); local edema; fatigue; falls; neck bruises; increased aspartate aminotransferase; decreased platelet count; prolonged activated partial thromboplastin time; weight loss; blood lactate dehydrogenase; metabolic and nutritional disorders (such as hyperglycemia, hypocalcemia, hypoalbuminemia, hypophosphatemia, hypernatremia, hypoglycemia, hyperkalemia, and hyponatremia); musculoskeletal and connective tissue disorders (such as pain in the upper back, neck, back, chest wall, and extremity); nervous system disorders (e.g., headache, involuntary movements, memory impairment, sleep disturbances-increased dreaming, sensory neuropathy, and hypersomnia); psychiatric disorders (e.g., hallucinations, depression, insomnia, and impulse control disorders); renal and urinary disorders (e.g., urinary incontinence); respiratory, thoracic, and mediastinal disorders (e.g., cough and productive cough); skin and subcutaneous tissue disorders (e.g., skin ulcers); surgical and medical procedures (e.g., carpal tunnel release); transient paresthesias; transient tremors; hypotension; and vascular disorders (e.g., hypertension). Nucleic Acids

[0214] In some aspects, the present disclosure provides Isolated nucleic acids expressing a GDNF gene or a GDNF gene product. A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, the nucleic acids disclosed herein and the proteins translated therefrom are isolated. As used herein, the term "isolated" refers to artificial production. As used herein with respect to nucleic acids, the term "isolated" refers to: (i) amplification in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinant production by cloning; (iii) purification, such as by cleavage and gel separation; or (iv) synthesis by, for example, chemical synthesis. An isolated nucleic acid is one that is readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for 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 that exists in its native state in a natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid isolated in a cloning or expression vector is not pure because it may constitute only a small fraction of the material in the cell in which it is located. However, as the term is used herein, such a nucleic acid is isolated because it is readily manipulated by standard techniques known to those of ordinary skill in the art. As used herein with respect to a protein or peptide, the term "isolated" refers to a protein or peptide that has been separated from its natural environment or artificially produced (eg, by chemical synthesis, by recombinant DNA technology, etc.).

[0215] Those of ordinary skill will also recognize that conservative amino acid substitutions can be made to provide functionally equivalent capsid protein variants or homologs. In some aspects, the present disclosure encompasses sequence changes that produce conservative amino acid substitutions. As used herein, conservative amino acid substitutions refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., ed., John Wiley & Sons, Inc, New York. Conservative substitutions of amino acids include substitutions between 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. Thus, conservative amino acid substitutions may be made to the amino acid sequences of the proteins and polypeptides disclosed herein.

[0216] The isolated nucleic acid described herein can be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described in the present disclosure comprises a region (e.g., a first region) containing a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) can be packaged into a capsid composed of capsid proteins and administered to a subject and / or delivered to a selected target cell. A "recombinant AAV (rAAV) vector" is typically composed of at least a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). As described elsewhere in the present disclosure, a transgene may include a region encoding, for example, a protein and / or an expression control sequence (e.g., a poly-A tail).

[0217] In general, the length of the ITR sequence is about 145bp. In various aspects, the left ITR and the right ITR are independently 145bp or less, or 130bp or less. "Independently" means that the length of the left ITR and the right ITR can be the same or different. As a non-limiting example, the left ITR and the right ITR can be independently 145bp, 130bp, 128bp, 124bp or 119bp. Although it is allowed to carry out a certain degree of minor modification of these sequences, it is preferred to use the entire sequence encoding ITR in the nucleic acid sequence. The ability to modify these ITR sequences is within the technical scope of the art. (For example, see textbooks, such as Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol, 70: 520 532 (1996)). Examples of such nucleic acid molecules are "cis-acting" plasmids containing transgenes in which the selected transgenic sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., rAAV vector) comprises at least one ITR, the serotype of which is selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

[0218] In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) containing a second AAV-ITR. In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal resolution site (TRS). The term "lack of a terminal resolution site" may refer to an AAV ITR comprising a mutation (e.g., a sense mutation such as a non-synonymous mutation or a missense mutation) that eliminates the function of the ITR terminal resolution site (TRS), or to a truncated AAV ITR (e.g., an ATRS ITR) lacking a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors comprising ITRs lacking functional TRSs are generated from self-complementary rAAV vectors, for example as described in McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0219] In addition to the elements identified above for recombinant AAV vectors, the vectors also contain conventional control elements that are operably linked to the transgenic elements to allow them to be transcribed, translated and / or expressed in cells transfected with the vector or infected with the virus produced by the methods described herein. As used herein, "operably linked" sequences include expression control sequences adjacent to the gene of interest (i.e., GDNF) and expression control sequences that control the gene of interest by trans or remote action. 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 sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. Many expression control sequences are known and available in the art, including natural, constitutive, inducible and / or tissue-specific promoters.

[0220] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are considered to be operably linked when they are covalently linked so that the expression or transcription of the nucleic acid sequence is placed under the influence or control of the regulatory sequence. If it is desired to translate the nucleic acid sequence into a functional protein, if induction of a promoter in a regulatory sequence (e.g., a 5' regulatory sequence) results in transcription of the coding sequence, and if the nature of the connection between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein, then the two DNA sequences are said to be operably linked. Thus, when the promoter region is able to affect the transcription of the DNA sequence, the promoter region is operably linked to the nucleic acid sequence so that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, when two or more coding regions are linked in such a way that they are transcribed from a common promoter so that two or more proteins translated in frame are expressed. In some embodiments, operably linked coding sequences produce fusion proteins. Promoter

[0221] In various embodiments, the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., promoters, etc.). 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 improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. Many expression control sequences are known and can be used in the art, including natural, constitutive, inducible, and / or tissue-specific promoters.

[0222] "Promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate specific transcription of a gene (e.g., the coding sequence of a gene). The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression.

[0223] For nucleic acids encoding proteins, polyadenylation sequences are usually inserted after the transgenic sequence, i.e., downstream of the transgenic sequence or 3' of the transgenic sequence, and before the 3'AAV ITR sequence. The rAAV constructs that can be used for the present disclosure can also include introns that are ideally located between the promoter / enhancer sequence and the transgenic. An exemplary intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. Another vector element that can be used is an internal ribosome entry site (IRES). The IRES sequence is used to produce more than one polypeptide from a single gene transcript. The IRES sequence will be used to produce proteins containing more than one polypeptide chain. The selection of these elements and other common vector elements is conventional, and there are many such sequences available [see Sambrook et al., and references cited therein, such as pages 3.18, 3.26, and 16.1716.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the polyprotein; this is a small peptide (about 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996, p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6:198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11:1921-1931; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

[0224] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41: 521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1 alpha promoter [Invitrogen]. In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is a U6 promoter.

[0225] Inducible promoters allow for 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 (such as the acute phase, a specific differentiation state of the cell, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of 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, and the T7 polymerase promoter system (WO98 / 10088), the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repressor system (Gossen ... USA (89: 5547-5551 (1992)), the tetracycline inducible system (Gosson et al., Science, 268: 1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2: 512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech, 15: 239-243 (1997) and Wang et al., Gene Ther., 4: 432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100: 2865-2872 (1997). Other types of inducible promoters that may be useful in this context are promoters that are regulated by specific physiological states (e.g., temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells).

[0226] In another embodiment, the natural promoter of the transgenic will be used. When it is desired that the expression of the transgenic should mimic natural expression, a natural promoter may be preferred. When the expression of the transgenic must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus, a natural promoter may be used. In additional embodiments, other natural expression control elements (such as enhancer elements, polyadenylation sites or Kozak consensus sequences) may also be used to simulate natural expression. As used herein, a "natural promoter" refers to the endogenous promoter of the transgenic.

[0227] In some embodiments, regulatory sequences confer tissue-specific gene expression ability. In some cases, tissue-specific regulatory sequences bind tissue-specific transcription factors, and the tissue-specific transcription factors induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (such as promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include but are not limited to the following tissue-specific promoters: liver-specific thyroxine binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synaptic protein-1 (Syn) promoter, creatine kinase (MCK) promoter, mammal desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter or cardiac troponin T (cTnT) promoter.

[0228] Other exemplary promoters include the β-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)), the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the 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 alpha 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 neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15: 373-84 (1995)), as well as those obvious to the skilled person.

[0229] Nervous system (NS) specific promoters contemplated for use in the present methods and compositions also include those described in International Patent Application Nos. WO / 2022 / 049385 and WO / 2021 / 214443, the contents of which are incorporated herein by reference in their entirety. In some embodiments, 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. In some embodiments, 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 maintaining the NS-specific promoter activity of the promoter of Table 1.

[0230] The 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 herein by reference in their entirety. In some embodiments, the CNS-specific promoter is a promoter of Table 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 2-4. In some embodiments, the CNS-specific promoter is a promoter of Table 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 2-4 and retaining a CNS-specific promoter activity of a promoter of Table 2-4.

[0231] In some embodiments, the nucleic acid comprises one or more cis-regulatory elements (CRE). In some embodiments, the nucleic acid comprises one or more NS-specific CRE or CNS-specific CRE. In some embodiments, the nucleic acid comprises one or more CRE in Table 4-6, or a CRE with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with the CRE in Table 4-6. In some embodiments, the CRE is a CRE in Table 4-6, or a CRE with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with the CRE in Table 4-6 and retains the CRE activity of Table 4-6.

[0232] In some embodiments, CRE may include one or more CRE known in the art. For example, in one embodiment, one or more CRE may be selected from SEQ ID NO: 19-SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 38 in International Patent Application No. WO / 2022 / 049385. For example, in one embodiment, one or more CRE may be selected from: SEQ ID NO: 1-SEQ ID NO: 8 from WO 2019 / 199867A1, SEQ ID NO: 1-SEQ ID NO: 7 from WO 2020 / 076614A1, and SEQ ID NO: 25-SEQ ID NO: 51, SEQ ID NO: 177-SEQ ID NO: 178, SEQ ID NO: 188 from WO 2020 / 097121. The above references are incorporated herein in their entirety by reference.

[0233] Table 1 - NS-specific promoters

[0234] Table 2 - CNS-specific promoters

[0235] Table 3 - Minimal / proximal promoters included in the promoters of Table 2

[0236] Table 4 - Overview of synthetic CNS-specific promoters

[0237] Table 5 - Exemplary CREs

[0238] Table 6 - Cis-regulatory elements (CRE) contained in the promoters of Table 2

[0239] In some embodiments, the promoter is a synapsin (Syn1) promoter (see, e.g., SEQ ID NO: 61). In one aspect, the promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 61, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical. In one aspect, provided herein is a composition comprising a recombinant viral vector comprising a promoter comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 61, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical.

[0240] Synapsin-1 (SEQ ID NO: 61)

[0241] In one embodiment, the nucleic acid further comprises an enhancer sequence that helps drive expression for the CNS, for example, for a specific CNS tissue or cell type. Exemplary enhancer sequences are described in, for example, U.S. Patent Application No. 17 / 283,232; US17 / 291,584; or International Patent Publication Nos. WO2020168279A2, WO2021195591A2, WO2021248085A2, WO2021216778A2; the contents of each are incorporated herein by reference in their entirety.

[0242] In some embodiments, the nucleic acid comprises a transgene encoding a protein. The protein can be a therapeutic protein (e.g., a peptide, protein, or polypeptide that can be used to treat or prevent a disease state in a mammalian subject) or a reporter protein. In some embodiments, the protein is GDNF. In some embodiments, the protein is human GDNF. In some embodiments, the GDNF gene encodes SEQ ID NO: 2 or a protein comprising SEQ ID NO: 2. In some embodiments, the GDNF gene encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity with SEQ ID NO: 2. In some embodiments, therapeutic proteins and genes encoding such proteins can be used to treat or slow the progression of PD.

[0243] Nucleic acids as described herein may further comprise a reporter sequence (e.g., a nucleic acid sequence encoding a reporter protein). The reporter sequence includes, but is not limited to, a DNA sequence encoding beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other substances well known in the art. When relevant to the regulatory element driving its expression, the reporter sequence provides a signal that can be detected by conventional means, including enzymes, radiography, colorimetry, fluorescence or other spectrometry, fluorescence activated cell sorting assays and immunoassays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry. For example, when the marker sequence is a LacZ gene, the presence of the carrier carrying the signal is detected by beta-galactosidase activity assay. When the transgenic is green fluorescent protein or luciferase, the carrier carrying the signal can be visually measured by light generation or color in a photometer. For example, such reporters can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of nucleic acids. GDNF

[0244] 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 motor neurons. In various animal models of Parkinson's disease (PD), GDNF can prevent neurotoxin-induced dopamine neuron death and can promote axonal sprouting, thereby restoring function. Two GDNF splice variants have been described, called pre-(α)pro-GDNF (formerly known as GDNFα) and pre-(β)pro-GDNF (formerly known as GDNFβ) (Suter-Crazzolara and Unsicker, Neuroreport, 5: 2486-2488 (1994)). These splice variants are generated by alternative splicing of GDNF mRNA.

[0245] Many secretory proteins, including neurotrophins, are synthesized as precursors, pre-pro-mature proteins. The pre region, which consists of the ER signal peptide, is cleaved off by signal peptidases during translation, and the pro-mature protein is released into the lumen of the ER immediately after synthesis. Proteolytic cleavage of the mature protein can occur either intracellularly or in the extracellular matrix, or both.

[0246] The pro-mature protein can also remain uncleaved and have different functions from the cleaved mature protein. For example, both mature brain-derived neurotrophic factor (BDNF) and pro-BDNF are secreted by neuronal cells. Mature BDNF binds to the TrkB receptor to induce neuronal survival, differentiation, and synaptic regulation, while pro-BDNF binds to p75 NTR and sortilin receptors to induce apoptosis (for review, see Thomas and Davies, Curr. Biol., 15:262-264 (2005); Teng et al., J. Neurosci., 25:5455-5463 (2005)).

[0247] In scientific texts, the names GDNF mRNA and GDNF protein are used for the full-length pre-(α)pro-GDNF mRNA as well as the mature GDNF protein produced by proteolytic cleavage of (α)pro-GDNF. This mature GDNF protein has been extensively studied, with over 2500 citations to GDNF in PubMed. 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 an effective factor in protecting substantia nigra dopaminergic neurons from toxin-induced degeneration in animal models of PD and can also be used to treat PD patients (reviewed in Airaksinen and Saarma, Nat. Rev. Neurosci. 3:383-394 (2002); and Bespalov and Saarman, Trends Pharmacol. Sci. 28:68-74 (2007)). In addition, GDNF has therapeutic effects in 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. Neurosci., 25: 619-628 (2005); Angelucci et al., Int. J. Neuropsychopharmacol., 6: 225-231 (2003)). GDNF also has important roles outside the nervous system. It acts as a morphogenetic factor in kidney development and regulates the differentiation of spermatogonia (reviewed in Sariola and Saarma, J. Cell Sci. 116: 3855-3862 (2003)).

[0248] In some embodiments, described herein are viral vectors for slowing or inhibiting the progression of PD, wherein the vector comprises a GDNF encoding nucleic acid. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector (eg, rAAV).

[0249] In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. In some embodiments, the viral vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to SEQ ID NO: 2.

[0250] In some embodiments, the viral vector comprises a sequence having 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.

[0251] The term "gene" refers to a polynucleotide containing at least one open reading frame, which is capable of encoding a specific polypeptide or protein after transcription or translation.

[0252] The term "coding sequence" or "sequence encoding a specific protein" refers to a nucleic acid sequence that 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' (carboxyl) terminus. Coding sequences may include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0253] An exemplary cDNA sequence of GDNF is disclosed in Genbank Access NM_000514.4 (SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2. The methods described herein utilize a nucleic acid construct comprising the sequence SEQ ID NO: 1 or a variant thereof to slow or inhibit the progression of PD. The variant includes, for example, naturally occurring variants due to allelic variation (such as polymorphism) between individuals, alternative splicing forms, etc. The term variant also includes GDNF gene sequences from other sources or organisms. The variant is preferably substantially homologous to SEQ ID NO: 1 and / or SEQ ID NO: 2, that is, the nucleotide sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 is generally at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95%. In some embodiments, the nucleic acid construct comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1, and it retains the activity of SEQ ID NO: 1 or SEQ ID NO: 2. Variants of the GDNF gene also include nucleic acid sequences that hybridize to the above sequences (or their complementary strands) under stringent hybridization conditions. Typical stringent hybridization conditions include a temperature above 30° C., preferably above 35° C., more preferably above 42° C., and / or a salinity of less than about 500 mM, preferably less than 200 mM. Those skilled in the art can adjust the hybridization conditions by changing the temperature, salinity and / or the concentration of other reagents (such as SDS, SSC), etc.

[0254] There are reports of variations in GDNF at nucleotide 277, nucleotide 633, and nucleotide 1389. 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 an A to G point mutation at nucleotide 1389. Other possible variants include codon optimized sequences and conservative changes. Conservative substitutions are well known in the art.

[0255] In one embodiment, the GDNF gene is codon optimized. In one embodiment, the GDNF nucleic acid sequence is codon optimized for any one or more of the following purposes, for example, (1) enhanced in vivo expression, (2) reduction of CpG islands or (3) reduction of innate immune response. A skilled artisan can codon optimize GDNF using standard techniques in the art.

[0256] In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 or a variant thereof. In some embodiments, the viral vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to SEQ ID NO: 2.

[0257] SEQ ID NO: 1GDNF mRNA

[0258] SEQ ID NO: 2GDNF amino acid sequence

[0259] SEQ ID NO: 62GDNF variant mRNA (C277T)

[0260] SEQ ID NO: 63GDNF variant mRNA (C633G) Carrier

[0261] In some embodiments, the vector is an adeno-associated virus (AAV) or a recombinant AAV. In some aspects, the present disclosure provides an isolated AAV. As used herein with respect to AAV, the term "isolated" refers to an AAV that is artificially produced or obtained. An isolated AAV can be produced using a recombinant method. Such AAVs are referred to herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities so that the nuclease and / or transgene of the rAAV will be specifically delivered to one or more predetermined tissues. The AAV capsid is an important element that determines these tissue-specific targeting capabilities. Therefore, rAAVs with capsids suitable for targeted tissues can be selected.

[0262] Methods for obtaining recombinant AAV with desired capsid proteins are well known in the art. (See, for example, US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Generally, the method involves culturing a host cell containing a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, a recombinant AAV vector consisting of an AAV inverted terminal repeat (ITR) and a transgene, and auxiliary functions sufficient to allow the recombinant AAV vector to be packaged into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. AAV contains three capsid proteins, viral particle proteins 1 to 3 (named VP1, VP2, and VP3), all of which are transcribed from a single cap gene by alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, after translation, the capsid protein forms a spherical 60-mer protein shell around the AAV genome. In some embodiments, the function of the capsid protein is to protect the viral genome, deliver the genome and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0263] In some embodiments, the recombinant AAV (rAAV) capsid protein is 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. In some embodiments, the AAV capsid protein is a serotype derived from a non-human primate, such as an AAVrh10 serotype. In some embodiments, the AAV capsid protein is an AAV9 serotype. In some embodiments, the capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV3b capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, or an AAV13 capsid protein, or a chimera thereof. In some embodiments, 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, AAVrh74, AAV10, or AAV11, or a chimera thereof.

[0264] In one embodiment, the AAV serotypes and / or capsids described herein are selected from Table 7.

[0265] In a specific embodiment, the rAAV comprises a chemically modified capsid as disclosed in WO2017 / 212019, for example, a mannose ligand is chemically coupled to AAV2. The rAAV with a chemically modified capsid disclosed in WO2017 / 212019 is incorporated herein by reference in its entirety. As a further embodiment, the AAV capsid proteins and viral capsids used herein can be polyploid (also referred to as reasonable haploids) because they can contain different combinations of VP1, VP2 and VP3 AAV serotypes in a single AAV capsid, as described in PCT / US18 / 22725, PCT / US2018 / 044632 or US10,550,405, which are incorporated herein by reference.

[0266] The components to be cultured in host cells to package the rAAV vector in the AAV capsid can be provided to the host cells in trans. Alternatively, any one or more required components (e.g., recombinant AAV vectors, rep sequences, cap sequences and / or auxiliary functions) can be provided by stable host cells, and the stable host cells have been engineered to contain one or more of the required components using methods known to those skilled in the art. Most suitably, such stable host cells will contain the required components under the control of an inducible promoter. However, the required components may be under the control of a constitutive promoter. In the discussion of regulatory elements suitable for use with transgenics, examples of suitable inducible and constitutive promoters are provided herein. In another alternative, the selected stable host cells may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell can be generated, the cell being derived from a 293 cell (which contains an E1 auxiliary function under the control of a constitutive promoter), but which contains rep and / or cap proteins under the control of an inducible promoter. Other stable host cells can be generated by those skilled in the art. In some embodiments, the disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a protein (e.g., wild-type huntingtin protein, optionally "hardened" wild-type huntingtin protein). In some embodiments, the disclosure relates to a composition comprising the above host cell. In some embodiments, the composition comprising the above host cell further comprises a cryoprotectant.

[0267] Any suitable genetic element (vector) can be used to deliver the recombinant AAV vector, rep sequence, cap sequence and auxiliary functions required for producing the rAAV of the present disclosure to the packaging host cell. The selected genetic element can be delivered by any suitable method (including those described herein). The method for constructing any embodiment of the present disclosure is known to those skilled in the art of nucleic acid manipulation and includes genetic engineering, recombinant engineering and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the selection of suitable methods is not a limitation of the present disclosure. See, for example, K. Fisher et al., J. Virol., 70: 520-532 (1993) and U.S. Patent No. 5,478,745.

[0268] In some embodiments, recombinant AAV can be produced using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV auxiliary function vector, and an accessory function vector. The AAV auxiliary function vector encodes an "AAV auxiliary function" sequence (i.e., rep and cap), which acts in trans for fruitful AAV replication and encapsidation. Preferably, the AAV auxiliary function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19 (described in U.S. Patent No. 6,001,650) and pRep6cap6 vectors (described in U.S. Patent No. 6,156,303), the entire contents of which are incorporated herein by reference. The accessory function vector encodes a nucleotide sequence of a non-AAV-derived viral and / or cellular function (i.e., "accessory function") on which AAV replication depends. Accessory functions include those required for AAV replication, including but not limited to those involved in: activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

[0269] In some aspects, the present disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art. See, for example, Graham et al. (1973) Virology, 52: 456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, 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 (e.g., nucleotide integration vectors and other nucleic acid molecules) into suitable host cells.

[0270] "Host cell" refers to any cell that contains or is capable of containing a substance of interest. Typically, the host cell is a mammalian cell. The host cell can be used as a recipient of the following: AAV auxiliary constructs, AAV minigene plasmids, accessory function vectors or other transfer DNAs associated with the production of recombinant AAV. The term includes the descendants of the transfected original cells. Therefore, "host cell" as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the descendants of a single parent cell may not necessarily be identical to the original parent in morphology or in the genome or total DNA complement due to natural, accidental or deliberate mutations.

[0271] As used herein, the term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that during storage or transfer of such clonal populations, karyotypes can undergo spontaneous or induced changes. Therefore, cells derived from a referenced cell line may not be exactly identical to the progenitor cell or culture, and referenced cell lines include such variants.

[0272] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced (eg, a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid (eg, RNA)).

[0273] As used herein, the term "vector" includes any genetic element, such as plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which can be replicated when associated with appropriate control elements, and it can transfer gene sequences between cells. Therefore, the term "vector" includes cloning tools and expression tools, and viral vectors. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, to which other DNA segments are connected. Another type of vector is a viral vector, in which other DNA segments are connected to the viral genome. Specific vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). In addition, specific vectors can guide the expression of genes operably connected to them. Such vectors are referred to as "expression vectors" in this article. In general, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this application document, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the technology described herein is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0274] A cloning vector is a vector capable of autonomous replication or integration in the genome of a host cell, further characterized by one or more endonuclease restriction sites, at which the vector can be cut in a determinable manner, and a desired DNA sequence can be connected thereto so that the new recombinant vector maintains its ability to replicate in a host cell. In the case of a plasmid, as the number of copies of the plasmid increases in a host cell (e.g., a host bacterium), the replication of the desired sequence can occur many times, or the replication of the desired sequence before the host is propagated by mitosis is only once in each host. In the case of a phage, replication can occur actively during the lysis phase or passively during the lysogenic phase.

[0275] An expression vector is a vector into which a desired DNA sequence can be inserted by restriction and connection so that it can be operably joined to a regulatory sequence and can be expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for identifying cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds; genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase); and genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In a specific embodiment, the vector used herein is capable of autonomous replication and expression of structural gene products present in the DNA segment to which it is operably joined.

[0276] In some aspects of the invention, a recombinant AAV (AAV2-GDNF) comprising a nucleic acid encoding GDNF is produced by a triple transfection method using a closed-end linear double-stranded DNA molecule lacking a bacterial backbone sequence, e.g., as described in PCT / US2021 / 013689, which is published as WO / 2021 / 146591, which is incorporated herein by reference in its entirety.

[0277] In some aspects of the invention, a recombinant AAV comprising a nucleic acid encoding GDNF (AAV2-GDNF) is prepared by the method described in PCT / US2022 / 013279 (published as WO2022159679), which is incorporated herein by reference in its entirety.

[0278] In some embodiments, useful vectors are considered to be those in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. If the coding sequence is desired to be translated into a functional protein, if induction of the promoter in the 5' regulatory sequence causes transcription of the coding sequence, and if the nature of the connection between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, then the two DNA sequences are said to be operably joined. Therefore, if the promoter region is able to effect transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide, then the promoter region will be operably joined to the coding sequence.

[0279] The precise nature of the regulatory sequences required for gene expression may vary between species or cell types, but may generally include 5' non-transcribed sequences and 5' non-translated sequences that are involved in transcription initiation and translation, respectively, if necessary, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region that contains a promoter sequence for transcriptional control of the operably joined gene. As desired, the regulatory sequence may also include an enhancer sequence or an upstream activator sequence. The vectors described herein may optionally include a 5' leader or signal sequence. The selection and design of appropriate vectors are within the capabilities and discretion of those of ordinary skill in the art.

[0280] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into the cells. The heterologous DNA (RNA) is placed under the operable control of transcriptional elements to allow the heterologous DNA to be expressed in the host cell.

[0281] The phrases "operably linked", "operably positioned", "under control" or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term "expression vector or construct" means any type of genetic construct comprising a nucleic acid in which part or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of a nucleic acid, for example to produce a biologically active polypeptide product or a functional RNA (e.g., a guide RNA) from a transcribed gene.

[0282] The foregoing methods for packaging a recombinant vector into a desired AAV capsid to produce the rAAV of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to the ordinarily skilled artisan.

[0283] In some embodiments, one or more of the recombinantly expressed genes may be integrated into the genome of the cell.

[0284] The nucleic acid molecules described herein can be introduced into one or more cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced by standard protocols (such as transformation, including chemical transformation and electroporation, transduction, particle bombardment, etc.). Expression of nucleic acid molecules described herein can also be achieved by integrating nucleic acid molecules into the genome.

[0285] In one embodiment, the genome packaged within AAV2-GDNF comprises the 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 consists 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.

[0286] In one embodiment, the AAV2-GDNF vector comprises an ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64). In one embodiment, the AAV2-GDNF vector consists of or essentially consists of an ITR to ITR portion of sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64). In one embodiment, the AAV2-GDNF vector comprises, consists of, or consists essentially of an ITR to ITR portion of the sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64) and is produced from a plasmid comprising, consisting of, or consisting essentially of SEQ ID NO: 64. In one embodiment, the AAV2-GDNF vector comprises, consists of, or consists essentially of an ITR to ITR portion of the sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64) and is produced from a plasmid comprising, consisting of, or consisting essentially of SEQ ID NO: 64. In some embodiments, the invention relates to 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 base pairs 12-2716 of NO:64. ... Fig.26As depicted, rAAV is made using the plasmid DNA described in SEQ ID NO: 64. In some embodiments, rAAV is made using end-blocked linear double-stranded DNA. In various embodiments, AAV2-GDNF comprises a plasmid comprising an ITR to ITR portion of the sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64). In other embodiments, AAV2-GDNF comprises an end-blocked linear double-stranded DNA comprising an ITR to ITR portion of the sequence SEQ ID NO: 64 (i.e., base pairs 12-2716 of SEQ ID NO: 64), a non-limiting example of which is DoggyboneDNA (dbDNA) as disclosed in U.S. Application No. 2018 / 0037943 and Karbowniczek et al., Bioinsights, 2017. TM ), which is incorporated herein by reference in its entirety. SEQ ID NO: 64

[0287] In one embodiment, Fig.26 The plasmids depicted were used to generate the AAV2-GDNF genome.

[0288] In one embodiment, the genome packaged in AAV2-GDNF is Fig.26 Depicted.

[0289] In one embodiment, using Fig.26 The depicted plasmids produced AAV2-GDNF.

[0290] In one embodiment, AAV2-GDNF comprises at least one component listed in Table 8. Modified capsid

[0291] In one embodiment, the capsids described herein are further modified to increase tropism for the CNS. In one embodiment, the capsids, and thus the AAVs, have tropism increased 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%, 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%, 100%, 101%, 102%, 103%, 104%, 105%, 10 %, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more, or at least 1×, 2×, 3×, 4×, 0×, 5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×, 70×, 75×, 80×, 85×, 90×, 95×, 100×, 250×, 500×, 750× or 1000× or more.

[0292] In one embodiment, the capsid is modified so that it has a reduced tropism for non-CNS tissues. For example, a capsid having a liver-specific tropism can be modified so that it no longer has such tropism. In one embodiment, the capsid is modified so that its tropism for non-CNS tissues is reduced 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%, 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 10 %, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0293] In another embodiment, the capsid is modified to increase its tropism for CNS tissues and reduce its tropism for non-CNS tissues. For example, a capsid with liver-specific tropism can be modified to exhibit CNS-specific tropism and reduce liver-specific tropism.In one embodiment, the capsid has a CNS tropism that 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%, 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%, 87%, 88%, 89%, %, 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, or at least 1×, 2×, 3×, 4×, 0×, 5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45× , 50×, 55×, 60×, 65×, 70×, 75×, 80×, 85×, 90×, 95×, 100×, 250×, 500×, 750×, or 1000× or more; and the tropism for non-CNS tissues is reduced 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%, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0294] Provided herein are compositions comprising modified viral capsids, the viral capsids comprising a payload, wherein the payload comprises a nucleic acid sequence targeting a central nervous system disorder flanked by a regulatory sequence and an inverted terminal repeat (ITR), and wherein the modification is a chemical, non-chemical or amino acid modification. In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a transgenic (e.g., GDNF). In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a GDNF protein.

[0295] In certain embodiments, the modified viral capsid comprises modifications that make it preferentially target the CNS. For example, the modified viral capsid has a tropism that increases for the CNS, and / or a tropism that decreases for at least a second position (e.g., liver). The preferential targeting of the CNS does not exclude targeting to other sites, but shows that it targets the CNS more highly than other sites.

[0296] In one embodiment, the modified viral capsid comprises a modification that enables it to target the CNS. For example, modifications to a capsid that is typically targeted to a non-CNS site (e.g., liver) can redirect the capsid to now target the CNS and non-CNS sites. In such embodiments, CNS targeting does not need to be a priority.

[0297] In one embodiment, the modification of the capsid is an amino acid modification, e.g., a deletion, insertion or substitution of an amino acid. In one embodiment, the amino acid modification increases tropism to the CNS. In one embodiment, the amino acid modification targets the modified capsid to the CNS.

[0298] In one embodiment, the modified viral capsid has, consists of, or consists essentially of a nucleic acid sequence that is 90% identical to SEQ ID NO: 1-SEQ ID NO: 4 of U.S. Patent Application No. 16 / 511,913, the contents of which are incorporated herein by reference in their entirety. This U.S. patent application describes chimeric AAV capsid sequences that exhibit a dominant tropism for oligodendrocytes and can be used to create AAV vectors that transduce oligodendrocytes in the CNS of a subject.

[0299] In one embodiment, the modified viral capsid is an AAV capsid protein comprising one or more amino acid substitutions, wherein the substitution introduces a new glycan binding site into the AAV capsid protein. In some embodiments, the amino acid substitutions are at amino acid 266, amino acid 463-475, and amino acid 499-502 in AAV2, or the corresponding amino acid positions in AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV10. Such AAV capsid proteins are further described in, for example, U.S. Patent Application No. 16 / 110,773; the contents of which are incorporated herein by reference in their entirety.

[0300] In one embodiment, the modified viral capsid is an AAV capsid protein, which comprises an AAV2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT / US2020 / 029493) (the contents of which are incorporated herein by reference in their entirety), consists of an AAV2.5 capsid protein, or consists essentially of an AAV2.5 capsid protein, which comprises one or more amino acid substitutions that introduce new glycan binding sites. Such amino acid substitutions can target the capsid to neurons and glial cells (e.g., astrocytes). In embodiments of the capsid proteins, capsids, viral vectors, and methods described in International Patent Application No. WO / 2020 / 219656, the one or more amino acid substitutions include A267S, SQAGASDIRDQSR464-476SX 1 AGX 2 SX 3 X 4 X 5 X 6 QX 7 R (SEQ ID NO: 153 and SEQ ID NO: 154, respectively) (wherein X 1-7 can be any amino acid), and EYSW 500-503 (SEQ ID NO: 155) EX 8 X 9 W, where X 8-9 Can be any amino acid. In the embodiments of the capsid protein, capsid, viral vector and method described herein, X 1 is V or a conservative substitution thereof; X 2 is P or a conservative substitution thereof; X 3 is N or a conservative substitution thereof; X 4 is M or a conservative substitution thereof; X 5 is A or a conservative substitution thereof; X 6 is V or a conservative substitution thereof; X 7 is G or a conservative substitution thereof; X 8is F or a conservative substitution thereof; and / or X 9 is A or a conservative substitution thereof. In the embodiments of the capsid protein, capsid, viral vector and method described herein, X 1 It is V, X 2 Yes P, X 3 It is N, X 4 It is M, X 5 It's A, X 6 It is V, X 7 It's G, X 8 is F, and X 9 is A, wherein the novel glycan binding site is a galactose binding site. Such AAV capsid proteins are further described, for example, in International Patent Application No. WO / 2020 / 219656; the contents of which are incorporated herein by reference in their entirety.

[0301] In one embodiment, 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., U.S. Patent Application No. 16 / 956,306; the contents of which are incorporated herein by reference in their entirety. Such AAV capsids allow delivery of (e.g., payload) across the blood-brain barrier.

[0302] In one embodiment, the modified viral capsid comprises an 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 replacement of a threonine residue with a non-threonine residue) at positions corresponding to: one or more, or each, of Y705, Y731, and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. patent application Ser. No. 16 / 565,191; the contents of which are incorporated herein by reference in their entirety); one or more, or each, of Y436, Y693, and Y719 of a wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. patent application Ser. No. 16 / 565,191); or one or more, or each, of Y705, Y731, and T492 of a wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. patent application Ser. No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0303] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5 or AAV6 capsid protein), which contains a Y to F (tyrosine to phenylalanine) modification or a T to V (threonine to valine) modification in the VP3 region of the capsid corresponding to the following positions: one or more, or each, of Y705F, Y731F and T492V of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. patent application Ser. No. 16 / 565,191); one or more, or each, of Y436F, Y693F and Y719F of the wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. patent application Ser. No. 16 / 565,191); or one or more, or each, of Y705F, Y731F and T492V of the wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. patent application Ser. No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0304] In one embodiment, the modified viral capsid comprises an 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 replacement of a threonine residue with a non-threonine residue) at positions corresponding to: one or more or each of Y705, Y731, and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); one or more or each of Y436, Y693, and Y719 of a wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); or one or more or each of Y705, Y731, and T492 of a wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0305] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5 or AAV6 capsid protein), which contains a Y to F (tyrosine to phenylalanine) modification or a T to V (threonine to valine) modification in the VP3 region of the capsid protein corresponding to the following positions: one or more or each of Y705F, Y731F and T492V of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. patent application Ser. No. 16 / 565,191); one or more or each of Y436F, Y693F and Y719F of the wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. patent application Ser. No. 16 / 565,191); or one or more or each of Y705F, Y731F and T492V of the wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. patent application Ser. No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0306] In one embodiment, the amino acid modification allows the modified capsid to escape neutralizing antibodies, such as neutralizing antibodies produced against a viral vector (e.g., a viral vector of the same serotype). In one embodiment, the amino acid modification allows the modified capsid to be used for repeated administration, for example, the modification will cause the capsid to have a therapeutic effect when it is administered again.

[0307] In one embodiment, the modified viral capsid is a chimeric capsid. As used herein, a "chimeric" capsid protein refers to an AAV capsid protein (e.g., any one or more of VP1, VP2, or VP3) that has been modified by substitution of 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 the wild type, and insertion or deletion 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 the wild type. In some embodiments, a complete or partial domain, functional region, epitope, 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. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a large number of chimeric capsid proteins are described in the literature and herein, which can be included in the capsid.

[0308] In one embodiment, the modified viral capsid is a haploid capsid. As used herein, the term "haploid AAV" shall refer to the AAV described in International Application WO2018 / 170310 or U.S. Application US2018 / 037149, which is incorporated herein by reference in its entirety. In some embodiments, the group of virions is a haploid AAV group, in which virion particles can be constructed, in which at least one viral protein from the group consisting of AAV capsid protein, VP1, VP2 and VP3 is different from at least one of the other viral proteins required to form virion particles capable of encapsulating the AAV genome. For each viral protein (VP1, VP2 and / or VP3) present, the protein is of the same type (e.g., all AAV2 VP1). In one example, at least one of the viral proteins is a chimeric viral protein, and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric, and only VP3 is non-chimeric. For example, the viral particle consists only of: VP1 / VP2 from chimeric AAV2 / 8 (N-terminus of AAV2 and C-terminus of AAV8) paired with VP3 from AAV2 alone; or only chimeric VP1 / VP2 28m-2P3 (N-terminus of AAV8 and C-terminus of AAV2 without VP3 start codon mutation) paired with VP3 from AAV2 alone. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1 / VP2 28m-2P3 is paired with VP3 from AAV3 alone. In another example, no chimeric protein is present.

[0309] In some embodiments of the technology described herein, the modified viral capsid comprises one or more modifications, such as chemical modifications, non-chemical modifications or amino acid modifications to the capsid. Such modifications can, for example, modify the tissue tropism or cell tropism of the modified capsid and others.

[0310] Modifications may directly alter the properties of the capsid (including biochemical properties, such as receptor binding), such that the modification itself alters the behavior of the capsid, or may allow for further modifications (such as the attachment of a ligand, which in turn modifies the behavior of the capsid in a desired manner).

[0311] In one embodiment, chemical modification of cysteine ​​residues (which may be naturally occurring or introduced by genetic modification of the capsid polypeptide encoding sequence) allows for covalent attachment of ligands via disulfide bond formation (see, e.g., WO 2005 / 106046, the contents of which are incorporated herein by reference).

[0312] A variety of ligands are contemplated, including but not limited to, for example, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by target cells (see, for example, WO 2000 / 002654, which is incorporated herein by reference).

[0313] WO 2015 / 062516 (the contents of which are also incorporated herein by reference) describes the insertion of an amino acid comprising an azido group by genetic modification of the capsid gene, followed by chemical coupling of a ligand via the azido group.

[0314] Horowitz et al., Bioconjugate Chem., 22:529-532 (2011) describe the tropism for modifying AAV capsids by glycosylation or chemical coupling of sugar moieties. This method and similar methods are contemplated for modification of capsids as described herein.

[0315] In other embodiments, coating of the viral capsid with a polymer such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl)methacrylamide (pHPMA) is specifically contemplated. Such modifications can, for example, reduce specific and nonspecific interactions with non-target tissues.

[0316] In other embodiments, carbodiimide coupling is specifically contemplated. See, for example, Joo et al., ACS Nano 5, entitled "Enhanced Real-time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates" (2011).

[0317] In other embodiments, the viral capsid can be modified, for example, as described in WO 2017 / 212019, see also US National Phase USSN 16 / 308,740, the contents of which are each incorporated herein by reference. The method described therein couples the viral capsid with a ligand by a bond comprising -CSNH- and an aromatic moiety. Although the genetically modified viral capsid can be further modified by this method, the modification described therein does not require the genetic modification of the viral capsid. The ligand described therein includes, for example, a targeting agent, a spatial shielding agent for avoiding neutralizing antibody interactions, a labeling agent or a magnetic agent. The targeting ligand described therein includes, for example, cell type-specific ligands, proteins, monosaccharides or polysaccharides, steroid hormones, RGD motif peptides (e.g., Arg-Gly-Asp, a cell adhesion motif that can simulate cell adhesion proteins and bind to integrins), vitamins and small molecules.

[0318] In one embodiment, the chemical modification described herein is a modification described in international patent application WO / 2017 / 212019, the contents of which are incorporated herein by reference in their entirety.

[0319] In one embodiment, the chemical modification described herein is a modification described in international patent application WO / 2021 / 005210, the contents of which are incorporated herein by reference in their entirety.

[0320] In one embodiment, the capsid has at least one chemically modified tyrosine residue in its capsid, wherein the chemically modified tyrosine residue is of formula (I):

[0321] in:

[0322] -X 1 Selected from the group consisting of:

[0323] -Ar is an optionally substituted aryl or heteroaryl moiety.

[0324] In one embodiment, the capsid has at least one chemically modified tyrosine residue, which is of formula (Ia):

[0325] in:

[0326] -X i , and Ar are as defined above,

[0327] - a spacer is a group used to connect the "Ar" group to the functional moiety "M", which preferably contains up to 1000 carbon atoms and which is preferably in the form of a chemical chain optionally containing heteroatoms and / or cyclic moieties,

[0328] -n is 0 or 1; and

[0329] -M is a functional moiety comprising a steric hindering agent, a labeling agent, a cell type specific ligand, or a drug moiety.

[0330] In one embodiment, Xi is of formula (a) and / or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl and anthracenyl.

[0331] In one embodiment, the capsid has at least one chemically modified tyrosine, which is of formula (Ic):

[0332] in:

[0333] -X 2is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)-, or -(C=S)-NH-, preferably -(C=O)-NH- or -(C=O)-O-,

[0334] -X 2 In the para-, meta- or ortho-position of the phenyl group, preferably in the para-position,

[0335] - Spacer, n and M are as defined above.

[0336] In one embodiment, when present, the "spacer" is selected from the group consisting of: a saturated or unsaturated, linear or branched C 2 -C 40 hydrocarbon chains, optionally substituted polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-hydroxypropyl)methacrylamide), polylactic-co-glycolic acid (PLGA), polymers of alkyl diamines, and combinations thereof, and / or

[0337] "M" comprises or consists of a cell type targeting ligand, which is preferably selected from monosaccharides or polysaccharides, hormones (including steroid hormones), peptides (such as RGD peptides (such as Arg-Gly-Asp, a cell adhesion motif that can mimic cell adhesion proteins and bind to integrins), muscle targeting peptides (MTP) or Angiopep-2), proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies (including heavy chain antibodies) and fragments thereof (such as antigen binding fragments (Fab), Fab' (which are antigen binding fragments further comprising free thiol groups)) and VHH), single-chain variable fragments (ScFv), spiegelmers, peptide aptamers, vitamins and drugs (such as cannabinoid receptor 1 (CB1) and / or cannabinoid receptor 2 (CB2) ligands).

[0338] In one embodiment, the "spacer" (when present) is selected from the group consisting of: a linear or branched C 2 -C 20Polymers of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyl diamines, and combinations thereof, wherein the polymer having 2 to 20 monomers and / or "M" comprises or consists of a cell type-specific ligand, wherein the cell type-specific ligand is derived from: a protein selected from transferrin, epidermal growth factor (EGF) and basic fibroblast growth factor 13FGF; a monosaccharide or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, a GalNac bridge or mannose-6-phosphate; an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7; and a vitamin (e.g., folic acid).

[0339] In one embodiment, the capsid further has at least one additional chemically modified amino acid residue in the capsid, which is different from the tyrosine residue, and the amino acid residue preferably has an amino group chemically modified with a group of formula (V):

[0340] in:

[0341] -N* is the nitrogen of the amino group of an amino acid residue (e.g. a lysine residue or an arginine residue), and

[0342] -Ar, spacer, n and M have the same definitions as Ar, spacer, n and M of formula (II) of claim 2.

[0343] In one embodiment, the capsid is incubated with a chemical reagent bearing a reactive group selected from aryldiazo and 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moieties under conditions that favor reaction of the reactive group with tyrosine residues present in the capsid, thereby forming a covalent bond.

[0344] In one embodiment, the capsid is incubated with a chemical reagent of Formula VId to obtain at least one chemically modified tyrosine residue in the capsid of Formula Ic. Pharmaceutical composition

[0345] The expression cassette, vector or virosome of the present invention can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable excipient (i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc.). The pharmaceutical composition can be provided in the form of a kit.

[0346] Thus, a further aspect of the invention provides a pharmaceutical composition comprising an expression cassette, vector or virosome as described herein.

[0347] In various aspects, the pharmaceutical composition comprises a phosphate buffer. The phosphate buffer comprises about 1mM to about 50mM phosphate, for example, a concentration of phosphate of about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM or about 50mM. The phosphate is selected from dibasic phosphates (e.g., Na 2 HPO 4 , K 2 HPO 4 ) and monobasic phosphates (such as NaH 2 PO 4 , KH 2 PO 4 ) is prepared with a molar ratio of dibasic phosphate:monobasic phosphate of about 1:10 to about 10:1. For example, in various exemplary embodiments, the 10mM phosphate includes 9.5mM dibasic phosphate and 0.5mM monobasic phosphate, 9mM dibasic phosphate and 1mM monobasic phosphate, 8.5mM dibasic phosphate and 1.5mM monobasic phosphate, 8mM dibasic phosphate and 2mM monobasic phosphate, 7.5mM dibasic phosphate and 2.5mM monobasic phosphate, or 7mM dibasic phosphate and 3mM monobasic phosphate. The pH of the phosphate buffer is about 6.5 to about 7.5, for example, the pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In one embodiment, the pH of the phosphate buffer is 7.2-7.3. In one embodiment, the phosphate buffer has a pH of 7.22. The phosphate buffer may also include NaCl at a concentration of about 50 mM to about 200 mM, such as 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 may also include KCl at a concentration of about 0.5 mM to about 10 mM, such as 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 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 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. The phosphate buffer may also include CaCl at a concentration of about 0.20 mM to about 10 mM. 2, 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. The phosphate buffer may also contain MgCl at a concentration of about 0.10 mM to about 1 mM. 2 , such as 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 may also include poloxamer 188 (e.g., Pluronic TMF-68 nonionic surfactant), for example, at a concentration of about 0.0001wt%, about 0.0002wt%, about 0.0003wt%, about 0.0004wt%, about 0.0005wt%, about 0.0006wt%, about 0.0007wt%, about 0.0008wt%, about 0.0009wt%, about 0.001wt%, about 0.0015wt%, about 0.002wt%, about 0.0025wt%, about 0.003wt%, about 0.0035wt%, about 0.004wt%, about 0.0045wt% or about 0.005wt%. The phosphate buffer may also include sorbitol in a concentration of about 0.005 wt % to about 10 wt %, for example, 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 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt %, about 9 wt % or about 10 wt %. The titer of the rAAV comprising a nucleic acid (e.g., AAV2 comprising a CMV promoter and a nucleic acid comprising a sequence that is 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) in phosphate buffer can be about 1×10 12 vg / mL to about 4×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12 vg / mL, 1×10 12 vg / mL to about 3×10 12 vg / mL, 1×10 12 vg / mL to about 2×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12 vg / mL, 8×10 11 vg / mL to about 9×10 12 vg / mL, 9×10 11 vg / mL to about 9×10 12 vg / mL, 1×10 12 vg / mL to about 9×10 12 vg / mL, 2×10 12vg / mL to about 9×10 12 vg / mL, 3×10 12 vg / mL to about 9×10 12 vg / mL, 4×10 12 vg / mL to about 9×10 12 vg / mL, 5×10 12 vg / mL to about 9×10 12 vg / mL, 6×10 12 vg / mL to about 9×10 12 vg / mL, 7×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 11 vg / mL to about 8×10 12 vg / mL, 8×10 11 vg / mL to about 7×10 12 vg / mL, 8×10 11 vg / mL to about 6×10 12 vg / mL, 8×10 11 vg / mL to about 5×10 12 vg / mL, 8×10 11 vg / mL to about 4×10 12 vg / mL, 8×10 11 vg / mL to about 3×10 12 vg / mL, 8×10 11 vg / mL to about 2×10 12 vg / mL, 8×10 11 vg / mL to about 1×10 12 vg / mL, 8×10 11 vg / mL to about 9×10 11 vg / mL, 1×10 12 vg / mL to about 7×10 12 vg / mL, 3×10 12 vg / mL to about 6×10 12 vg / mL, 4×10 12 vg / mL to about 5×10 12 vg / mL, 3.1×10 12 vg / mL to about 4×10 12 vg / mL, 3.2×10 12 vg / mL to about 4×10 12 vg / mL, 3.3×10 12vg / mL to about 4×10 12 vg / mL, 3.4×10 12 vg / mL to about 4×10 12 vg / mL, 3.5×10 12 vg / mL to about 4×10 12 vg / mL, 3.6×10 12 vg / mL to about 4×10 12 vg / mL, 3.7×10 12 vg / mL to about 4×10 12 vg / mL, 3.8×10 12 vg / mL to about 4×10 12 vg / mL, 3.9×10 12 vg / mL to about 4×10 12 vg / mL, 3×10 12 vg / mL to about 3.9×10 12 vg / mL, 3×10 12 vg / mL to about 3.8×10 12 vg / mL, 3×10 12 vg / mL to about 3.7×10 12 vg / mL, 3×10 12 vg / mL to about 3.6×10 12 vg / mL, 3×10 12 vg / mL to about 3.5×10 12 vg / mL, 3×10 12 vg / mL to about 3.4×10 12 vg / mL, 3×10 12 vg / mL to about 3.3×10 12 vg / mL, 3×10 12 vg / mL to about 3.2×10 12 vg / mL, and 3×10 12 vg / mL to about 3.1×10 12 vg / mL.

[0348] In one embodiment, the pharmaceutical composition comprises, consists essentially of, or consists of a composition described in Table 9.

[0349] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of phosphate (monophosphate and dibasic acid salts), NaCl, and poloxamer; pH 7.2-7.3.

[0350] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of about 10 mM phosphate (monophosphate and dibasic acid salts), about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.

[0351] In some embodiments, 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.

[0352] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM Na 2 HPO 4 , about 2 mM NaH 2 PO 4 , about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.

[0353] In some embodiments, 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 1×10 12 vg / mL to about 3.1×10 13 vg / mL of AAV2-GDNF.

[0354] In some embodiments, 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 1×10 12 vg / mL of AAV2-GDNF.

[0355] In some embodiments, 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 5×10 12vg / mL of AAV2-GDNF.

[0356] In some embodiments, 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 1×10 13 vg / mL of AAV2-GDNF.

[0357] In some embodiments, 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 3×10 13 vg / mL of AAV2-GDNF.

[0358] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM K 2 HPO 4 , about 2mM KH 2 PO 4 , about 180 mM NaCl and about 0.001% poloxamer; pH 7.2-7.3; and about 1×10 12 vg / mL to about 3.1×10 13 vg / mL of AAV2-GDNF.

[0359] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM K 2 HPO 4 , about 2mM KH 2 PO 4 , about 180 mM NaCl and about 0.001% poloxamer; pH 7.2-7.3; and at least 1×10 12 vg / mL of AAV2-GDNF.

[0360] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM K 2 HPO 4 , about 2mM KH 2 PO 4 , about 180 mM NaCl and about 0.001% poloxamer; pH 7.2-7.3; and at least 5×10 12 vg / mL of AAV2-GDNF.

[0361] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM K 2 HPO 4 , about 2mM KH 2 PO 4 , about 180 mM NaCl and about 0.001% poloxamer; pH 7.2-7.3; and at least 1×10 13 vg / mL of AAV2-GDNF.

[0362] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of: about 8 mM K 2 HPO 4 , about 2mM KH 2 PO 4 , about 180 mM NaCl and about 0.001% poloxamer; pH 7.2-7.3; and at least 3×10 13 vg / mL of AAV2-GDNF. Application

[0363] The rAAV of the present disclosure can be delivered to a subject in a composition according to any suitable method known in the art. For example, the rAAV preferably suspended in a physiologically compatible carrier (i.e., in a composition) can be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include a human.

[0364] It may be desirable to deliver the rAAV described herein directly to the CNS of a subject. "CNS" refers to all cells and tissues of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Recombinant AAV can be delivered directly to the CNS or brain by injection with a needle, catheter, or related device using neurosurgical techniques known in the art (e.g., 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. Gene Ther. 11:2315-2329, 2000), for example, into the ventricular region and the striatum (e.g., the putamen or caudate nucleus of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule. In some embodiments, rAAV as described in the present disclosure is administered by intravenous injection. In some embodiments, rAAV is administered by intracerebral injection. In some embodiments, rAAV is administered by intrathecal injection. In some embodiments, rAAV is administered by intrastriatal injection. In some embodiments, the rAAV is delivered by intracranial injection. In some embodiments, the rAAV is delivered by cisterna magna injection. In some embodiments, the rAAV is delivered by intracerebroventricular injection.

[0365] In one embodiment, rAAV or a composition thereof is delivered locally to the CNS, eg, directly to the putamen, via a stepped cannula, for example, as described in US Pat. Nos. 7,815,623, 8,337,458, and 9,302,070, the contents of each of which are incorporated herein by reference in their entirety.

[0366] In one embodiment, rAAV or a composition thereof is delivered locally to the CNS, for example, directly to the putamen, via a SmartFlow cannula connected to an MRI-compatible infusion pump (eg, a Medfusion syringe pump, Smiths Medical Inc.).

[0367] In one embodiment, the compositions described herein are topically applied to, for example, the putamen, via a cannula at a flow rate of 1-30 μL / min. In one embodiment, the flow rate is about 1-25 μL / min, 1-20 μL / min, 1-15 μL / min, 1-10 μL / min, 1-5 μL / min, 5-30 μL / min, 10-30 μL / min, 15-30 μL / min, 20-30 μL / min, 25-30 μL / min, 5-25 μL / min, 10-20 μL / min, 15-25 μL / min, 5-15 μL / min, 5-25 μL / min, or 10-15 μL / min.

[0368] In one embodiment, the flow rate is about 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, 10 μL / min, 11 μL / min, 12 μL / min, 13 μL / min, 14 μL / min, 15 μL / mi n, 16μL / min, 17μL / min, 18μL / min, 19μL / min, 20μL / min, 21μL / min, 22μL / min, 23μL / min, 24μL / min, 25μL / min, 26μL / min, 27μL / min, 28μL / min, 29μL / min, or 30μL / min.

[0369] In addition, in some cases, it may be desirable to deliver rAAV to a mammalian subject, for example, by intramuscular injection or by administration into the bloodstream of a mammalian subject. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream by means of isolated limb perfusion, a technique well known in the surgical field, which essentially enables a technician to isolate a limb from the systemic circulation prior to administering rAAV virions. Technicians may also use a variant of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 to administer virions into the vasculature of an isolated limb, thereby potentially enhancing transduction into muscle cells or tissues.

[0370] In one embodiment, the rAAV or composition thereof is administered during a subject's "off" period.

[0371] In one embodiment, the rAAV or composition thereof is administered while the subject is "on."

[0372] Aspects of the present disclosure relate to compositions for slowing or inhibiting the progression of PD in a subject, the compositions comprising any one of a recombinant adeno-associated virus (rAAV) and a pharmaceutically acceptable carrier, the recombinant rAAV comprising a genome containing a glial cell-derived neurotrophic factor (GDNF) gene operably connected to a promoter as described herein. In one aspect, the composition comprises any one of the viral vectors described herein, and an optional pharmaceutically acceptable carrier. The compositions of the present disclosure may comprise a single rAAV, or an rAAV in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0373] The compositions of the present disclosure may further comprise a second therapeutic agent, such as an anti-Parkinson's disease therapeutic agent described herein. The compositions of the present disclosure may further comprise any immunomodulator described herein. The compositions of the present disclosure may further comprise a second therapeutic agent, such as an anti-Parkinson's disease therapeutic agent described herein and any immunomodulator described herein.

[0374] Those skilled in the art can easily select a suitable carrier according to the indications for which rAAV is intended. For example, a suitable carrier includes saline, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrose, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of the present disclosure.

[0375] Optionally, in addition to rAAV and carrier, the compositions of the present disclosure may also include other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0376] rAAV is administered in sufficient amounts to transfect cells of the desired tissue and provide sufficient levels of gene transfer and expression without excessive 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 administration, inhalation administration (including intranasal and intratracheal delivery), intraocular administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, and other parenteral administration routes. If desired, the route of administration can be combined. In some embodiments, all or at least one nucleic acid sequence disclosed herein is delivered via a non-viral DNA construct comprising at least one DD-ITR. For example, one or more of the nucleic acids described herein can be delivered using a non-viral DNA construct as described in WO 2019 / 246554. WO 2019 / 246554 is incorporated herein by reference in its entirety.

[0377] The dosage of rAAV virions required to achieve a particular "therapeutic effect" (e.g., dosage units in terms of genome copies per kilogram of body weight (GC / kg)) will vary based on several factors, including, but not limited to: the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. Based on the above factors and other factors well known in the art, one skilled in the art can readily determine the dosage range of rAAV virions to treat a patient with a particular disease or disorder.

[0378] In one embodiment, rAAV is expressed at 5×10 12 vg is about 1.5×10 13 In another embodiment, rAAV is administered at a total dose within the range of 1×10 12 vg is about 6.5×10 13 vg, 2×10 12 vg is about 6.5×10 13 vg, 3×10 12 vg is about 6.5×10 13 vg, 4×10 12 vg is about 6.5×10 13 vg, 6×10 12 vg is about 6.5×10 13 vg, 7×10 12 vg is about 6.5×10 13 vg, 8×10 12 vg is about 6.5×10 13 vg, 9×10 12 vg is about 6.5×10 13 vg, 1×10 13vg is about 6.5×10 13 vg, 1×10 12 vg is about 1.5×10 13 vg, 2×10 12 vg is about 1.5×10 13 vg, 3×10 12 vg is about 1.5×10 13 vg, 4×10 12 vg is about 1.5×10 13 vg, 6×10 12 vg is about 1.5×10 13 vg, 7×10 12 vg is about 1.5×10 13 vg, 8×10 12 vg is about 1.5×10 13 vg, 9×10 12 vg is about 1.5×10 13 vg, 1×10 13 vg is about 1.5×10 13 vg, 1×10 12 vg is about 6.5×10 13 vg, 2×10 12 vg is about 6.5×10 13 vg, 3×10 12 vg is about 6.5×10 13 vg, 4×10 12 vg is about 6.5×10 13 vg, 6×10 12 vg is about 6.5×10 13 vg, 7×10 12 vg is about 6.5×10 13 vg, 8×10 12 vg is about 6.5×10 13 vg, 9×10 12 vg is about 6.5×10 13 vg, 1×10 13 vg is about 6.5×10 13 vg, 1×10 12 vg is about 1×10 13 vg, 1×10 12 vg is about 9×10 12 vg, 1×10 12 vg is about 8×10 12 vg, 1×10 12 vg is about 7×10 12 vg, 1×10 12 vg is about 6×10 12vg, 1×10 12 vg is about 5×10 12 vg, 1×10 12 vg is about 4×10 12 vg, 1×10 12 vg is about 3×10 12 vg, 1×10 12 vg is about 2×10 12 vg, 5×10 12 vg is about 1×10 13 vg, 5×10 12 vg is about 9×10 12 vg, 5×10 12 vg is about 8×10 12 vg, 5×10 12 vg is about 7×10 12 vg, 5×10 12 vg is about 6×10 12 vg, 5×10 12 vg is about 5.5×10 13 vg, 5×10 12 vg is about 4.5×10 13 vg, 5×10 12 vg is about 3.5×10 13 vg, and 5×10 12 vg is about 2.5×10 13 vg.

[0379] In one embodiment, rAAV is administered in a total amount of at least 5.1×10 12 vg, 5.2×10 12 vg, 5.3×10 12 vg, 5.4×10 12 vg, 5.5×10 12 vg, 5.6×10 12 vg, 5.7×10 12 vg, 5.8×10 12 vg, 5.9×10 12 vg, 6×10 12 vg, 6.1×10 12 vg, 6.2×10 12 vg, 6.3×10 12 vg, 6.4×10 12 vg, 6.5×10 12 vg, 6.6×10 12 vg, 6.7×10 12 vg, 6.8×10 12 vg, 6.9×1012 vg, 7×10 12 vg, 7.1×10 12 vg, 7.2×10 12 vg, 7.3×10 12 vg, 7.4×10 12 vg, 7.5×10 12 vg, 7.6×10 12 vg, 7.7×10 12 vg, 7.8×10 12 vg, 7.9×10 12 vg, 8×10 12 vg, 8.1×10 12 vg, 8.2×10 12 vg, 8.3×10 12 vg, 8.4×10 12 vg, 8.5×10 12 vg, 8.6×10 12 vg, 8.7×10 12 vg, 8.8×10 12 vg, 8.9×10 12 vg, 9×10 12 vg, 9.1×10 12 vg, 9.2×10 12 vg, 9.3×10 12 vg, 9.4×10 12 vg, 9.5×10 12 vg, 9.6×10 12 vg, 9.7×10 12 vg, 9.8×10 12 vg, 9.9×10 12 vg, 1.1×10 13 vg, 1.2×10 13 vg, 1.3×10 13 vg, and 1.4×10 13 In one embodiment, rAAV is administered in a total amount of at least 1×10 12 vg, at least 2×10 12 vg, at least 3×10 12 vg, at least 4×10 12 vg, at least 5×10 12 vg, at least 6×10 12 vg, at least 7×10 12 vg, at least 8×10 12 vg, at least 9×10 12 vg, at least 1×10 13vg, at least 2×10 13 vg, at least 3×10 13 vg, at least 4×10 13 vg, at least 5×10 13 vg, at least 6×10 13 vg, and at least 7×10 13 vg or more.

[0380] In one embodiment, wherein the subject's putamen is administered or induced locally, half of the total dose is administered to each putamen of the subject. In other words, the total dose is substantially evenly divided between the left and right putamen of the subject.

[0381] An effective amount of rAAV is an amount sufficient to target an infected animal, or to target a desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to generate a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health, and tissue to be targeted of the subject, and may therefore vary from animal to animal and tissue to tissue. For example, the effective amount of rAAV is generally present in an amount containing about 10 9 to 10 16 In some cases, about 10 11 to 10 13 A dosage of between 10 and 100 rAAV genome copies is appropriate. 12 or 10 13 rAAV genome copies are effective for targeting CNS tissues (ie, putamen). In some cases, stable transgenic animals are generated from multiple doses of rAAV.

[0382] In one embodiment, rAAV is introduced or administered in the form of a liquid composition. In one embodiment, the rAAV concentration of the liquid composition is about 3×10 12 vg / mL to about 4×10 12 In one embodiment, the rAAV concentration of the liquid composition is about 1×10 12 vg / mL to about 4×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12 vg / mL, 1×10 12 vg / mL to about 3×10 12 vg / mL, 1×10 12 vg / mL to about 2×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12vg / mL, 8×10 11 vg / mL to about 9×10 12 vg / mL, 9×10 11 vg / mL to about 9×10 12 vg / mL, 1×10 12 vg / mL to about 9×10 12 vg / mL, 2×10 12 vg / mL to about 9×10 12 vg / mL, 3×10 12 vg / mL to about 9×10 12 vg / mL, 4×10 12 vg / mL to about 9×10 12 vg / mL, 5×10 12 vg / mL to about 9×10 12 vg / mL, 6×10 12 vg / mL to about 9×10 12 vg / mL, 7×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 11 vg / mL to about 8×10 12 vg / mL, 8×10 11 vg / mL to about 7×10 12 vg / mL, 8×10 11 vg / mL to about 6×10 12 vg / mL, 8×10 11 vg / mL to about 5×10 12 vg / mL, 8×10 11 vg / mL to about 4×10 12 vg / mL, 8×10 11 vg / mL to about 3×10 12 vg / mL, 8×10 11 vg / mL to about 2×10 12 vg / mL, 8×10 11 vg / mL to about 1×10 12 vg / mL, 8×10 11 vg / mL to about 9×10 11 vg / mL, 1×10 12 vg / mL to about 7×10 12 vg / mL, 3×10 12 vg / mL to about 6×10 12 vg / mL, 4×1012 vg / mL to about 5×10 12 vg / mL, 3.1×10 12 vg / mL to about 4×10 12 vg / mL, 3.2×10 12 vg / mL to about 4×10 12 vg / mL, 3.3×10 12 vg / mL to about 4×10 12 vg / mL, 3.4×10 12 vg / mL to about 4×10 12 vg / mL, 3.5×10 12 vg / mL to about 4×10 12 vg / mL, 3.6×10 12 vg / mL to about 4×10 12 vg / mL, 3.7×10 12 vg / mL to about 4×10 12 vg / mL, 3.8×10 12 vg / mL to about 4×10 12 vg / mL, 3.9×10 12 vg / mL to about 4×10 12 vg / mL, 3×10 12 vg / mL to about 3.9×10 12 vg / mL, 3×10 12 vg / mL to about 3.8×10 12 vg / mL, 3×10 12 vg / mL to about 3.7×10 12 vg / mL, 3×10 12 vg / mL to about 3.6×10 12 vg / mL, 3×10 12 vg / mL to about 3.5×10 12 vg / mL, 3×10 12 vg / mL to about 3.4×10 12 vg / mL, 3×10 12 vg / mL to about 3.3×10 12 vg / mL, 3×10 12 vg / mL to about 3.2×10 12 vg / mL, and 3×10 12 vg / mL to about 3.1×10 12 vg / mL.

[0383] In one embodiment, the rAAV concentration of the liquid composition is about 8×10 11 vg / mL, 9×1011 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 3.1×10 12 vg / mL, 3.2×10 12 vg / mL, 3.3×10 12 vg / mL, 3.4×10 12 vg / mL, 3.5×10 12 vg / mL, 3.6×10 12 vg / mL, 3.7×10 12 vg / mL, 3.8×10 12 vg / mL, 3.9×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, and 9×10 12 vg / mL.

[0384] In some embodiments, the rAAV dose is administered to the subject no more than once, for example, no more than once to each putamen. In some embodiments, the dose of rAAV is administered to the subject no more than once per calendar day (e.g., during a 24-hour period). In some embodiments, the dose of rAAV is administered to the subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of rAAV is administered to the subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, the dose of rAAV is administered to the subject no more than once every two weeks (e.g., once during two calendar weeks). In some embodiments, the dose of rAAV is administered to the subject no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, the dose of rAAV is administered to the subject no more than once every six calendar months. In some embodiments, the dose of rAAV is administered to the subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0385] In one embodiment, rAAV or a composition thereof is administered by infusion through a frontal (e.g., bifrontal) track. The frontal track is at least one track substantially perpendicular to the AP axis of each putamen and entering through the frontal bone of the skull. Frontal administration may include infusing rAAV through more than one track substantially perpendicular to the AP axis of each putamen. Each track can enter through a single borehole through the frontal bone of the skull or through different individual boreholes. Therefore, each of the left and right putamen requires at least one borehole on the frontal bone. For example, a frontal track (such as a bifrontal track) can be used for administration, wherein a cannula is guided from the frontal bone at a first track substantially perpendicular to the AP axis of the first putamen, and rAAV is infused using the first track. The cannula is then positioned at a second track substantially perpendicular to the AP axis of the first putamen (the second track is different from the first track), and rAAV is infused using the second track. The process is then repeated in the second putamen. It is understood that each track causes the cannula to contact the putamen at different locations. Fig.13A An exemplary transfrontal trajectory is shown.

[0386] In one embodiment, rAAV or a composition thereof is administered by infusion via a dual occipital trajectory. The occipital trajectory is a single posterior trajectory that is substantially parallel to the AP axis of the putamen and enters through the occipital bone of the skull. Thus, each putamen requires a drill hole in the occipital bone. For example, a dual occipital trajectory can be used for administration, wherein a cannula is guided from the occipital bone using a trajectory substantially parallel to the AP axis of the first putamen, and rAAV is infused while the cannula is advanced toward the rostral end of the first putamen. The process is then repeated at the second putamen. In one variation, rAAV is infused into each putamen while the cannula is fixed and not advanced to the rostral end of each putamen. Fig. 13B An exemplary occipital trajectory is shown.

[0387] In one embodiment, each putamen is infused using the same trajectory technique, i.e., each putamen is infused via a bifrontal trajectory. In one embodiment, each putamen is infused using a different trajectory technique, i.e., one putamen is infused via a bifrontal trajectory and the other putamen is infused via a bioccipital trajectory.

[0388] In one embodiment, the infusion volume of a single shell is less than or equal to 2000 μL, and greater than 1800 μL. For example, the total volume that can be infused to a single shell is 1800 μL, 1810 μL, 1820 μL, 1830 μL, 1840 μL, 1850 μL, 1860 μL, 1870 μL, 1880 μL, 1890 μL, 1900 μL, 1910 μL, 1920 μL, 1930 μL, 1940 μL, 1950 μL, 1960 μL, 1970 μL, 1980 μL, 1990 μL or 2000 μL. In one embodiment, the infusion volume of each shell is the same. In one embodiment, the infusion volume of each shell is different (for example, a shell infusion volume of 1800uL, another shell infusion volume of 2000um).

[0389] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., -10 13 GC / mL or higher). Methods for reducing rAAV aggregation are well known in the art, including, for example, the addition of surfactants, pH adjustment, salt concentration adjustment, etc. (see, for example, Wright FR et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0390] Dosage forms of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for use of the specific compositions described herein in a variety of treatment regimens.

[0391] One aspect described herein is a formulation for slowing or inhibiting the progression of PD in a subject, the formulation comprising any one of the viral vectors described herein (e.g., AAV) and a pharmaceutically acceptable carrier, wherein the concentration of the viral vector is 3×10 12 vg to 4×10 12 In one embodiment, the concentration of the viral vector is about 1×10 12 vg / mL to about 4×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12 vg / mL, 1×10 12 vg / mL to about 3×10 12 vg / mL, 1×10 12 vg / mL to about 2×10 12 vg / mL, 2×10 12 vg / mL to about 4×10 12vg / mL, 8×10 11 vg / mL to about 9×10 12 vg / mL, 9×10 11 vg / mL to about 9×10 12 vg / mL, 1×10 12 vg / mL to about 9×10 12 vg / mL, 2×10 12 vg / mL to about 9×10 12 vg / mL, 3×10 12 vg / mL to about 9×10 12 vg / mL, 4×10 12 vg / mL to about 9×10 12 vg / mL, 5×10 12 vg / mL to about 9×10 12 vg / mL, 6×10 12 vg / mL to about 9×10 12 vg / mL, 7×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 12 vg / mL to about 9×10 12 vg / mL, 8×10 11 vg / mL to about 8×10 12 vg / mL, 8×10 11 vg / mL to about 7×10 12 vg / mL, 8×10 11 vg / mL to about 6×10 12 vg / mL, 8×10 11 vg / mL to about 5×10 12 vg / mL, 8×10 11 vg / mL to about 4×10 12 vg / mL, 8×10 11 vg / mL to about 3×10 12 vg / mL, 8×10 11 vg / mL to about 2×10 12 vg / mL, 8×10 11 vg / mL to about 1×10 12 vg / mL, 8×10 11 vg / mL to about 9×10 11 vg / mL, 1×10 12 vg / mL to about 7×10 12 vg / mL, 3×10 12 vg / mL to about 6×10 12 vg / mL, 4×1012 vg / mL to about 5×10 12 vg / mL, 3.1×10 12 vg / mL to about 4×10 12 vg / mL, 3.2×10 12 vg / mL to about 4×10 12 vg / mL, 3.3×10 12 vg / mL to about 4×10 12 vg / mL, 3.4×10 12 vg / mL to about 4×10 12 vg / mL, 3.5×10 12 vg / mL to about 4×10 12 vg / mL, 3.6×10 12 vg / mL to about 4×10 12 vg / mL, 3.7×10 12 vg / mL to about 4×10 12 vg / mL, 3.8×10 12 vg / mL to about 4×10 12 vg / mL, 3.9×10 12 vg / mL to about 4×10 12 vg / mL, 3×10 12 vg / mL to about 3.9×10 12 vg / mL, 3×10 12 vg / mL to about 3.8×10 12 vg / mL, 3×10 12 vg / mL to about 3.7×10 12 vg / mL, 3×10 12 vg / mL to about 3.6×10 12 vg / mL, 3×10 12 vg / mL to about 3.5×10 12 vg / mL, 3×10 12 vg / mL to about 3.4×10 12 vg / mL, 3×10 12 vg / mL to about 3.3×10 12 vg / mL, 3×10 12 vg / mL to about 3.2×10 12 vg / mL, and 3×10 12 vg / mL to about 3.1×10 12 In one embodiment, the concentration of the viral vector is about 8×10 11 vg / mL, 9×10 11 vg / mL, 1×1012 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 3.1×10 12 vg / mL, 3.2×10 12 vg / mL, 3.3×10 12 vg / mL, 3.4×10 12 vg / mL, 3.5×10 12 vg / mL, 3.6×10 12 vg / mL, 3.7×10 12 vg / mL, 3.8×10 12 vg / mL, 3.9×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, and 9×10 12 vg / mL.

[0392] Typically, these dosage forms may contain at least about 0.1% active compound or more, although the percentage of active ingredient may vary of course and may conveniently be between about 1% or 2% to about 70% or 80% or more of the total dosage form weight or volume. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a manner that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical dosage forms will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and as such, a variety of dosages and treatment regimens may be desirable.

[0393] In certain cases, it will be desirable to deliver rAAV-based therapeutic constructs in appropriately formulated pharmaceutical compositions disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the modes of administration described in U.S. Pat. Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety) can be used to deliver rAAV. In some embodiments, the preferred mode of administration is injection via the portal vein.

[0394] The pharmaceutical form suitable for injectable use includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under general storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and flowing, reaching the extent of easy injection. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures and / or vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of microbial action can be brought about by a variety of antibacterial and antifungal agents (such as parabens, trichlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents (such as sugars or sodium chloride) will preferably be included. 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.

[0395] For example, for the use of injectable aqueous solutions, if necessary, the solution can be appropriately buffered, and the liquid diluent is first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this respect, operable sterile aqueous media will be known to those skilled in the art. For example, a dosage can be dissolved in 1mL isotonic NaCl solution, and added to 1000mL of subcutaneous perfusion (hypodermoclysis) fluid or injected at the recommended site of infusion (see, for example, " Remington's Pharmaceutical Sciences " the 15th edition, 1035-1038 pages and 1570-1580 pages). According to the situation of the host, some changes in dosage will definitely occur. In any case, the personnel responsible for administration will determine the appropriate dosage for individual hosts.

[0396] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV with the various other ingredients listed herein as required in an appropriate solvent followed by filtered sterilization. Dispersions are generally prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0397] The rAAV compositions disclosed herein may also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or with such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., hydroxides of sodium, potassium, ammonium, calcium or iron), and such organic bases as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be administered in a manner compatible with the formulation and in an amount such as therapeutically effective. The formulation is easily administered in a variety of formulation forms (e.g., injectable solutions, drug release capsules, etc.).

[0398] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspending agents, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic reaction or similar adverse reaction when administered to a host.

[0399] Delivery vehicles (e.g., liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, vesicles, etc.) can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, the rAAV vectors delivering the transgene can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0400] Such dosage forms may be preferably used for introducing a pharmaceutically acceptable dosage form of a nucleic acid or rAAV construct disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-life have been developed (U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like formulations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0401] Liposomes have been successfully used with many cell types that are usually resistant to other program transfections. In addition, liposomes are not limited by DNA length (typical in viral-based delivery systems). Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into various cultured cell lines and animals. In addition, several successful clinical trials have been completed to detect the effectiveness of liposome-mediated drug delivery.

[0402] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 Å to 500 Å, containing an aqueous solution in their core.

[0403] Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are considered.

[0404] In addition to the above delivery methods, the following technologies are also considered as alternative methods for delivering rAAV compositions to the host. Ultrasound introduction (i.e., ultrasound) has been used and is described in U.S. Patent No. 5,656,016 as a device for increasing the rate and efficacy of drug penetration into and through the circulatory system. Other drug delivery alternatives considered are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic dosage forms (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0405] In some embodiments, the methods described herein relate to treating a subject suffering from or diagnosed with PD with a nucleic acid described herein. For example, as described above, a subject suffering from PD can be identified by a clinician using current methods for diagnosing such diseases and disorders. Symptoms and / or complications of PD that characterize this condition and will aid in diagnosis, as well as clinical tests for making a diagnosis, are well known in the art and are described above. A family history of PD can also help determine whether a subject is likely to suffer from PD or diagnose PD.

[0406] In one embodiment, the subject has been diagnosed with PD prior to receiving a treatment as described herein. In one embodiment, the subject was diagnosed with 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.

[0407] In one embodiment, the subject has been diagnosed as being at risk for PD prior to receiving a treatment described herein.

[0408] In one embodiment, the subject has not been diagnosed as having or being at risk for PD prior to receiving a treatment described herein.

[0409] In one embodiment, the subject is diagnosed as having PD prior to receiving a treatment described herein. In one embodiment, the subject is diagnosed as being at risk for PD prior to receiving a treatment described herein.

[0410] In one embodiment, prior to administering the treatment, the person administering the treatment receives the results of a diagnostic analysis that diagnoses the subject as having PD. In one embodiment, prior to administering the treatment, the person administering the treatment receives the results of an assay that diagnoses the subject as being at risk for PD.

[0411] The compositions and methods described herein can be applied to subjects suffering from or diagnosed with PD. In some embodiments, the methods described herein include administering an effective amount of a composition described herein (e.g., a nucleic acid described herein) to a subject to alleviate the symptoms of PD. As used herein, "alleviating symptoms" refers to improving any condition or symptom associated with PD. Such reductions are at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more compared to an equivalent untreated control, measured according to any standard technique.

[0412] The effective amount, toxicity and therapeutic effect can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, for determining the minimum effective dose and / or the maximum tolerated dose. The dosage can vary according to the dosage form used and the route of administration used. The therapeutically effective dose can be estimated initially from the cell culture assay. In addition, the dosage can be formulated in an animal model to obtain a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dose can be monitored by appropriate bioassays (e.g., neuronal degradation or functional assays, etc.). The dosage can be determined by a doctor and adjusted if necessary to accommodate the observed therapeutic effect. Combination therapy

[0413] In one embodiment, at least one anti-PD therapeutic agent is administered to the subject prior to the introduction or administration of any of the rAAV described herein.

[0414] In one embodiment, following introduction or administration of any of the rAAVs described herein, at least one anti-PD therapeutic is administered to the subject.

[0415] In one embodiment, the at least one anti-PD therapeutic agent does not include Duopa.

[0416] In one embodiment, the rAAV described herein is used as a monotherapy. In one embodiment, the rAAV described herein can be used in combination with other known PD agents and therapies. "Combined" administration as used herein refers to delivering two (or more) different treatments to a subject during the subject's illness, for example, after the subject is diagnosed with PD and before the disease is cured or eliminated, or the treatment is stopped for other reasons, providing two or more treatments. In some embodiments, when the second delivery begins, the delivery of one treatment is still ongoing, so there is overlap in the administration. This is sometimes referred to as "simultaneous" or "concurrent delivery" in this article. In other embodiments, the delivery of one treatment ends before the start of another treatment. In some embodiments of either case, due to combined administration, the treatment is more effective. For example, the second treatment is more effective, for example, an equivalent effect can be seen with less second treatment than when the second treatment is administered without the first treatment, or the second treatment can alleviate symptoms to a greater extent, or a similar situation can be seen with the first treatment. In some embodiments, delivery reduces symptoms or other parameters associated with the disease more than observed when one treatment is delivered without providing another treatment. The effects of the two treatments can be partially added, fully added, or greater than added. Delivery can be such that the effect of the first treatment delivered is still detectable when the second is delivered. The rAAV and at least one additional therapy described herein can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the agent described herein can be administered first, followed by the additional agent, or the order of administration can be reversed. The agent and / or other therapeutic agents, procedures or modalities can be administered during an active disorder, or during remission or a less active disease. The agent can be administered before another treatment, simultaneously with treatment, after treatment, or during remission of a disorder.

[0417] Exemplary therapeutic agents for treating PD are described above.

[0418] When rAAV and an additional therapeutic agent (e.g., a second or third anti-PD therapeutic agent) are co-administered, they can be administered in an amount or dose that is higher, lower, or the same as the amount or dose of each therapeutic agent used alone (e.g., as a monotherapy). In certain embodiments, the additional therapeutic agent is administered in an amount or dose that 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 less) than the amount or dose of each additional therapeutic agent used alone.

[0419] In other embodiments, the amount or dosage of the additional therapeutic agent that produces a desired effect (e.g., inhibiting or slowing the progression of PD) 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 less) than the amount or dosage of each additional therapeutic agent required to achieve the same therapeutic effect alone.

[0420] In one embodiment, following the introduction or administration of any of the rAAVs described herein, the subject maintains the same amount or dosage of at least one anti-PD therapeutic agent.

[0421] In one embodiment, after the introduction or administration of any of the rAAVs described herein, the subject reduces the amount or dosage of at least one anti-PD therapeutic agent. In one embodiment, the amount or dosage of the at least one anti-PD therapeutic agent is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the amount or dosage taken before the introduction or administration of any of the rAAVs.

[0422] In one embodiment, following the introduction or administration of any of the rAAVs described herein, no anti-PD therapeutic is administered to the subject. Immunomodulators

[0423] In some embodiments, the compositions described herein include an immunomodulator, and the method further includes administering the immunomodulator. The immunomodulator can be administered at the time of administration, before administration, or after administration. In the case where the subject is administered at least a second composition again, the immunomodulator can be administered before, at the same time, or after at least the second administration.

[0424] In a preferred embodiment, the immunomodulator is used before the recombinant viral vector is used. In various embodiments, the immunomodulator is used at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours or longer before the recombinant viral vector is used. In one embodiment, the immunomodulator is used no more than 24 hours before the recombinant viral vector is used.

[0425] In one embodiment, the immunomodulator is administered substantially simultaneously with the recombinant viral vector, for example, administered slightly prior to administration of the recombinant viral vector disclosed herein (i.e., within 6 hours, or within 5 hours, or within 4 hours, or within 3 hours, or within 2 hours, or within 1 hour). In some embodiments, the immunomodulator is administered simultaneously with administration of the viral vector 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 the disclosed viral vector composition).

[0426] In some embodiments, the immunomodulator allows the recombinant viral vector to be administered to a subject, and the subject is not a good candidate for receiving this vector. A subject that is not a good candidate for receiving this vector is, for example, a subject that has previously received the administration of a recombinant viral vector and / or has previously been exposed to a recombinant viral vector and subsequently developed an antibody response to the vector. Typically, when the titer of the subject's viral vector binding antibody is less than 1:5 (e.g., 1:1, 1:2, 1:3 or 1:4), the subject is considered to be a candidate (i.e., a good candidate) for administering a recombinant viral vector. On the contrary, when the titer of the subject's viral vector binding antibody is 1:5 or higher (e.g., 1:6, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:50, 1:100, 1:1000 or higher), the subject is considered to be a candidate that is not suitable for administering a recombinant viral vector. Those skilled in the art can assess the antibody titer of a subject using standard techniques in the art, for example, by collecting a biological sample (e.g., blood from a subject), stimulating the biological sample with a known antigen, and detecting the presence of virus-binding antibodies to the known antigen. Antibody titer is a measure of how much a sample can be diluted before 50% viral vector neutralization can be detected in a sample. Antibody titers are usually expressed as ratios, such as 1:100, which means that one serum and 100 parts of saline solution (i.e., diluent) cause 50% antibody neutralization in the sample, that is, the mutual dilution of serum required to inhibit 50% viral infection can be designated as the neutralizing antibody titer at 50% inhibition. Therefore, a viral vector antibody titer of 1:10 indicates that the viral vector antibody level is lower than a 1:100 titer.

[0427] Thus, in one embodiment, the subject is assessed for the presence of anti-AAV antibodies to the AAV vector of the gene therapy prior to administration of the gene therapy. In one embodiment, the subject is assessed for the presence of neutralizing anti-AAV antibodies to the AAV vector of the gene therapy prior to administration of the gene therapy. Methods for detecting neutralizing anti-AAV antibodies are further described, for example, in Kasprzyk T. et al., Mol Therapy. Methods & Clinical Dev. January 6, 2022, the contents of which are incorporated herein by reference in their entirety.

[0428] In one embodiment, an immunomodulator is administered to a subject having a titer of viral vector binding antibodies present in a 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), wherein 1 part of the biological sample diluted in 10,000 parts of buffer produces 50% neutralization of the viral vector.

[0429] In one embodiment, the immunomodulator is administered to a subject having a titer of viral vector binding antibodies present in a blood product or biological sample 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), wherein dilution of 1 part of the biological sample or blood product in 10,000 parts of buffer produces 50% neutralization of the viral vector, to expand the pool of subjects that can be effectively treated with AAV gene therapy. Currently, potential patients with viral neutralizing antibody levels of 1:5 or higher are excluded from such treatment, i.e., they are not good candidates. Administration of an immunomodulator to a subject with an antibody titer greater than or equal to 1:5 but less than 1:10 is expected to reduce the existing antibody titer in the subject to less than 1:5, thereby qualifying the subject as a candidate for administration of a recombinant viral vector (e.g., a gene therapy vector).

[0430] In one embodiment, the immunomodulator is administered to a subject and the subject is found to have a titer of viral vector binding antibodies present in a 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, and 1:24), wherein 1 part of the biological sample or blood product is diluted in 10,000 parts of buffer to produce 50% neutralization of the viral vector. For example, administration of an immunomodulator to a subject having a titer greater than or equal to 1:5 but less than 1:15 is expected to reduce the existing antibody titer in the subject to less than 1:5, thereby qualifying the subject as a candidate for administration of a recombinant viral vector (e.g., a gene therapy vector). In one embodiment, the immunomodulator is administered to a subject having an antibody titer of viral vector binding antibodies present in a 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), wherein 1 part of the biological sample or blood product is diluted in 10,000 parts of the buffer to produce 50% neutralization of the viral vector. Administration of an immunomodulator to a subject with an antibody titer greater than or equal to 1:5 but less than 1:25 is expected to reduce the existing antibody titer in the subject to less than 1:5, thereby qualifying the subject as a candidate for administration of a recombinant viral vector (e.g., a gene therapy vector).

[0431] In some embodiments, the immunomodulator enables repeated administration or repeated use of the AAV vector disclosed herein. For example, the administration of the viral vector (e.g., AAV vector disclosed herein) and the immunomodulator (substantially simultaneously, before or after the administration of the AAV vector) can be administered multiple times (i.e., more than once) within a specified time period. For exemplary purposes, the AAV vector can be administered several times (i.e., more than once) within a few weeks (e.g., 2 weeks) to several months (e.g., 2 months). Without wishing to be limited by theory, as a non-limiting example, the administration of the AAV vector and the immunomodulator according to the method disclosed herein can be once a month within a 6-month period, 3 times-4 times within a 6-week period, and once a week within a 1 month (or about 4 weeks) or 2 months (or about 8 weeks) period. In some embodiments, when an AAV vector disclosed herein is administered multiple times (e.g., repeated administration) with an immunomodulator (at substantially the same time, before, or after administration of the AAV vector), the dose of the viral vector (e.g., AAV vector) is lower than the dose typically used in a single dose regimen, e.g., the dose is lower than the single dose regimen described herein. For example, the dose of the AAV vector can be less than or equal to about 10 12 , or less than about 10 12 For example, the dose may be about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 or about 10 12 , or 10 7 and 10 12 In some embodiments, when repeated doses of an AAV vector disclosed herein are administered to a subject together with an immunomodulator according to the methods disclosed herein, the immunomodulator can be changed between doses, i.e., the same or different immunomodulators can be used in repeated doses. For example, when a first dose of AAV is co-administered with immunomodulator A, the second immunomodulator administered with the second or third dose of AAV is different from immunomodulator A, e.g., the second immunomodulator is immunomodulator B. For exemplary purposes, a dosing regimen according to the disclosed methods can be administering a concentration of 10 12 or less than 10 12AAV vector, wherein the immunomodulator is administered as ABCD, or AABC, or ABAC, wherein A, B, C, and D are different immunomodulators disclosed herein. In other words, the dosing regimen may include multiple doses of immunomodulators within a time period, wherein each of the multiple doses includes an immunomodulator independently selected from immunomodulator A, immunomodulator B, immunomodulator C, immunomodulator D, and combinations thereof (ie, each dose may contain more than one immunomodulator). Such varying immunomodulators enable repeated administration of the AAV vectors disclosed herein. In some embodiments, for example, as disclosed herein, immunomodulator "A" may be, for example, IdeS, and immunomodulator "B" may be ImmTOR TM In some embodiments, an AAV vector disclosed herein is administered with an IdeS immunomodulator at a first time point, and an AAV vector disclosed herein is administered with a different immunomodulator (e.g., an immunoglobulin degradation protein or small molecule, such as ImmTOR) at a second time point. TM ) or vice versa. For example, the AAV vector disclosed herein can be administered at a first time point with ImmTOR TM An immunomodulatory agent may be administered together with the AAV vector disclosed herein, and at a second time point, the AAV vector may be administered together with IdeS.

[0432] One aspect of the present invention provides a method of administering a recombinant viral vector (e.g., a gene therapy vector) to a subject who has previously received a recombinant viral vector (e.g., the same recombinant viral vector or another viral vector with a similar serotype), the method comprising administering an immunomodulator to the subject prior to administering the recombinant viral vector. In one embodiment, the previously received recombinant viral vector elicits an immune response that produces anti-AAV antibodies that target (i.e., recognize and bind) the administered recombinant viral vector.

[0433] Another aspect of the present invention provides a method of administering a recombinant viral vector (e.g., a gene therapy vector) to a subject previously exposed to a viral vector, wherein the exposure elicits an immune response to produce anti-AAV antibodies that target the recombinant viral vector to be administered, and wherein the subject has an anti-AAV antibody titer of at least 1:5-1:15, 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 administering an immunomodulator to the subject prior to administering the recombinant viral vector.

[0434] In one embodiment, the immunomodulatory agent is administered systemically.

[0435] In some embodiments, the immunomodulator crosses the blood-brain barrier. In alternative embodiments, the immunomodulator does not cross the blood-brain barrier.

[0436] In one embodiment, the immunomodulator is administered topically. For example, when the recombinant viral vector is administered topically to brain tissue and the immunomodulator does not cross the blood-brain barrier, the immunomodulator is preferably administered topically to the brain tissue, for example, via a suitable catheter, either directly to the brain tissue or indirectly to the brain tissue via the cerebrospinal fluid circulating around the spinal cord (i.e., spinal puncture).

[0437] In one embodiment, the immunomodulator is administered topically 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, etc. Any composition described herein can be delivered directly to the CNS or brain by injection into, for example, the ventricular region and striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction or the cerebellar lobule.

[0438] In one embodiment, the immunomodulator is administered topically to any of the following: a neural pathway, a somatosensory system, a visual system, an auditory system, a nerve, a neuroendocrine system, a neurovascular system, a brain neurotransmitter system, a dura mater system, or a combination thereof.

[0439] In one embodiment, the immunomodulator is administered topically to the eye, such as the vitreous, retina, or sclera.

[0440] In one embodiment, the immunomodulatory agent is administered systemically.

[0441] In some embodiments, the immunomodulator is an immunoglobulin degrading enzyme, such as IdeS, IdeZ, IdeS / Z, Endo S, or a functional variant thereof. Non-limiting examples of such 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 is incorporated by reference in its entirety.

[0442] In some embodiments, the immunomodulators disclosed herein can be administered to a subject at any suitable dose, such as a suitable dose determined by a medical professional. For example, a suitable dose can be from about 0.05 mg / kg subject weight to about 5 mg / kg subject weight, or from about 0.1 mg / kg subject weight to about 4 mg / kg subject weight.

[0443] In some embodiments, an immunomodulator disclosed herein (e.g., IdeZ) is administered at a dose of about 0.01 mg / kg to about 10 mg / kg of subject weight. For example, a suitable dose can be about 0.05 mg / kg to about 5 mg / kg of subject weight, or about 0.1 mg / kg to about 4 mg / kg of subject weight.

[0444] In some embodiments, an immunomodulator disclosed herein (e.g., IdeS) is administered at a dose of about 0.01 mg / kg subject weight to about 10 mg / kg subject weight. For example, a suitable dose can be about 0.05 mg / kg subject weight to about 5 mg / kg subject weight, or about 0.1 mg / kg subject weight to about 4 mg / kg subject weight.

[0445] In some embodiments, an immunomodulator disclosed herein (e.g., EndoS) is administered at a dosage of about 0.01 mg / kg subject weight to about 10 mg / kg subject weight. For example, a suitable dosage can be about 0.05 mg / kg subject weight to about 5 mg / kg subject weight, or about 0.1 mg / kg subject weight to about 4 mg / kg subject weight.

[0446] Further, with respect to numerical ranges, for example, "0.01 to 10" includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, 10, etc., and the like. For example, a dose of "about 0.01 mg / kg to about 10 mg / kg of subject weight" 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 the like.

[0447] In various embodiments, prior to administering the recombinant viral vector to the subject, a protease and / or glycosidase is administered to inhibit, reduce or prevent an immune response (e.g., a humoral immune response) to the recombinant viral vector or an antibody that binds to a heterologous polynucleotide or a peptide or protein encoded by a heterologous polynucleotide, which is encapsulated by the viral vector. For example, the protease and / or glycosidase may be administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours; or at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days prior to administering the viral vector. , at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days; or at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months; or at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years or more.

[0448] In one embodiment, administration of a recombinant viral vector to a subject is performed simultaneously with administration of a protease and / or glycosidase to inhibit, reduce or prevent an immune response (e.g., a humoral immune response) to the recombinant viral vector or antibodies that bind to a heterologous polynucleotide or a peptide or protein encoded by a heterologous polynucleotide that is packaged by the viral vector.

[0449] In a specific embodiment, a protease and / or glycosidase is administered to a subject prior to an immune response (e.g., a humoral immune response) (e.g., prior to the development of neutralizing antibodies or antibodies that bind to a heterologous polynucleotide, a peptide or protein encoded by a heterologous polynucleotide, which is packaged by a viral vector). In one embodiment, the 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 after administration of the recombinant viral vector; or within 1 day, 2 days, 3 days, 4 days, 5 days or more.

[0450] In some embodiments, the immunomodulator is a proteasome inhibitor. In some embodiments, the immunomodulator is a protease or a glycosidase. In particular aspects, the proteasome inhibitor is bortezomib. In some aspects of the embodiments, the immunomodulator includes bortezomib and an anti-CD20 antibody, such as rituximab. In other aspects of the embodiments, the immunomodulator includes 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 US10,028,993, US 9,592,247, and US 8,809,282, each of which is incorporated herein by reference in its entirety.

[0451] In alternative embodiments, the immunomodulator is an inhibitor of NF-κB pathways. In particular aspects of the embodiments, the immunomodulator is rapamycin or a functional variant thereof. Non-limiting examples of the use of rapamycin are described below, each of which is incorporated herein by reference in its entirety: US10,071,114, US20160067228, US20160074531, US 20160074532, US20190076458, US10,046,064. In other aspects of the embodiments, the immunomodulator is a synthetic nanocarrier comprising an immunosuppressant. Non-limiting examples of immunosuppressants, immunosuppressants coupled to synthetic nanocarriers, synthetic nanocarriers comprising rapamycin and / or tolerogenic synthetic nanocarriers, their dosages, administrations, and uses are described in US20150320728, US20180193482, US20190142974, US20150328333, US20160243253, US10,039,822, US20190076522, US20160022650, US 10,441,651, US10,420,835, US20150320870, US2014035636, US10,434,088, US10,335,395, US20200069659, US10,357,483, US20140335186, US 10,668,053, US10,357,482, US20160128986, US20160128987, US 20200038462, US20200038463, each of which is incorporated herein by reference in its entirety.

[0452] In some embodiments, the immunomodulatory agent comprises a synthetic nanocarrier comprising rapamycin (ie, ImmTOR TM Nanoparticles), as disclosed in Kishimoto et al., 2016, Nat Nanotechnol, 11(10):890-899; Maldonado et al., 2015, PNAS, 112(2):E156-165 and US20200038463, U.S. Patent 9,006,254, each of which is incorporated herein by reference in its entirety. In some embodiments, the immunomodulator is an engineered cell, such as an immune cell modified using the SQZ technology described in WO2017192786 (incorporated herein by reference in its entirety).

[0453] In some embodiments, the immunomodulator is selected from the group consisting of poly-ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, carrier system, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys and Aquila's QS21 stimulators and combinations thereof. In another further embodiment, the immunomodulator or poly-ICLC.

[0454] In some embodiments, the immunomodulator is a small molecule that inhibits the innate immune response in the cell, such as chloroquine (TLR signaling inhibitor) and / or 2-aminopurine (PKR inhibitor), which can also be administered in combination with a composition comprising at least one rAAV as disclosed herein. Some non-limiting examples of commercially available TLR signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available from INVIVOGEN). In addition, inhibitors of pattern recognition receptors (PRRs) (which are involved in innate immune signaling) (e.g., 2-aminopurine, BX795, chloroquine, and H-89) can also be used in compositions and methods comprising at least one rAAV vector as disclosed herein to perform in vivo protein expression as disclosed herein.

[0455] In some embodiments, the immunomodulator is photopheresis, also known as extracorporeal photochemotherapy or ECP. Photopheresis treatment is performed on the blood of the subject. Using IV or catheter, blood is transported from the subject by a device that separates some white blood cells (leukocytes). The separated white blood cells are treated with a naturally occurring photosensitive chemical called 8-methoxypsoralen (8-MOP) and then exposed to ultraviolet (UVA) light of a specific wavelength. After exposure to UVA light, the blood is administered back to the subject. Photopheresis can be performed at least once a day. In one embodiment, before administering the recombinant viral vector, photopheresis is performed at least once a week, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times. In one embodiment, prior to administration of the recombinant viral vector, photopheresis is performed at least once a week, at least twice a week, at least three times a week, at least four times a week, at least five times a week, at least six times a week, at least seven times a week, for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least ten weeks, at least ten weeks, at least ten months ...

[0456] In some embodiments, the rAAV vector with a modified viral capsid may also encode a negative regulator of innate immunity (e.g., NLRX1). Thus, in some embodiments, the rAAV vector may also optionally encode one or more of NLRX1, NS1, NS3 / 4A, or A46R. In addition, in some embodiments, the composition comprising at least one rAAV vector as disclosed herein may also comprise a synthetic, modified RNA encoding inhibitor of the innate immune system to avoid an innate immune response generated by a tissue or subject.

[0457] In some embodiments, the immunomodulator used in the method for administration disclosed herein is an immunosuppressive drug or an immunosuppressant. As used herein, the term "immunosuppressive drug or immunosuppressant" refers to an agent that inhibits or interferes with normal immune function. Examples of immunosuppressive drugs or immunosuppressants suitable for methods disclosed herein include agents that inhibit T-cell / B-cell costimulatory pathways (e.g., agents that interfere with T-cells and B-cells through CTLA4 and B7 pathway coupling), as disclosed in U.S. Patent Publication No. 2002 / 0182211, which are incorporated herein by reference in their entirety. In one embodiment, the immunosuppressant is cyclosporin A. Other examples of immunosuppressants include myophenylatemofetil, rapamycin, and antithymocyte globulin. In each embodiment, the immunosuppressive drug is administered in a composition comprising at least one rAAV vector as disclosed herein, or administered with a separate composition, but administered simultaneously with, or before, or after the administration of a composition comprising at least one rAAV vector according to the method for administration disclosed herein. The immunosuppressive drug is administered in a formulation compatible with the route of administration and is administered to the subject at a dose sufficient to achieve the desired therapeutic effect. In some embodiments, the immunosuppressive drug is administered transiently for a sufficient period of time to induce tolerance to the rAAV vector disclosed herein.

[0458] In any embodiment of the methods and compositions disclosed herein, the subject to whom the compositions disclosed herein are administered is also administered an immunosuppressant. It is known that a variety of methods can achieve immunosuppression of the immune response in patients administered AAV. Methods known in the art include administering an immunosuppressant (e.g., a proteasome inhibitor) to a patient. One such proteasome inhibitor known in the art is bortezomib, as disclosed in, for example, U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15 / 796,137, both of which are incorporated herein by reference as a whole. In some embodiments, the immunosuppressant is an antibody, including polyclonal, monoclonal, scfv or other antibody-derived molecules capable of suppressing an immune response, for example, by eliminating or suppressing cells producing antibodies. In a further embodiment, the immunosuppressive element is a short hairpin RNA (shRNA). In this embodiment, the coding region of the shRNA is contained in the rAAV box and is generally located downstream, i.e., 3' of the poly-A tail. shRNAs can be targeted to reduce, decrease or eliminate the expression of immunostimulants such as cytokines, growth factors including transforming growth factor β1 and β2, TNF and other growth factors known in the art.

[0459] The use of such immunomodulators facilitates the ability to use multiple dosing (e.g., multiple administrations) over months and / or years. This allows the use of multiple agents, such as rAAV vectors encoding multiple genes, or multiple administrations to a subject as discussed below. definition

[0460] For convenience, the meanings of some terms and phrases used in this specification, examples and appended claims are provided below. Unless otherwise specified or implied in the context, the following terms and phrases include the meanings provided below. The definitions are provided to help describe specific embodiments and are not intended to limit the technology described herein as claimed, as the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the technology belongs. If there is a clear difference between the use of a term in the art and its definition provided herein, the definition provided in this specification shall prevail.

[0461] For convenience, certain terms used herein, in the specification, examples and appended claims, are collected here.

[0462] An "effective amount" of a substance refers to an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect in the case of rAAV-mediated delivery) a sufficient number of target cells in a subject's target tissue. In some embodiments, the target tissue is a central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, an effective amount of an isolated nucleic acid (e.g., which can be delivered by rAAV) can be an amount sufficient to have a therapeutic benefit in a subject (e.g., reducing or stabilizing the subject's MDS-UPDRS score, extending the subject's lifespan, improving one or more disease symptoms (e.g., PD symptoms), etc.). The effective amount will depend on various factors, such as the subject's species, age, weight, health status, and tissue to be targeted, and may therefore vary from subject to subject and tissue, as described elsewhere in this disclosure.

[0463] The terms "decrease", "reduced / reduction", or "inhibit" are all used herein to indicate a statistically significant amount of reduction. In some embodiments, "decrease" or "reduction" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more reduction. As used herein, "reduction" or "inhibit" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The reduction may preferably be down to a level that is acceptable as being within the normal range for individuals not having a given disorder.

[0464] The terms "increased / increase", "enhance" or "activate" are all used herein to indicate an increase in a statistically significant amount. In some embodiments, the terms "increase", "enhance" or "activate" may represent an increase of at least 10% compared to a reference level, such as 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% compared to a reference level, or up to and including a 100% increase or any increase between 10%-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in the level.

[0465] As used herein, "subject" means a person or non-human animal. Typically, non-human animals are vertebrates, such as primates, rodents, livestock or hunting animals. Primates include chimpanzees, crab-eating monkeys, spider monkeys and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Livestock and hunting animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches) and fish (e.g., trout, catfish and salmon). In some embodiments, the subject is a mammal, such as a primate (e.g., human). The terms "individual," "patient," and "subject" are used interchangeably herein.

[0466] Preferably, the subject is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of PD. The subject may be male or female.

[0467] The subject may be a subject who has previously been diagnosed with or identified as suffering from or having a condition (e.g., PD) or one or more complications associated with such a condition that requires treatment, and optionally has undergone treatment for the condition or one or more complications associated with the condition. Alternatively, the subject may also be a subject who has not previously been diagnosed with the condition or one or more complications associated with the condition. For example, the subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition or a subject who does not exhibit such risk factors.

[0468] A "subject in need" of treatment for a particular disorder (eg, PD) may be a subject having the disorder, diagnosed as having the disorder, or at risk of developing the disorder.

[0469] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino groups and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to small polypeptides, but the usage of these terms in the art overlaps. When it comes to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologues, paralogues, fragments, and other equivalents, variants, fragments and analogs described above.

[0470] 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 the native or reference sequence. For example, the degree of homology (percent identity) between the native sequence and the mutant sequence can be determined by comparing the two sequences using a commonly used free available computer program for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0471] The change of the native amino acid sequence can be achieved by any of a variety of techniques known to those skilled in the art. For example, oligonucleotides containing the mutant sequence can be synthesized and flanked by restriction sites to be able to be connected to fragments of the native sequence, thereby introducing mutations at a specific locus. After connection, the resulting reconstructed sequence encodes an analog with a desired amino acid insertion, displacement or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis program can be used to provide a changed nucleotide sequence with a specific codon that changes according to a desired displacement, deletion or insertion. The technology for making such changes is well established and includes, for example, the technology disclosed by Walder et al., (Gene 42: 133, 1986); Bauer et al., (Gene 37: 73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al., (Genetic Engineering: Principles and Methods, Plenum Press, 1981) and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine ​​residue that is not involved in maintaining the correct conformation of the polypeptide may also be replaced, usually with serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to a polypeptide to increase its stability or promote oligomerization.

[0472] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or their analogs. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid strand of a denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid may be DNA. In another aspect, the nucleic acid may be RNA. Suitable DNA may include, for example, genomic DNA or cDNA. Suitable RNA may include, for example, mRNA, miRNA.

[0473] In some embodiments of any aspect, a polypeptide, nucleic acid, or cell as described herein may be engineered. As used herein, "engineered" refers to aspects that have been artificially manipulated. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide (e.g., its sequence) has been artificially manipulated so that it is different from the aspect that exists in nature. As is commonly practiced and understood by those skilled in the art, the descendants of engineered cells are still generally referred to as "engineered", even if the actual manipulations were performed on a previous entity.

[0474] 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 the native or reference sequence. For example, the degree of homology (percent identity) between the native sequence and the mutant sequence can be determined by comparing the two sequences using a commonly used free available computer program for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0475] The term "exogenous" refers to a substance present in a cell that is not its natural source. The term "exogenous" when used herein may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced into a biological system (e.g., a cell or an organism) in which it is not normally present through a process involving human intervention, and people wish to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that has been introduced into a biological system (e.g., a cell or an organism) in which it is present in a relatively low amount through a process involving human intervention, and people wish to increase the amount of the nucleic acid or polypeptide in the cell or organism (e.g., to produce ectopic expression or level). In contrast, the term "endogenous" refers to a natural substance of a biological system or cell. As used herein, "ectopic" refers to a substance that exists in an unusual position and / or amount. Ectopic substances may be substances that are normally present in a given cell, but exist in much less amount and / or at different times. Ectopic also includes substances, such as polypeptides or nucleic acids that are not naturally present or expressed in a given cell in its natural environment.

[0476] As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral. The term "vector" includes any genetic element that can replicate and transfer a gene sequence to a cell when associated with appropriate control elements. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, cosmids, chromosomes, viruses, virions, etc.

[0477] In some embodiments of any aspect, the vector is recombinant (e.g., it comprises sequences derived from at least two different sources). In some embodiments of any aspect, the vector comprises sequences derived from at least two different species. In some embodiments of any aspect, the vector comprises sequences derived from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product, the nucleic acid being operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, a repressor, an activator, an enhancer, a response element, etc.).

[0478] In some embodiments of any aspect, the vectors or nucleic acids described herein are 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 the altered or engineered nucleic acid encodes a polypeptide expression product identical to the native / wild-type sequence, but will be transcribed and / or translated with improved efficiency in the desired expression system. In some embodiments of any aspect, the expression system is an organism (or a cell obtained from such an organism) other than the source of the native / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon optimized for expression in mammals or mammalian cells (e.g., mice, murine cells, or human cells). In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon optimized for expression in bacterial cells. In some embodiments of any of the aspects, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in E. coli cells.

[0479] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides by a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is usually (but not necessarily) heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms (e.g., in human cells for expression and in prokaryotic hosts for cloning and amplification).

[0480] As used herein, the term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may comprise a nucleic acid encoding a polypeptide as described herein instead of a non-essential viral gene. The vector and / or particle may be used for the purpose of transferring any nucleic acid to a cell in vitro or in vivo. Various forms of viral vectors are known in the art. Non-limiting examples of viral vectors described herein include AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, herpes virus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0481] It should be understood that, in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is an additional type. Using a suitable additional type vector provides a means for maintaining high copy number extrachromosomal DNA of nucleotides of interest in a subject, thereby eliminating the potential impact of chromosome integration.

[0482] As used herein, the term "treat / treatment / treating" or "amelioration" refers to a therapeutic treatment, wherein the purpose is to reverse, alleviate, relieve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., PD). The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as assessed by reducing or stabilizing the initial (i.e., before administration) MDS-UPDRS score of the subject. If one or more symptoms or clinical markers are reduced, the treatment is generally "effective". Alternatively, if the progression of the disease is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing down of the progression or deterioration o...

Claims

1. A method for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising: introducing into the subject a recombinant adeno-associated virus (rAAV), wherein the recombinant rAAV comprises a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein at least 30% of the subject's putamen volume is transduced with the GDNF transgene, and wherein the subject does not show an increase in PD-related symptoms for at least 6 months after introduction compared to before introduction.

2. The method of claim 1, wherein: The rAAV is introduced by systemic introduction.

3. The method of claim 1, wherein: The rAAV is introduced by topical introduction.

4. The method of claim 3, wherein: Topical introduction is introduction directly into the putamen of said subject.

5. The method according to claim 3 or 4, wherein: The local introduction comprises introducing the rAAV directly into each of the putamen of the subject.

6. The method according to any one of claims 3 to 5, wherein: The local introduction is performed simultaneously with non-invasive imaging.

7. The method of claim 6, wherein: The 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); electroencephalogram (EEG); magnetoencephalogram (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof.

8. The method according to any one of claims 3 to 7, wherein: The local introduction includes introducing approximately half of the rAAV vector into each putamen by convection-enhanced delivery (CED) guided by intraoperative magnetic resonance imaging (iMRI).

9. The method according to any one of claims 3 to 8, wherein: The local introduction further comprises introducing an MRI contrast agent substantially simultaneously with the AAV vector.

10. The method of claim 8, wherein: The MRI contrast agent is gadoteridol.

11. The method according to claim 9 or 10, wherein: The MRI contrast agent is introduced into the subject in the same composition as the rAAV.

12. The method according to claim 9 or 10, wherein: The MRI contrast agent is introduced into the subject in separate compositions from the rAAV.

13. The method of claim 1, wherein: The rAAV is introduced by systemic introduction.

14. The method according to any one of claims 1 to 13, wherein: Transduction and / or coverage of the putamen is assessed by magnetic resonance imaging.

15. The method according to any one of claims 1 to 14, wherein: At least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the subject's putamen volume is transduced with the GDNF transgene.

16. The method according to any one of claims 1 to 15, wherein: The subject does not show a significant increase in PD-related symptoms for at least 12 months immediately following introduction compared to before introduction.

17. The method according to any one of claims 1 to 15, wherein: The subject exhibits a reduction in PD-related symptoms for at least 6 months or more immediately following introduction compared to before introduction.

18. The method of any one of claims 1 to 15, wherein: The subject exhibits a reduction in PD-related symptoms for at least 12 months or more immediately following introduction compared to before introduction.

19. The method of claim 1, wherein: Prior to introduction, the subject had an initial Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) score of less than 32.

20. The method of claim 19, wherein: The slowing or inhibiting the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score 6 months following introduction being not substantially higher than the initial MDS-UPDRS score.

21. The method of claim 19 or 20, wherein: The slowing or inhibiting the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score about 12 months following introduction being not substantially higher than the initial MDS-UPDRS score.

22. The method of claim 1, wherein: Prior to introduction, the subject's initial MDS-UPDRS score was greater than or equal to 32.

23. The method of claim 22, wherein: The subject exhibits a reduction in the initial MDS-UPDRS score at least 6 months immediately following introduction compared to before introduction.

24. The method of claim 22 or 23, wherein: The slowing or inhibiting the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score about 6 months following introduction that is at least about 20% lower than the initial MDS-UPDRS score.

25. The method of any one of claims 21 to 23, wherein: The slowing or inhibiting the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score about 12 months following introduction that is at least about 30% lower than the initial MDS-UPDRS score.

26. The method of claim 1, further comprising determining the subject's initial MDS-UPDRS score prior to introduction.

27. The method of claim 1, further comprising, prior to introduction, receiving an assay result providing an initial MDS-UPDRS score for the subject.

28. The method of claim 1, wherein: Slowing or inhibiting the progression of PD in the subject is characterized by a decrease in the initial MDS-UPDRS score following introduction.

29. The method of claim 28, wherein: The reduction is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more reduction in the initial MDS-UPDRS score 6 months after introduction.

30. The method of claim 28, wherein: The reduction is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more reduction in the initial MDS-UPDRS score 12 months after introduction.

31. The method of claim 1, wherein: Slowing or inhibiting the progression of PD in the subject is characterized by stabilization of the initial MDS-UPDRS score following introduction.

32. The method of claim 31, wherein: The stabilization is characterized by an increase or decrease of no more than 10% in the initial MDS-UPDRS score.

33. The method of claim 31 or 32, wherein: Stabilization occurs for at least 6 months or longer.

34. The method of claim 1, wherein: The subject was mildly affected by PD.

35. The method of claim 34, wherein: Prior to the introduction of rAAV, the subjects mildly affected by PD had an initial MDS-UPDRS score of less than 32 and were diagnosed with PD less than 5 years prior to the introduction.

36. The method of claim 1, further comprising, prior to introduction, diagnosing the subject as mildly affected by PD.

37. The method of claim 1, further comprising, prior to introducing, receiving an assay result diagnosing that the subject is mildly affected by PD.

38. The method of claim 1, wherein: The subject was moderately affected by PD.

39. The method of claim 38, wherein: Prior to the introduction of rAAV, the subjects moderately affected by PD had an initial MDS-UPDRS score equal to or greater than 32 and were diagnosed with PD less than 4 years prior to the introduction.

40. The method of claim 1, further comprising, prior to introduction, diagnosing the subject as moderately affected by PD.

41. The method of claim 1, further comprising, prior to introduction, receiving an assay result diagnosing that the subject is moderately affected by PD.

42. The method of claim 1, wherein: The promoter is a cytomegalovirus (CMV) promoter.

43. The method of claim 1, wherein: The promoter is a nervous system (NS) or central nervous system (CNS) specific promoter.

44. The method of claim 43, wherein: The NS-specific promoter is selected from the NS-specific promoters in Table 1.

45. The method of claim 43, wherein: The CNS-specific promoter is selected from the CNS-specific promoters in Table 2.

46. ​​The method of claim 1, wherein: The GDNF transgene comprises the 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.

47. The method of claim 1, wherein: The rAAV is AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10 or a reasonable haploid thereof.

48. The method of claim 47, wherein: The rAAV is AAV2.

49. The method of claim 1, wherein: The rAAV exhibits brain-specific tropism.

50. The method of claim 1, wherein: The rAAV comprises modifications that increase its brain-specific tropism.

51. The method of claim 50, wherein: The brain-specific tropism is increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to unmodified AAV.

52. The method of claim 1, wherein: 5×10 12 vg is about 1.5×10 13 The rAAV was introduced at a total dose ranging from 1:1 to 1:

1.

53. The method of claim 52, wherein: Approximately half of the total dose is administered to each putamen of the subject.

54. The method of claim 3, wherein: The introduction was performed at a flow rate of about 1 μL / min to about 30 μL / min.

55. The method of claim 1, wherein: The rAAV is introduced in a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier.

56. The method of claim 55, wherein: The rAAV concentration of the liquid composition is about 3×10 12 vg / mL to about 4×10 12 vg / mL.

57. The method of claim 1, wherein: Prior to introducing the rAAV, at least one anti-PD therapeutic agent is administered to the subject.

58. The method of claim 1, wherein: At least one anti-PD therapeutic agent is administered to the subject before and after the introduction of the rAAV.

59. The method of claim 57 or 58, wherein: The at least one anti-PD therapeutic agent is selected from the group consisting of: levodopa, Sinemet, Rytary, Darlingfor, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, and entacapone.

60. The method of any one of claims 57-59, wherein: The subject maintains or reduces the dose of the at least one anti-PD therapeutic agent after introduction.

61. The method of claim 60, wherein: The dose of the at least one anti-PD therapeutic is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.

62. A method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising: locally introducing into the putamen of the subject a recombinant adeno-associated virus (rAAV) vector comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein at least 30% of the putamen volume of the subject is transduced with the GDNF transgene.

63. A method of slowing or inhibiting the progression of PD in a subject in need thereof, the method 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) transgene, wherein the subject shows no significant increase in PD-related symptoms for at least 6 months after transduction.

64. The method of claim 63, wherein: The transduction is performed by administering a rAAV comprising the GDNF transgene to each putamen of the subject.

65. A method of reducing or stabilizing an initial Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) score in a subject with Parkinson's disease (PD), the method comprising: administering to the putamen of the subject a recombinant adeno-associated virus (rAAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein, at 6 months after the administration, the subject has a second MDS-UPDRS score that is reduced or stabilized compared to the subject's initial MDS-UPDRS score before the administration.

66. The method of claim 65, further comprising the step of obtaining or receiving an initial MDS-UPDRS score from the subject prior to administration.

67. The method of claim 65, wherein: Twelve months after administration, the second MDS-UPDRS score is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the initial MDS-UPDRS score.

68. The method of claim 65, wherein: Stabilization was an increase or decrease of no more than 10% in the initial MDS-UPDRS score.

69. A method of treating a subject mildly affected by Parkinson's disease (PD), the method comprising: administering to each of the putamen of the subject a recombinant adeno-associated virus (rAAV), the recombinant rAAV comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein at least 30% of the putamen of the subject is transduced with the GDNF transgene, and wherein, at 6 months after the administration, the subject has a second MDS-UPDRS score that is stable compared to the initial MDS-UPDRS score.

70. The method of claim 69, wherein: At 12 months after the administration, the subject had a stable MDS-UPDRS score compared to the initial MDS-UPDRS score before the administration.

71. The method of claim 69 or 70, wherein: Stabilization was an increase or decrease of no more than 10% in the initial MDS-UPDRS score.

72. A method of treating a subject moderately affected by Parkinson's disease (PD), the method comprising: administering to each of the putamen of the subject a recombinant adeno-associated virus (AAV), the recombinant AAV comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein at least 30% of the putamen of the subject is transduced with the GDNF transgene, and wherein at 6 months after the administration, the subject has a second MDS-UPDRS score that is at least about 20% lower than the initial MDS-UPDRS score.

73. The method of claim 72, wherein: The reduction is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the initial MDS-UPDRS score.

74. A method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising: introducing into each local area of ​​the putamen of the subject a recombinant adeno-associated virus (rAAV) vector comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter; as well as locally introducing an MRI contrast agent into each of the putamen of the subject substantially simultaneously with the rAAV, wherein at least 30% of the putamen volume of the subject is transduced with the transgene, and wherein the subject does not show a significant increase in PD-related symptoms for at least 6 months immediately following the introduction compared to before the introduction.

75. A method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the method comprising: introducing into the subject a recombinant adeno-associated virus (rAAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, wherein at least 30% of the subject's putamen volume is transduced with the GDNF transgene, and wherein the subject does not show a significant increase in PD-related symptoms for at least 6 months immediately following the introduction compared to before the introduction.

76. A composition for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, the composition comprising: a recombinant adeno-associated virus (rAAV) comprising a genome comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter; and Pharmaceutically acceptable carrier.

77. The composition of claim 76, wherein The rAAV concentration of the composition is 3×10 12 vg / mL to 4×10 12 vg / mL.

78. The composition of claim 76, wherein The composition comprises a concentration of 3.3×10 12 vg / mL of rAAV.

79. A preparation for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, the preparation comprising: a concentration of 3×10 12 vg / mL pharmaceutically acceptable carrier to 4×10 12 vg / mL adeno-associated virus (AAV) of a pharmaceutically acceptable carrier, in, The rAAV comprises a genome comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter.

80. A method as claimed in any preceding claim, wherein: The subject did not experience any serious adverse events for at least 6 months immediately following introduction or administration.

81. Use of a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, hereinafter referred to as a GDNF transgene, in the preparation of a medicament for slowing, inhibiting or stabilizing the progression of Parkinson's disease (PD) in a subject or treating a subject with a mild form of PD, in, At least 30% of the putamen volume is transduced with the GDNF transgene, and wherein the subject does not exhibit an increase in PD-related symptoms for at least 6 months immediately following the introduction of the agent compared to before the introduction of the agent.

Images