Methods and Compositions for the Treatment of Parkinson's Disease

Abstract

The 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 GDNF nucleic acid sequence.In some embodiments, administration is localized to the putamen of a subject.In some embodiments, administration is systemic, for example, via a viral vector comprising modified viral capsid, for example, to preferentially target cells in the CNS or PNS.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 63 / 323,830, filed March 25, 2022; 63 / 326,236, filed March 31, 2022; 63 / 341,841, filed May 13, 2022; 63 / 393,196, filed July 28, 2022; and 63 / 438,164, filed January 10, 2023, the contents of each of which are incorporated herein by reference in their entirety.

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

[0003] government support This invention was made in the 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 this invention. [Background technology]

[0004] background Parkinson's disease (PD) is a progressive neurodegenerative disorder that progresses relentlessly over a 10-30 year period until disability and death occur. Pharmacotherapy, generally those that help correct known striatal dopamine deficiencies, can provide substantial clinical benefit for the cardinal motor signs of PD: resting tremor, rigidity, bradykinesia, and postural instability. However, because dopamine replacement and other medical treatments do not affect the underlying neurodegenerative process, disease progression continues. Clinical response to antiparkinsonian medications diminishes over time, and a variety of drug-related complications ensue, including motor fluctuations, dyskinesia, and neuropsychiatric symptoms.

[0005] Deep brain stimulation (DBS) is a reasonable and effective symptomatic treatment option for specific cardinal 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. More recently, Duopa® has been approved for more advanced patients with severe motor fluctuations. Duopa® is a levodopa / carbidopa enteral gel administered via a gastrostomy tube connected to an external portable pump to provide constant medication. While this avoids intracranial surgery, Duopa® requires the need to maintain a stoma site and the inconvenience of carrying external components. Due to Duopa® oxidation, this therapy is approved for 16 hours per day, and therefore, some patients remain inadequately treated overnight.

[0006] Research efforts have pointed to several potential mechanisms that may underlie the neurodegenerative process in PD. Oxidative stress, mitochondrial dysfunction, and abnormalities in intracellular protein processing are commonly postulated mechanisms. Experimental therapy studies are designed to correct such pathological disturbances with the intent of slowing, preventing, or reversing the neurodegenerative process. Neurotrophic factors such as GDNF have the potential to modify the course of PD, not just treat specific clinical features.

[0007] PD is a progressive, multicentric neurodegenerative disease characterized by resting tremor, rigidity, bradykinesia, and postural instability. Most cases of PD are idiopathic and are the second most common neurodegenerative disorder after Alzheimer's disease. Patients struggle with emotional symptoms, including depression and anxiety, as well as characteristic motor features and movement disorders. Currently, there is no cure for PD, and treatment options are limited to ameliorating disease symptoms. Summary of the Invention [Means for solving the problem]

[0008] Abstract One aspect provided herein describes a method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, 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 (sometimes referred to as a transgene), and wherein the subject does not exhibit an increase in PD-related symptoms for at least 6 months after the introduction compared to before the introduction.

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

[0010] In one embodiment of any aspect herein, the rAAV is introduced via local introduction.

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

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

[0013] In one embodiment of any aspect of the present specification, localized introduction is carried out 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); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof.

[0014] In one embodiment of any aspect of the present specification, the local introduction comprises introducing approximately half of the total delivered dose of the rAAV vector into each putamen via intraoperative magnetic resonance imaging (iMRI)-guided convection-enhanced delivery (CED).

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

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

[0017] In one embodiment of any aspect herein, the MRI contrast agent is introduced to the subject in the same composition as the rAAV. In one embodiment of any aspect herein, the MRI contrast agent is introduced to the subject in a different composition than the rAAV.

[0018] In one embodiment of any aspect herein, the rAAV is introduced via systemic (e.g., intravenous) introduction.

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

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

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

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

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

[0024] In one embodiment of any aspect herein, slowing or inhibiting the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score immediately after induction, 6 months after induction, that is not substantially higher than the initial MDS-UPDRS score.

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

[0026] In one embodiment of any aspect herein, the subject has an initial MDS-UPDRS score of greater than or equal to 32 prior to induction.

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

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

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

[0030] In one embodiment of any aspect herein, the method further comprises, prior to the introducing, determining an initial MDS-UPDRS score for the subject.

[0031] In one embodiment of any aspect herein, the method further includes, prior to the introducing, receiving results of an assay that provides an initial MDS-UPDRS score for the subject.

[0032] In one embodiment of any aspect herein, slowing or inhibiting the progression of PD in a subject is characterized by a reduction in the initial MDS-UPDRS score after induction.

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

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

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

[0036] In one embodiment of any aspect herein, the stabilization is characterized by an increase or decrease of 10% or less in the initial MDS-UPDRS score. In one embodiment of any aspect herein, the stabilization occurs for at least 6 months or longer.

[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 the introduction.

[0038] In one embodiment of any aspect herein, the method further comprises, prior to the 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 results of an assay that diagnoses the subject as being 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 had 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 aspect herein, the method further comprises, prior to the introducing, diagnosing the subject as being moderately affected by PD.

[0042] In one embodiment of any aspect herein, the method further comprises, prior to the introducing, receiving results of an assay that diagnoses the subject as being 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 specification, the promoter is a nervous system (NS) or central nervous system (CNS) specific promoter. In one embodiment of any aspect of the present specification, the NS specific promoter is selected from the NS specific promoters in Table 1. In one embodiment of any aspect of the present specification, 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 of the present specification, 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 of the present specification, the rAAV is AAV2.

[0047] In one embodiment of any aspect of the present specification, the rAAV exhibits brain-specific tropism. In one embodiment of any aspect of the present specification, the rAAV comprises a modification that increases its brain-specific tropism. In one embodiment of any aspect of the present specification, 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, the rAAV is 5×10 12 vg~approx. 1.5×10 13 It is introduced at a total dose in the range of vg.

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

[0050] In one embodiment of any aspect 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 as a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier.

[0052]

[0023] In one embodiment of any aspect herein, the liquid composition comprises about 3 x 10 12 vg / mL ~ approx. 4×10 12 with an rAAV concentration of 1000 mg / mL.

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

[0054] In one embodiment of any aspect herein, the subject is administered at least one anti-PD therapeutic agent before and after rAAV introduction. In one embodiment of any aspect herein, 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 the at least one anti-PD therapeutic agent after induction. 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, comprising locally introducing into the subject's putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the volume of the subject's putamen is transduced with the GDNF gene.

[0057] Another aspect provided herein describes a method of slowing or inhibiting the progression of PD in a subject in need thereof, comprising transducing greater than or equal to about 30% of the volume of the subject's putamen with a glial cell line-derived neurotrophic factor (GDNF) gene, wherein the subject does not exhibit a substantial increase in PD-related symptoms for at least six months after transduction. In one embodiment of any aspect herein, the transduction is carried out by administering an rAAV containing the GDNF gene to each of the subject's putamen.

[0058] Another embodiment provided herein describes a method of reducing or stabilizing an initial Movement Disorder Society Unified Parkinson's Disease Rating Scale-Part 1 (MDS-UPDRS) score in a subject with Parkinson's disease (PD), comprising administering to the subject's putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the subject has a second MDS-UPDRS score at 6 months after administration that is reduced or stabilized compared to the subject's initial MDS-UPDRS score before the administration.

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

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

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

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

[0063] Another aspect provided herein describes a method of treating a subject moderately affected by Parkinson's disease (PD), comprising administering to each of the subject's putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject's putamen is transduced with the transgene, and the subject has a second MDS-UPDRS score 6 months after administration that 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 of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, comprising: locally introducing into each of the subject's putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing into each of the subject's putamen an MRI contrast agent 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 the subject does not exhibit a substantial increase in PD-related symptoms for at least six months immediately following the introduction compared to before the introduction.

[0065] Another aspect provided herein describes a method of slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, 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 wherein the subject does not exhibit a substantial increase in PD-related symptoms for at least six months immediately following the introduction 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 that includes 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 composition comprises 3 x 10 12 vg~4×10 12 With an rAAV concentration of 1000 mg / mL.

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

[0069] Another aspect provided herein is a formulation for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, comprising 3 x 10 hydroxybenzoates per mL of a pharmaceutically acceptable carrier. 12 vg~4×10 12 A formulation is described that contains adeno-associated virus (AAV) at a concentration of 1000 ng / vg, where the rAAV contains a genome that includes the glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter. [Brief explanation of the drawings]

[0070] [Figure 1]Figure 1 shows a schematic of the clinical study schedule. Subjects cycle "on" and "off" their prescribed antiparkinsonian medications, as indicated by the "off" (off-drug therapy) and "on" (on-drug therapy) arrows. MRI, FDG, and DaT scans are administered, as indicated by crosses. Blood studies are obtained, as indicated by drops. Subject activity is monitored, as indicated by hexagons. For visits requiring evaluation in a defined off-drug state, participants are asked to stop all PD medications (e.g., carbidopa / levodopa, Sinemet, Rytary, Stalevo, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, entacapone) from the night before the visit and must abstain for at least 12 hours.

[0071] [Figure 2] Figure 2 shows a summary of subjects included in Cohort A (mildly affected by PD).

[0072] [Figure 3] Figure 3 shows a summary of subjects included in Cohort B (moderately affected by PD).

[0073] [Figure 4] Figure 4 shows a summary of the percent volume of putamen in subjects transduced with GDNF after local administration of AAV2-GDNF. The mean volume of putamen transduced with GDNF is 63%.

[0074] [Figure 5] 5 depicts a bar graph showing the percent volume of putamen in subjects transduced with GDNF after local administration of AAV2-GDNF operably linked to a CMV promoter. The average volume of putamen transduced with GDNF is approximately 63%.

[0075] [Figure 6A-B]Figures 6A and 6B show representative postoperative MRIs of subjects in Cohort A. Figure 6A shows an MRI T1 (pre-contrast) image after a period of therapeutic drug administration. Figure 6B shows an MRI T2 image after a period of therapeutic drug administration. Various manifestations of putamen hyperintensity are observed.

[0076] [Figure 7A-B] Figures 7A and 7B show representative postoperative MRI scans of subjects in Cohort B. Figure 7A shows an MRI T1 (pre-contrast) image after a period of therapeutic drug administration. Figure 7B shows an MRI T2 image after a period of therapeutic drug administration. Various manifestations of putamen hyperintensity are observed.

[0077] [Figure 8A-B] Figures 8A-8H present data assessing PD progression at various time points after administration. Figures 8A-8D present line graphs showing changes in MDS-UPDRS aggregate motor skills either on (Figure 8A) or off (Figure 8B) antiparkinsonian medication. Stabilized MDS-UPDRS scores are observed in Cohort A (represented by the "mild PD cohort") over 12 months. A significant decrease in MDS-UPDRS scores is 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 scores (Figure 8C) and MDS-UPDRS II scores (Figure 8D) follow the same trends over the indicated times after surgery. Figures 8E-8H show stable motor measures over 18 months after AAV2-GDNF administration in the mild PD cohort (Figures 8E and 8F) and motor improvement over 18 months after AAV2-GDNF administration in the moderate PD cohort (Figures 8G and 8H). In Figures 8E and 8F, (A) shows the stability demonstrated over 18 months in mild PD. (B) shows the limited window for measuring significant functional improvement in mild PD. (C) shows one outlier identified as having a TH mutation. [Figure 8C-D]Figures 8A-8H present data assessing PD progression at various time points after administration. Figures 8A-8D present line graphs showing changes in MDS-UPDRS aggregate motor skills either on (Figure 8A) or off (Figure 8B) antiparkinsonian medication. Stabilized MDS-UPDRS scores are observed in Cohort A (represented by the "mild PD cohort") over 12 months. A significant decrease in MDS-UPDRS scores is 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 scores (Figure 8C) and MDS-UPDRS II scores (Figure 8D) follow the same trends over the indicated times after surgery. Figures 8E-8H show stable motor measures over 18 months after AAV2-GDNF administration in the mild PD cohort (Figures 8E and 8F) and motor improvement over 18 months after AAV2-GDNF administration in the moderate PD cohort (Figures 8G and 8H). In Figures 8E and 8F, (A) shows the stability demonstrated over 18 months in mild PD. (B) shows the limited window for measuring significant functional improvement in mild PD. (C) shows one outlier identified as having a TH mutation. [Figure 8E-F]Figures 8A-8H present data assessing PD progression at various time points after administration. Figures 8A-8D present line graphs showing changes in MDS-UPDRS aggregate motor skills either on (Figure 8A) or off (Figure 8B) antiparkinsonian medication. Stabilized MDS-UPDRS scores are observed in Cohort A (represented by the "mild PD cohort") over 12 months. A significant decrease in MDS-UPDRS scores is 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 scores (Figure 8C) and MDS-UPDRS II scores (Figure 8D) follow the same trends over the indicated times after surgery. Figures 8E-8H show stable motor measures over 18 months after AAV2-GDNF administration in the mild PD cohort (Figures 8E and 8F) and motor improvement over 18 months after AAV2-GDNF administration in the moderate PD cohort (Figures 8G and 8H). In Figures 8E and 8F, (A) shows the stability demonstrated over 18 months in mild PD. (B) shows the limited window for measuring significant functional improvement in mild PD. (C) shows one outlier identified as having a TH mutation. [Figure 8G-H]Figures 8A-8H present data assessing PD progression at various time points after administration. Figures 8A-8D present line graphs showing changes in MDS-UPDRS aggregate motor skills either on (Figure 8A) or off (Figure 8B) antiparkinsonian medication. Stabilized MDS-UPDRS scores are observed in Cohort A (represented by the "mild PD cohort") over 12 months. A significant decrease in MDS-UPDRS scores is 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 scores (Figure 8C) and MDS-UPDRS II scores (Figure 8D) follow the same trends over the indicated times after surgery. Figures 8E-8H show stable motor measures over 18 months after AAV2-GDNF administration in the mild PD cohort (Figures 8E and 8F) and motor improvement over 18 months after AAV2-GDNF administration in the moderate PD cohort (Figures 8G and 8H). In Figures 8E and 8F, (A) shows the stability demonstrated over 18 months in mild PD. (B) shows the limited window for measuring significant functional improvement in mild PD. (C) shows one outlier identified as having a TH mutation.

[0078] [Figure 9A-B] Figures 9A-9D present data showing PD exercise diary data. (Figures 9A-9B) Bar graphs showing PD exercise diary data for Cohort A (Figure 9A) and Cohort B (Figure 9B). Diary "on / off" time is normalized to 16 hours of waking time. (Figure 9C) Pie chart showing significant improvement in subjects in Cohort B at 12 and 18 months post-dose. Good "on" time improved by 27% from baseline, and "off" time improved by 52% from baseline. (Figure 9D) Pie chart showing significant improvement in subjects in Cohort A at 12 and 18 months post-dose. Good "on" time decreased by 12% from baseline, and "off" time decreased by 46% from baseline. [Figure 9C]Figures 9A-9D present data showing PD exercise diary data. (Figures 9A-9B) Bar graphs showing PD exercise diary data for Cohort A (Figure 9A) and Cohort B (Figure 9B). Diary "on / off" time is normalized to 16 hours of waking time. (Figure 9C) Pie chart showing significant improvement in subjects in Cohort B at 12 and 18 months post-dose. Good "on" time improved by 27% from baseline, and "off" time improved by 52% from baseline. (Figure 9D) Pie chart showing significant improvement in subjects in Cohort A at 12 and 18 months post-dose. Good "on" time decreased by 12% from baseline, and "off" time decreased by 46% from baseline. [Figure 9D] Figures 9A-9D present data showing PD exercise diary data. (Figures 9A-9B) Bar graphs showing PD exercise diary data for Cohort A (Figure 9A) and Cohort B (Figure 9B). Diary "on / off" time is normalized to 16 hours of waking time. (Figure 9C) Pie chart showing significant improvement in subjects in Cohort B at 12 and 18 months post-dose. Good "on" time improved by 27% from baseline, and "off" time improved by 52% from baseline. (Figure 9D) Pie chart showing significant improvement in subjects in Cohort A at 12 and 18 months post-dose. Good "on" time decreased by 12% from baseline, and "off" time decreased by 46% from baseline.

[0079] [Figure 10A-B] Figures 10A and 10B represent plot graphs showing NMSS (circles) and PDQ-39 (squares) for Cohort A (mildly affected; Figure 10A) and Cohort B (moderately affected; Figure 10B). NMSS and PDQ-39 scores were assessed at pre-treatment screening, 6 months post-surgery, 12 months post-surgery, 18 months post-surgery, and 24 months post-surgery.

[0080] [Figure 11]Figure 11 depicts a line graph showing the dose response of AAV2-GDNF in Cohort B (moderately affected) for Phase 1 of the clinical trial. The data demonstrate a correlation of dose and putamen coverage (>50%) with clinical response in mid-stage PD. The magnitude of functional motor improvement in mid-stage PD exceeded the expected placebo effect (-5 pt).

[0081] [Figure 12] Figure 12 shows a graph depicting the volume distribution of putaminal infusions containing gene therapy. As predicted in animal models, this is highly dependent on the infusion volume delivered. For clinical PD cases, the average unilateral putaminal volume is approximately 4200 cubic mm. As represented below the dotted horizontal line in this figure, early gene therapy products infused into the putamen provided a limited volume of distribution, significantly less than 50% of the total putaminal volume. This initial limitation in volume of distribution was primarily a result of the small infusion volume delivered and the use of a standard bifrontal trajectory to the putamen. The standard bifrontal approach provides a trajectory nearly perpendicular to the long axis of the putamen; such trajectory volume coverage of the putamen is limited by the small dorsoventral putaminal dimensions and requires multiple trajectories to expand volume coverage. Deploying gene therapy infusions parallel to the long axis of the putamen provided much larger infusion volumes (up to 1800 microliters / putamen) and achieved >50% putaminal coverage.

[0082] [Figure 13A-B]Figures 13A and 13B show schematic diagrams of the bilateral frontal and bilateral occipital trajectory techniques. (Figure 13A) Bilateral frontal trajectory—One or more frontal burr holes are created on each side. A minimum of two trajectories per putamen is required to cover the anterior and posterior commissural putamen. The trajectories are approximately 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, volume coverage of the putamen is <50%. (Figure 13B) Bilateral occipital trajectory—A single occipital burr hole is created per putamen. This technique requires a single trajectory per putamen to cover the anterior and posterior commissural putamen. The trajectory is parallel to the long axis of the putamen, and volume coverage is limited primarily by perivascular leakage from within the putamen. Typically, volume coverage of the putamen is >50%.

[0083] [Figure 14A]Figures 14A and 14B present summaries of previously completed and currently ongoing clinical trials. Figure 14A presents a chart showing clinical experience with both bilateral frontal and bilateral occipital delivery methods for AAV2-GDNF gene therapy to the putamen in Parkinson's disease. Figure 14A provides details from previously completed Phase 1 clinical trials and ongoing clinical trials testing the safety and tolerability of different vector doses and putamen coverage in advanced, intermediate, and early-stage PD. The current clinical trial (described in Examples 1-3 herein below) is the first human gene therapy trial approved to test the safety of a gene therapy product in participants with early-stage 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 450 microliters of infusion volume (9 x 1010 vg - 9 x 1011 vg) to each putamen in 13 participants, resulting in a mean putaminal coverage of 26%. Bilateral occipital delivery in the clinical trials described herein has provided a maximum of 1800 microliters of infusion in each putamen to date in 11 participants, providing a mean putaminal distribution of 62.5%. Figure 14B presents a summary of the putaminal coverage achieved in the previously completed Phase 1 trial and the current clinical trial (Phase 1b; as described in Examples 1-3 herein below) for the indicated cohort. [Figure 14B]Figures 14A and 14B present summaries of previously completed and currently ongoing clinical trials. Figure 14A presents a chart showing clinical experience with both bilateral frontal and bilateral occipital delivery methods for AAV2-GDNF gene therapy to the putamen in Parkinson's disease. Figure 14A provides details from previously completed Phase 1 clinical trials and ongoing clinical trials testing the safety and tolerability of different vector doses and putamen coverage in advanced, intermediate, and early-stage PD. The current clinical trial (described in Examples 1-3 herein below) is the first human gene therapy trial approved to test the safety of a gene therapy product in participants with early-stage 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 450 microliters of infusion volume (9 x 1010 vg - 9 x 1011 vg) to each putamen in 13 participants, resulting in a mean putaminal coverage of 26%. Bilateral occipital delivery in the clinical trials described herein has provided a maximum of 1800 microliters of infusion in each putamen to date in 11 participants, providing a mean putaminal distribution of 62.5%. Figure 14B presents a summary of the putaminal coverage achieved in the previously completed Phase 1 trial and the current clinical trial (Phase 1b; as described in Examples 1-3 herein below) for the indicated cohort.

[0084] [Figure 15] Figure 15 presents a schematic diagram of AAV-GDNF. CMV, cytomegalovirus; hGDNF, human glial cell line-derived neurotrophic factor; hGH, human growth hormone; ITR, inverted terminal repeat.

[0085] [Figure 16] Figure 16 presents a schematic of the study design. AAV2-GDNF was administered via a single MRI-monitored CED into both putamen (maximum dose of 1.2 x 10 vg, maximum 1.8 mL per putamen) and contrast agent (2 mM gadoteridol) to visualize distribution.

[0086] [Figure 17-1] Figure 17 presents a summary of post-operative adverse effects (ie, investigational treatment-emergent adverse events (TEAEs)) observed more than one month after surgery. [Figure 17-2] Figure 17 presents a summary of post-operative adverse effects (ie, investigational treatment-emergent adverse events (TEAEs)) observed more than one month after surgery.

[0087] [Figure 18A] Figures 18A and 18B present a summary of individual post-treatment changes across exercise and non-exercise assessments for the mild (Figure 18A) and moderate (Figure 18B) cohorts. [Figure 18B] Figures 18A and 18B present a summary of individual post-treatment changes across exercise and non-exercise assessments for the mild (Figure 18A) and moderate (Figure 18B) cohorts.

[0088] [Figures 19A-F] Figures 19A-19F present data showing AAV2-GDNF expression 3.5 years after administration. (Figure 19A) MRI image of a participant who received intraputaminar administration of AAV2-GDNF. (Figures 19B-19D) Tyrosine hydroxylase staining of a putamen biopsy sample showing enrichment of dopaminergic neurons in the putamen. Figure 19B is the area in Figure 19A indicated by the arrow. Figure 19C shows a condensed, enlarged image of the area in Figure 19B indicated by the arrow. Figure 19D shows a condensed, enlarged image of the area in Figure 19C indicated by the arrow. (Figure 19E) Location of biopsy 6 performed on the sample. Biopsy locations 1 and 5 are the injection sites used during surgery. Biopsy location 6 is located outside the putamen in the white matter tract. (Figure 19F) GDNF transgene levels (pg GDNF / mg protein) at the indicated biopsy locations. The highest levels of GDNF were found in locations 1 and 5. GDNF expression was not identified in biopsy site #6.

[0089] [Figure 20]Figure 20 presents data showing longitudinal MRI monitoring for safety readout. T1 (top row) and T2 (bottom row) weighted MRI brain scans in the left column show gadoteridol distribution (bright white signal from T1 images) after bilateral injection into the putamen (lateral). Matched MRI scans obtained at 6 and 18 months demonstrate no residual gadoteridol signal or tissue abnormalities in the putamen or other brain structures.

[0090] [Figure 21] Figure 21 presents a chart depicting the response of the moderate PD cohort. Stronger, more progressive repair and motor function were found compared with previous CGT. The 18-month clinical data demonstrate: (1) stronger improvement than previous neurotrophic CGT; (2) AAV2-GDNF effects are more progressive than previous neurotrophic GTx, with continued improvement after 6 months, unlike the short-term improvements seen in other CGTs; and (3) clinically meaningful improvement over 6 months, consistent with the expected mechanism of action, e.g., terminal sprouting and progressive repair of dopamine function.

[0091] [Figure 22A-B] Figures 22A and 22B present charts showing the Unified Dyskinesia Rating Scale historical, objective, and total scores up to 18 months post-treatment for the mild (Figure 22A) and moderate (Figure 22B) cohorts.

[0092] [Figure 23A-B] 23A and 23B present charts showing the mean levadopa equivalent daily dose (LEDD) for the mild (FIG. 23A) and moderate (FIG. 23B) cohorts up to 18 months post-treatment.

[0093] [Figure 24A-C]Figures 24A-24C present data showing a preliminary analysis of functional imaging by DaT scans in the mild and moderate cohorts. Figure 24A presents a bar graph of values. Figures 24B and 24C show tables presenting the values ​​presented in Figure 24A for the mild (Figure 24B) and moderate (Figure 24C) cohorts. A preliminary analysis of changes in DAT binding over time is shown. Decreased binding in the caudate nucleus is observed in both the mild and moderate cohorts. Relatively stable or increased put him of DaT signal in both cohorts is shown.

[0094] [Figure 25A-D] Figures 25A-25D present data showing changes in F-dopa uptake at the injection site 6 and 18 months after gene therapy administration. (Figures 25A-25C) MRI images show gadoteridol distribution in the axial (left column) and coronal (right column) planes after bilateral injections into the medial (anterior commissural) and posterior (posterior commissural) putamen (Figure 25A). F-dopa Ki parametric maps in the axial and coronal planes from one patient at baseline (Figure 25B) and 18 months after surgery (Figure 25C) show increases in Ki in the regions corresponding to the injection sites visualized as gadoteridol signals on MRI.

[0095] [Figure 26] FIG. 26 provides a schematic diagram of the plasmid used to generate the AAV2-GDNF vector, e.g., SEQ ID NO:64. DETAILED DESCRIPTION OF THE INVENTION

[0096] Detailed Description Aspects of the technology disclosed herein relate to the administration, for example, local or systemic administration, of a glial cell line-derived neurotrophic factor (GDNF) gene such that at least 30% of a subject's putamen is covered and / or transduced with the gene. This level of coverage and / or transduction indicates effectiveness in reducing, slowing down, or inhibiting the progression of symptoms related to PD. Thus, the methods and compositions described by the present disclosure are useful, in some embodiments, for the treatment of PD.

[0097] Parkinson's disease (PD) The 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 progressively worsening shaking and stiffness, and increasing problems with balance, walking, and coordination. The majority of PD is considered idiopathic in nature, typically due to a combination of genetic predisposition and environmental influences acting in epigenetic regulation, including transcription factors such as synuclein alpha (SNCA; NCBI gene ID: 6622), leucine-rich repeat kinase 2 (LRRK2 / PARK8; NCBI gene ID 120892), glucosylceramidase beta (GBA1; NCBI gene ID 2629), parkin RBR E3 ubiquitin (PRKN; NCBI gene ID 5071), PTEN-induced kinase 1 (PINK1; NCBI gene ID 65018), parkinsonism-associated deglycase (DJ1 / PARK7; NCBI gene ID 11315), VPS35 retromer complex component (VPS35; NCBI gene ID 55737), and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1; NCBI gene ID 11315). Several genetic mutations can contribute to or increase the risk of PD, including mutations in the PD-associated genes PD1 (DnaJC13; NCBI Gene ID 23317), 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). The sequences of such PD-associated genes are known in several species; for example, human mRNA and protein sequences are available in the NCBI database using the provided Gene ID numbers.These PD-associated genes and others, as well as their PD-associated alleles (e.g., mutations, duplications, SNPs, etc.), are known in the art and are described in, e.g., D'Souza et al. Acta Neuropsychiatrica 2020 32:10-22; Sardi et al. Parkinsonism & Related Disorders 2019 59:32-38; Hardy et al. Current Opinion in Genetics & Development 2009 19:254-65; Ferreria et al. 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 the development of PD include, but are not limited to, age, heredity, exposure to certain toxins, and gender. Early-onset and young-adult PD diagnoses are rare. The risk of developing Parkinson's increases with age, beginning in middle to late life, with subjects typically developing the disease around the age of 60 or older. Having a close relative (e.g., a close family member, uncle, aunt, or grandparent) with PD increases a subject's chance of developing the disease. However, unless multiple blood relatives have been diagnosed with PD, the risk is still considered small. Continuous exposure to certain herbicides and pesticides has been shown to slightly increase a subject's risk of PD. Finally, men are more likely to develop PD than women.

[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, tremor (e.g., shaking that usually begins in the limbs, often the hands or fingers, when the body is at rest); pill-rolling tremor (e.g., rubbing the thumb and pointing finger back and forth when the body is at rest); slow movements (bradykinesia); stiff muscles (i.e., muscle stiffness in any part of the body that may be painful and limit the range of motion); impaired posture and balance (e.g., posture may become frozen or balance problems may be present); loss of automatic movements (e.g., decreased ability to perform involuntary movements, including blinking, smiling, or swinging the arms while walking); changes in speech (e.g., speaking more smoothly and faster, slurring, or hesitation before speaking, or changes in tone and loss of inflection); and changes in handwriting (e.g., handwriting may appear smaller and crowded).

[0100] Complications of PD include, but are not limited to, cognitive problems (dementia) and difficulty thinking in later stages of PD; depression and emotional changes (i.e., fear, anxiety, or loss of motivation) in early and late stages of Parkinson's; swallowing problems (e.g., difficulty swallowing, saliva accumulation, and drooling) as the condition progresses; chewing and eating problems in later stages of PD, which can lead to shortness of breath and nutritional deficiencies; sleep problems and disorders (i.e., frequent awakenings, waking early, and daytime dozing); REM sleep behavior disorder; bladder problems (i.e., not being able to control urination or having difficulty urinating); constipation; orthostatic hypotension (i.e., a sudden drop in blood pressure); olfactory dysfunction (e.g., loss of smell or difficulty identifying certain odors or the difference between odors); fatigue; pain, i.e., either in a specific area of ​​the body or throughout the body; and sexual dysfunction.

[0101] There is no single specific test for diagnosing the presence of Parkinson's disease; rather, a skilled clinician diagnoses a subject through a subject's medical records, family history, existing signs and / or symptoms, and neurological and physical examination. A specific single-photon emission computed tomography (SPECT) scan, called a dopamine transporter scan (DaTscan), is unlikely to be critical for diagnosis but may be performed to support 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 disorders but are not useful in diagnosing PD. Furthermore, subjects suspected of having Parkinson's disease may be administered sufficient (i.e., high) doses of antiparkinsonian medications (e.g., carbidopa-levodopa) and monitored for symptomatic improvement; improvement after administration would indicate / confirm a diagnosis of PD.

[0102] Treatments for PD include, but are not limited to, therapeutic agents designed to treat ongoing symptoms of the disease, including, but not limited to, carbidopa-levodopa, inhaled carbidopa-levodopa, infused carbidopa-levodopa, dopamine agonists, MAO B inhibitors, catechol O-methyltransferase (COMT) inhibitors, anticholinergics, and amantadine.

[0103] Levodopa, the most effective PD medication, is a natural chemical that travels to the brain and is converted to dopamine. Levodopa is typically combined with carbidopa (e.g., Lodosyn® carbidopa), which protects levodopa from premature conversion of dopamine outside the brain and prevents or reduces side effects such as nausea. As the disease progresses to later stages, the benefits from levodopa may wax and wane and become more erratic (i.e., "wearing off"). Involuntary movements (dyskinesias) are associated with higher doses of levodopa. Inbrija® levodopa inhalation powder is a therapeutic drug that delivers an inhaled form of levodopa. Duopa™ carbidopa / levodopa suspension is a brand of medication consisting of carbidopa and levodopa administered via a gastrostomy tube so that the medication is delivered directly to the small intestine in gel form. Duopa™ carbidopa / levodopa suspension is for patients with more advanced Parkinson's who still respond to carbidopa-levodopa, but whose response fluctuates considerably. Duopa™ is given as a continuous infusion so that blood levels of the two drugs (carbidopa and levodopa) remain constant.

[0104] Unlike levodopa, dopamine agonists do not convert to dopamine, but rather mimic the action of dopamine in the patient's brain.Dopamine agonists are less effective than levodopa in treating PD symptoms, but they last longer and can be used together with levodopa to support the off- and on-effects of levodopa.Exemplary dopamine agonists include pramipexole (e.g., Mirapex® pramipexole), ropinirole (e.g., Requip® ropinirole), rotigotine (e.g., Neupro® rotigotine transdermal system, administered as a patch), and apomorphine (e.g., Apokyn® apomorphine), a short-acting injectable dopamine agonist.

[0105] MAO B inhibitors help prevent the breakdown of dopamine in the brain by inhibiting the brain enzyme monoamine oxidase B (MAO B), which metabolizes brain dopamine. Exemplary MAO B inhibitors include selegiline (e.g., Zelapar® selegiline hydrochloride), rasagiline (e.g., Azilect® rasagiline), and safinamide (e.g., Xadago® safinamide). Administration of selegiline and levodopa has been shown to help prevent wearing-off.

[0106] Catechol O-methyltransferase (COMT) inhibitors moderately prolong the effects of levodopa therapy by blocking the enzyme that destroys dopamine. Exemplary COMT inhibitors include entacapone (e.g., Comtan® entacapone), opicapone (e.g., Ongentys® opicapone), and tolcapone (e.g., Tasmar® tolcapone). Tolcapone is rarely prescribed due to the risk of serious liver damage and liver failure.

[0107] Anticholinergic drugs have previously been administered to help control the tremors associated with PD. Exemplary anticholinergic drugs include antipsychotics (clozapine, quetiapine), atropine, benztropine (e.g., Cogentin® benztropine mesylate), biperiden, chlorpheniramine, certain SSRIs (paroxetine), dicyclomine (dicycloverine), dimenhydrinate, diphenhydramine, doxepin (Doxepi), doxylamine, flavoxate, glycopyrrolate, glycopyrronium, hyoscyamine, ipratropium, orphenadrine, oxitropium, oxybutynin, promethazine, propantheline bromide, scopolamine, solifenacin, tolterodine, tiotropium, tricyclic antidepressants, trihexyphenidyl, tropicamide, and umeclidinium.

[0108] Amantadine (e.g., Gocovri® amantadine) is an antidyskinetic medication prescribed as monotherapy to provide short-term relief of symptoms in mild, early-stage PD. It is additionally prescribed along with carbidopa-levodopa therapy during later stages of PD to control carbidopa-levodopa-induced involuntary movements (dyskinesias).

[0109] Patients with PD can also undergo surgery to implant deep brain stimulation (DBS) to reduce disease-related symptoms. DBS involves implanting electrodes in specific parts of the patient's brain, 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 an inconsistent, fluctuating response to levodopa or for controlling dyskinesias that do not improve with adjustments to medication. DBS is more commonly used in later-stage patients who exhibit an unstable response to medication, such as levodopa.

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

[0111] One aspect provided herein relates to a method of slowing or inhibiting the progression of PD in a subject in need thereof, 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 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.

[0112] One aspect provided herein relates to a method of slowing or inhibiting the progression of PD in a subject in need thereof, 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 introducing the rAAV results in at least 30% coverage of the subject's putamen with 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 comprises two bilaterally symmetric, oval-shaped subcortical lobes elongated longitudinally along the anterior-posterior (AP) axis. As used herein, and as may be determined by context, the term "putamen" may refer to a single putamen (i.e., the left or right putamen) or both putamen together. The putamen is located within the deep periventricular white matter of the forebrain of each cerebral hemisphere (telencephalon) and contains multiple nerve cell (neuron) bodies. Together with the adjacent caudate nucleus, it forms the striatum, which is one component of the basal ganglia of each cerebral hemisphere. Various pathways connect the putamen to many regions of the cerebral cortex, as well as the substantia nigra (including the pars compacta and pars reticularis), globus pallidus, claustrum, and thalamus. The putamen's primary function is to regulate the preparation and execution of physical movements and plays a role in various types of learning. The putamen also plays a role in the development of degenerative neurological disorders, such as PD. Retrograde axonal transport of GDNF protein and / or AAV2 vector from the putamen to the substantia nigra is possible; however, anterograde axonal transport of GDNF protein and / or AAV2 vector to the pars reticularis is more likely in PD. 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 of slowing or inhibiting the progression of PD in a subject in need thereof, 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 at least 30% of the subject's putamen volume is covered by the rAAV.

[0115] In one embodiment, the rAAV is introduced via local introduction. In one embodiment, the AAV capsid is a monoclonal AAV, for example, the capsid is AAV8, AAV9, and contains at least one capsid protein from the rhesus monkey AAV system.

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

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

[0118] In one embodiment, the local introduction further comprises introducing, at the same time or substantially simultaneously with the AAV vector, an MRI contrast agent. Exemplary MRI contrast agents include gadoteric acid, gadobutrol, gadoteridol, gadopentetic acid, gadobenic acid, gadopentetate dimeglumine, gadoxentate, gadoversetamide, gadodiamide, gadofosveset, gadocholetic acid, gadomelitol, and gadomer.

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

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

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

[0122] Another aspect provided herein is a method for slowing or inhibiting the progression of PD in a subject in need thereof, comprising transducing greater than or equal to about 30% of the volume of the subject's putamen with a glial cell line-derived neurotrophic factor (GDNF) gene, wherein the subject does not exhibit a substantial increase in PD-related symptoms for at least six months after the transduction. In one embodiment, the transduction is carried out by administering an rAAV containing the GDNF gene to the putamen of each brain hemisphere of the subject. In one embodiment, transducing the putamen is transducing a putaminal neuronal population. In one embodiment, at least 30% of the volume of the subject's putaminal neuronal 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, comprising administering to the subject's putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein the subject has a second MDS-UPDRS III score at 6 months after the administration that is reduced or stabilized compared to the subject's initial MDS-UPDRS III score before the administration.

[0124] Another embodiment provided herein relates to a method of treating a subject mildly affected by PD, comprising administering to each of the subject's putamen a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject's putamen is transduced with GDNF and / or at least 30% of the subject's putamen volume is covered by the rAAV, and wherein the subject has a second MDS-UPDRS III score at 6 months after the administration that is stabilized compared to the initial MDS-UPDRS III score.

[0125] Another embodiment provided herein relates to a method of treating a subject moderately affected by PD, comprising administering to each of the subject's putamen a recombinant adeno-associated virus (AAV) comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter, wherein at least 30% of the subject's putamen is transduced with the transgene and / or at least 30% of the subject's putamen volume is covered by the rAAV, and wherein the subject has a second MDS-UPDRS III score at 6 months after administering that is at least about 20% lower than the initial MDS-UPDRS III score.

[0126] Another aspect provided herein relates to a method of slowing or inhibiting the progression of PD in a subject in need thereof, comprising: locally introducing into each of the subject's putamen a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding glial cell line-derived neurotrophic factor (GDNF) operably linked to a promoter; and locally introducing into each of the subject's putamen, substantially simultaneously with the rAAV, an MRI contrast agent, wherein at least 30% of the volume of the subject's putamen is transduced with the nucleic acid and / or at least 30% of the subject's putamen volume is covered by the rAAV, and wherein the subject does not exhibit a substantial increase in PD-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 of slowing or inhibiting the progression of PD in a subject in need thereof, 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 / or at least 30% of the subject's putamen volume is covered by the rAAV, and wherein the subject does not exhibit a substantial increase in PD-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 subject's putamen is transduced with a GDNF gene. In one embodiment, the transduction of the subject's putamen is assessed via non-invasive imaging, for example, via MRI. One skilled in the art can assess the transduction of rAAV by measuring the total volume of the putamen containing rAAV (e.g., as assessed by an injected MRI contrast agent) compared to the total volume not containing rAAV.

[0129] In one embodiment, transducing the putamen comprises transducing intrinsic medium spiny neurons (MSNs) of the putamen. In one embodiment, transducing 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 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 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 11 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 (e.g., GDNF).

[0130] In one embodiment, transducing the putamen comprises transducing a putaminal neuron population. In one embodiment, at least 30% of the volume of the subject's putaminal neuron population is transduced with a 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 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 85%, at least 90%, at least 95%, at least 99% or more.

[0131] 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 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, 3%, 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. In some embodiments, the coverage of the subject's putamen is assessed via non-invasive imaging, for example, via MRI. Those skilled in the art can assess the coverage of rAAV by measuring the total volume of the putamen containing rAAV (e.g., as assessed by an injected MRI contrast agent) relative to the total volume of the putamen.

[0132] In one embodiment, an MRI contrast agent is co-administered or co-transduced with any of the rAAVs described herein to provide enhanced real-time intraoperative MRI monitoring of CED distribution and to assess the volume of transduction.

[0133] Non-invasive imaging In one embodiment, local administration is performed simultaneously with non-invasive imaging, e.g., to guide local delivery to a preferred or predetermined location, e.g., the putamen, and / or to visualize transduction after administration. In one embodiment, the non-invasive imaging is intraoperative magnetic resonance imaging (iMRI)-guided convection-enhanced delivery (CED). As used herein, "intraoperative magnetic resonance imaging (iMRI)" refers to MRI images, e.g., of the brain, obtained during a neurosurgical procedure. iMRI technology can be relied upon to produce precise, real-time pictures of the brain for guidance during neurosurgical procedures, e.g., tumor removal, or placement of a therapeutic agent in a desired location (e.g., the putamen). As used herein, "convection-enhanced delivery (CED)" refers to a drug delivery technique that uses positive hydrostatic pressure to deliver a fluid containing a therapeutic substance by bulk flow directly into the interstitial space within a localized region of the brain parenchyma. Direct intracerebral CED circumvents the blood-brain barrier and provides a wider, more uniform distribution than bolus deposition (local injection) or other diffusion-based direct delivery approaches. CED is further described, for example, in Rogawski MA, Neurotherapeutics. 2009 Apr; 6(2): 344-351 and Mehta AM, et al. Neurotherapeutics. 2017 Apr; 14(2): 358-371, the contents of each of which are incorporated herein by reference in their entirety.

[0134] In one embodiment, iMRI is used to monitor the administration of a rAAV or composition thereof described herein using T1-weighted sequences to visualize an MRI contrast agent, e.g., a gadolinium-based contrast agent, that is co-injected with the rAAV or composition thereof.

[0135] In one embodiment, the local introduction further comprises introducing an MRI contrast agent substantially simultaneously with the AAV vector. In this case, the MRI contrast agent is utilized to improve the visibility of internal brain structures captured in MRI images. Gadolinium(III)-containing paramagnetic contrast agents, known in the art as gadolinium-based MRI contrast agents (GBCAs), are preferred; see "Gadolinium(III) Chelates as MRI Contrast Agents Structure, Dynamics, and Applications" by P. Caravan et al., Chem. Rev. 99, 2293-2352 (1999), which is incorporated herein by reference in its entirety. Other contrast agents that may be used include gadoxetate disodium (e.g., Eovist™ gadoxetate disodium; Schering AG); the contrast agents disclosed in U.S. Pat. Nos. 5,798,092 and 5,695,739; gadobenate dimeglumine (e.g., MultiHance™ gadobenate dimeglumine, Bracco SpA); and the contrast agents disclosed in U.S. Pat. No. 5,733,528. "Blood pool" MRI contrast agents are particularly preferred; see "Blood Pool Contrast Agents for Cardiovascular MR Imaging" by LJM Kroft et al. JMRI 10, 395-403 (1999), which is incorporated herein by reference, and "The Future of Contrast-Enhanced Magnetic Resonance Angiography: Are Blood Pool Agents Needed?" by A. Muhler Invest. Radiol. 33, 709-714 (1998), which is also incorporated herein by reference.Examples of blood pool contrast agents include MP-2269 (Mallinckrodt, Inc.); contrast agents disclosed in U.S. Pat. No. 5,888,576; MS-325 (EPIX Medical, Inc.); contrast agents disclosed in PCT Publication No. WO 96 / 23526; P760 (Geurbet); gadolinium-diethylenetriaminepentaacetic acid (GD-DTPA; e.g., Gadomer-17™, 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™ gadoterate meglumine, Nycomed Amersham; contrast agents disclosed in PCT Publication Nos. WO96 / 09840 and WO9725073; B22956 / 1 (Bracco SpA); and contrast agents disclosed in PCT Publication Nos. WO00 / 30688, WO98 / 05625, WO98 / 05626, WO95 / 32741, WO98 / 38738, WO95 / 32741, and U.S. Patent No. 5,649,537. Other examples of such blood pool agents include, but are not limited to, ferucarbotran (e.g., Resovist™ ferucarbotran) or SHU 555 A and C (Schering). The contents of all patents and patent applications mentioned herein above are expressly incorporated herein by reference in their entirety.

[0136] Exemplary MRI contrast agents include gadoteric acid; gadobutrol; gadoteridol; gadopentetic acid; gadobenic acid; gadopentetate dimeglumine; gadoxetic acid; gadoversetamide; gadodiamide; gadofosveset; gadocholetic acid; gadomelitol, and gadomel.

[0137] In one embodiment, the MRI contrast agent is gadoteridol (e.g., ProHance® gadoteridol). In one embodiment, gadoteridol (e.g., ProHance® gadoteridol) is administered in a 2 mM solution.

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

[0139] MRI brain scans can be performed preoperatively as part of the screening process, as well as during the gene therapy infusion procedure and at 6 and 18 months after dosing. Scans may also be obtained at other points if deemed necessary by the investigator. MRI brain scans can 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 the 18-month screening may also include functional assessment with extended diffusion-weighted sequences, resting state, and image acquisition while participants perform simple tasks (i.e., finger tapping or hand grasping). Total imaging time, including functional and resting-state imaging, is 90 minutes per session.

[0140] Exemplary non-invasive imaging techniques that may be utilized in the methods described herein include ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); iMRI; positron emission tomography (PET); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); DaTscan dopamine transporter imaging; FDG imaging, and combinations thereof.

[0141] Iofulpane I-123 (e.g., DaTscan™ Iofulpane I23) selectively binds to presynaptic dopamine transporters, providing a method for imaging nigrostriatal terminals in the striatum. DaTscan™ Iofulpane I23 is an FDA-approved radiopharmaceutical used in conjunction with single-photon emission computed tomography (SPECT) scans for use in adults. Iodine-123 is released by electron capture. 123 It is a cyclotron-generated radionuclide that decays to Te and has a physical half-life of 13.2 hours. The recommended dose is 111–185 MBq (3–5 mCi) administered intravenously in adults. The effective dose obtained from DaTscan administration at an administered activity of 185 MBq (5 mCi) is 3.94 mSv in adults. DaTscan injections may contain up to 6% free iodide (iodine-123). To reduce thyroid accumulation of iodine-123, potassium iodide oral solution or Lugol's solution is administered in doses up to 100 mg.

[0142] Fluoro-2-deoxyglucose (FDG) is a common FDA-approved radiopharmaceutical tracer used in positron emission topography (PET) imaging to measure glucose metabolism in the brain and other organs. PD-specific brain metabolic patterns, not present in other Parkinson's disease-like disorders, have been characterized. FDG PET is utilized to confirm PD diagnosis during screening. FDG has a half-life of 110 minutes and is a fluorescein-like substance. 18The recommended dose is 111-185 MBq (3-5 mCi) administered intravenously in adults. The effective dose obtained from an FDG scan with an administered activity of 185 MBq (5 mCi) is 3.51 mSv in adults.

[0143] In one embodiment, the coverage or transduction of the putamen is assessed by non-invasive imaging, for example, intraoperative MRI. The volume of the putamen transduced with GDNF is indirectly determined by measuring the percentage of the putamen that shows CED-infused MRI contrast within the putamen compared to the total volume of the putamen. In one embodiment, the volume of the transduced putamen is assessed by F-DOPA PET imaging, which correlates with the localization of intraoperative CED-infused MRI contrast.

[0144] As used herein, "coverage" refers to the volume of the putamen occupied by the injected rAAV relative to the total volume of the putamen. Coverage provides that putaminal cells are exposed to the rAAV. There are many methods for determining coverage by a delivered therapeutic agent after administration. For example, in one embodiment, putamen coverage can be assessed via non-invasive imaging, such as co-injection with a visualized MRI contrast agent. For example, putamen coverage after introduction / administration can be determined by measuring the area or volume of the putamen displaying the co-injected MRI agent and comparing it to the area or volume of the putamen that does not display the agent. Similarly, coverage is assessed via F-DOPA PET imaging. Furthermore, in one embodiment, the rAAV can further comprise a reporter gene, such as a fluorescent tag, that can be visualized postmortem using standard histological methods, such as microscopy.

[0145] As used herein, "transduction" or "transduced" refers to cells in the putamen that contain the administered rAAV or composition thereof. In one embodiment, the transduced cells contain the genome of the rAAV and have the potential to express a GDNF transgene. In one embodiment, the transduced cells contain the genome of the rAAV and need not contain the ability to express a 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 methods for determining transduction after administration, and these are typically indirect inference (or indirect) approaches. For example, transduction can be assessed via co-injection with a visualized MRI contrast agent. The percentage of transduced cells in the putamen can be determined, for example, by measuring the percentage of cells in the putamen containing rAAV or a composition compared to the total volume of the putamen. Transduction can be assessed via non-invasive imaging, for example, co-injection with a visualized MRI contrast agent. In one embodiment, the rAAV can further comprise a reporter gene, e.g., a fluorescent tag, that can be visualized using standard methods, e.g., microscopy, and such a reporter gene can be used to determine the transduction percentage. Probes designed to target the rAAV (e.g., capsid protein) can be used to assess whether cells are transduced with the rAAV. Furthermore, probes designed to target the GDNF nuclear function can be used to assess whether cells express the GDNF transgene. In one embodiment, the volume of the transduced putamen is assessed via F-DOPA PET imaging. Additionally, in one embodiment, the rAAV can further comprise a reporter gene, e.g., a fluorescent tag, that can be visualized using standard methods, e.g., microscopy.

[0147] Diagnostic Assays for Parkinson's Disease and Disease Progression The progression and severity of PD are often measured using various clinical statistics that evaluate various symptoms and mental states of subjects who have or are suspected of having PD.These clinical statistics can be completed by subjects, their caregivers, and / or trained physicians.In many cases, these statistics are used by clinicians and researchers to evaluate the long-term course of PD during the course of treatment or in clinical research.The results of such statistics can help clinicians or researchers, for example, in determining the best course of action for treating subjects, i.e., changing the type of therapeutic drug, the dosage of therapeutic drug, or the frequency of administration of therapeutic drug.

[0148] In one embodiment, the method described herein further comprises 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 assays include the Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS), Non-Motor Symptom Scale (NMSS), Parkinson's Disease Questionnaire (PDQ-39) score; MDS-UPDRS Part III; Modified Hoehn and Yahr; Stand-Walk-Sit; 9-Hole Pegboard Dexterity Test; and Standing Balance Test; and Global Impression (CGI) score. PGI); Brief Smell Discrimination Test (BSIT); Parkinson's Disease Sleep Assessment Scale-2 (PDSS-2); Parkinson's Disease Outcome Assessment Scale-Autonomic Nervous System (SCOPA-AUT); Global Cognitive Assessment via the Montreal Cognitive Assessment (MoCA); 30-item Boston Naming Test (BNT); Verbal Fluency Test; Cambridge Neuropsychological Test Automated Battery (CANTAB); Beck Depression Inventory-II (BDI-II); Beck Anxiety Inventory (BAI); and Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease (QUIP-RS), or any combination thereof.

[0149] In one embodiment, the methods described herein further include receiving an initial score of at least one diagnostic assay described herein for the subject prior to introducing any of the rAAVs described herein.

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

[0151] The Unified Parkinson's Disease Rating Scale (UPDRS) is a rating scale for assessing the short-term (<1 year) and long-term (>1 year) progression of PD. The UPDRS is a standardized and accepted assay utilized in clinical settings that allows clinicians to track the progression of patients' symptoms in an objective manner. This test consists of six parts, both self-administered and clinician / researcher-administered. The six parts include: Part I: Assessment of mental status, behavior, and mood, including intellectual impairment, thought disorder, motivation / initiative, and depression; Part II: Self-assessment of activities of daily living (ADLs): speech, drooling, swallowing, handwriting, cutting food, dressing, hygiene, turning in bed, falling, freezing, gait, tremor, and sensory complaints; and Part III: Speech, facial expression, resting tremor, action tremor, rigidity, finger tapping, hand movements, hand pronation and supination, leg agility, rising from a chair, and posture. Part IV: Complications of treatment including dyskinesia-duration, dyskinesia-disability, dyskinesia-pain, early monitoring dystonia, off-predictable, off-unpredictable, off-sudden, off-duration, anorexia-nausea-vomiting, sleep disturbance, symptomatic orthostatic effect; Part V: Hoehn and Yahr staging of PD severity; and Part VI: Schwab and England ADL scale.

[0152] The Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) is a rating scale (i.e., 0–272) for assessing short-term (<1 year) and long-term (>1 year) progression of Parkinson's Disease (PD). Historically, researchers have used the UPDRS to measure treatment benefit from a given therapy in a unified and accepted rating system. The MDS-UPDRS is an updated version of the UPDRS that removes non-motor aspects of function from each subcategory. MDS-UPDRS Part I is titled "Non-Motor Symptom Experience in Daily Living" and includes Part IA (which concerns several behaviors assessed by the investigator using all relevant information from the patient and caregiver) and Part IB (which is independent of the investigator but completed by the patient with or without the assistance of a caregiver). MDS-UPDRS Part II is identical to the second part of the original UPDRS, but has been renamed "Motor Symptom Experience in Daily Living" to separate it from the new title of Part I. MDS-UPDRS Part III is titled "Motor Examination." MDS-UPDRS Part IV is condensed compared to the UPDRS to include only "motor complications." The total MDS-UPDRS score is the sum of Parts I, II, III (OFF state), and IV, which provides both functional and evaluator-derived subscores, thereby providing a score of disease severity and progression. Those skilled in the art will be able to properly administer the MDS-UPDRS survey and guide the subject through the self-administered portion of the MDS-UPDRS survey to determine, for example, an initial MDS-UPDRS score (i.e., before administration of the rAAV described herein) and a second MDS-UPDRS score (i.e., an MDS-UPDRS score at least 6 months or at least 12 months after administration of the rAAV described herein). It is understood that additional MDS-UPDRS scores (e.g., a third, fourth, fifth, etc.) can be determined if deemed necessary, for example, by a clinician.It is further understood that the initial and second MDS-UPDRS scores can be any individual part of the MDS-UPDRS survey (e.g., Part I, Part II, Part III, Part IV) or the total MDS-UPDRS score.

[0153] In one embodiment, the methods described herein further comprise determining an initial MDS-UPDRS score for the subject prior to administering any of the rAAVs described herein. In one embodiment, the methods described herein further comprise receiving an initial MDS-UPDRS score for the subject prior to administering any of the rAAVs described herein, i.e., receiving an initial MDS-UPDRS score previously determined by a person skilled in the art who has not administered the rAAV.

[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, subjects mildly affected by PD had an initial MDS-UPDRS score of less than 32 prior to the introduction of rAAV and were diagnosed with PD less than 5 years prior to the introduction of rAAV.

[0156] In one embodiment, 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, a subject moderately affected by PD had an initial MDS-UPDRS score equal to or greater than 32 (e.g., an MDS-UPDRS III score) 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 determining a second MDS-UPDRS score at least 6 months or at least 12 months immediately following administration / introduction of the rAAV.

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

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

[0162] In one embodiment, a subject mildly affected by PD does not show an increase in their initial MDS-UPDRS score for at least 12 months immediately after introduction of any of the rAAVs described herein. In one embodiment, a subject mildly affected by PD does not show an increase in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after introduction of any of the rAAVs described herein.

[0163] In one embodiment, a subject who is mildly affected by PD does not show a substantial increase in their initial MDS-UPDRS score for at least 12 months immediately after the introduction of any of the rAAVs described herein. As used herein, "substantial increase" refers to an increase of 10% or less of the initial MDS-UPDRS score. In one embodiment, a subject who is mildly affected by PD does not show a substantial increase in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after the introduction of any of the rAAVs described herein.

[0164] In one embodiment, a subject who is mildly affected by PD shows stabilization of their initial MDS-UPDRS score for at least 6 months immediately after the introduction of any of the rAAVs described herein. As used herein, "stabilization" refers to an initial MDS-UPDRS score that does not increase or decrease by more than 10%, i.e., the second MDS-UPDRS score is not more than + / - 10% of the initial MDS-UPDRS score. In one embodiment, a subject who is mildly affected by PD shows stabilization of their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after the introduction of any of the rAAVs described herein.

[0165] In one embodiment, subjects mildly affected by PD exhibit a reduction in their initial MDS-UPDRS score for at least 12 months immediately after introduction of any of the rAAVs described herein. In one embodiment, subjects mildly affected by PD exhibit a reduction in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 6 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after introduction of any of the rAAVs described herein. In one embodiment, the reduction in the initial MDS-UPDRS score is at least 1 point; 2 points; 3 points; 4 points; 5 points; 6 points; 7 points; 8 points; 9 points; 10 points; 11 points; 12 points; 13 points; 14 points; 15 points; 16 points; 17 points; 18 points; 19 points; 20 points; 21 points; 22 points; 23 points; 24 points; 25 points; 26 points; 27 points; 28 points; 29 points; 30 points; or 31 points; or 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, subjects moderately affected by PD show a reduction in their initial MDS-UPDRS score for at least 6 months immediately after introduction of any of the rAAVs described herein. In one embodiment, subjects moderately affected by PD show a reduction in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after introduction of any of the rAAVs described herein. In one embodiment, the reduction in the initial MDS-UPDRS score is at least 20%. In one embodiment, the reduction in initial MDS-UPDRS score 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%;91%;92%;93%;94%;95%;96%;97%;98%;99% or greater, or 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 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 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;121 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 t;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;156 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;1 95 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;214 points;215 points;216 points;217 points;218 points;219 points;220 points;221 points;222 points;223 points;224 points;225 points;226 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 nt;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 their initial MDS-UPDRS score for at least 6 months immediately after introduction of any of the rAAVs described herein. In one embodiment, a subject moderately affected by PD does not show an increase in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after introduction of any of the rAAVs described herein.

[0168] In one embodiment, a subject who is moderately affected by PD does not show a substantial increase in their initial MDS-UPDRS score for at least 6 months immediately after the introduction of any of the rAAVs described herein. As used herein, "substantial increase" refers to an increase of 10% or less of the initial MDS-UPDRS score. In one embodiment, a subject who is moderately affected by PD does not show a substantial increase in their initial MDS-UPDRS score for at least 1 month; 2 months; 3 months; 4 months; 5 months; 7 months; 8 months; 9 months; 10 months; 11 months; 12 months; 13 months; 14 months; 15 months; 16 months; 17 months; 18 months; 19 months; 20 months; 21 months; 22 months; 23 months; 24 months; or longer immediately after the introduction of any of the rAAVs described herein.

[0169] Non-Motor Symptom Scale (NMSS) The Nonmotor Symptom Scale (NMSS) is a 30-item self-administered survey rating scale for assessing a wide range of nonmotor symptoms in subjects with PD. Nonmotor symptoms in PD commonly include neuropsychiatric symptoms, sleep disturbances, autonomic dysfunction, gastrojejunal-colonic symptoms, and sensory symptoms, which can significantly reduce quality of life. The NMSS measures the severity and frequency of nonmotor symptoms across nine dimensions: cardiovascular, sleep / fatigue, mood / cognition, sensory problems, attention / memory, gastrojejunal-colonic, urinary, sexual function, and miscellaneous. This rating scale can be used for patients at all stages of PD. The score for each item is based on a combination of severity (0-3) and frequency scores (1-4) to capture severe but relatively infrequent symptoms or less severe but persistent symptoms. The total NMSS score ranges from 0 to 360.

[0170] In one embodiment, the methods described herein further comprise determining an initial NMSS score for the subject prior to administering any of the rAAVs described herein. In one embodiment, the methods described herein further comprise receiving an initial NMSS score for the subject prior to administering any of the rAAVs described herein, i.e., receiving an initial NMSS score previously determined by a clinician / researcher who has not administered the rAAV.

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

[0172] In one embodiment, the subject shows a reduction in its initial NMSS score for at least 6 months after being introduced with any of the rAAVs described herein.In one embodiment, the subject shows a reduction 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 longer after being introduced with any of the rAAVs described herein.In one embodiment, the reduction in initial NMSS score is at least 20%. In one embodiment, the reduction in initial NMSS score 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%;91%;92%;93%;94%;95%;96%;97%;98%;99% or greater, or 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 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 points;113 points;114 points;115 points;116 points;117 points;118 points;119 points;120 points;121 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 t;144 points;145 points;146 points;147 points;148 points;149 points;150 points;151 points;152 points;153 points;154 points;155 points;156 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;20 6 points;207 points;208 points;209 points;210 points;211 points;212 points;213 points;214 points;215 points;216 points;217 points;218 points;219 points;220 points;221 points;222 points;223 points;224 points;225 points;226 points;227 points;228 points;229 points;230 points;231 points;232 points;233 points;234 points;235 points;236 points;237 points 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 To;269 points;270 points;271 points;272 points;273 points;274 points;275 points;276 points;277 points;278 points;279 points;280 points;281 points;282 points;283 points;284 points;285 points;286 points;287 points;288 points;289 points;290 points;291 points;292 points;293 points;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;327 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 being introduced with any of the rAAVs 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 longer after being introduced with any of the rAAVs described herein.

[0174] In one embodiment, the subject does not show a substantial increase in its initial NMSS score for at least 6 months after being introduced with any of the rAAVs described herein.As used herein, "substantial increase" refers to an increase of 10% or less of the initial NMSS score.In one embodiment, the subject does not show a substantial 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 longer after being introduced with any of the rAAVs described herein.

[0175] In one embodiment, the subject shows stabilization of its initial NMSS score for at least 6 months immediately after introducing any of the rAAVs described herein.As used herein, "stabilization" refers to an initial NMSS score that does not increase or decrease by more than 10%.In one embodiment, the subject shows stabilization 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 after introducing any of the rAAVs described herein.

[0176] Parkinson's Disease Questionnaire (PDQ-39) score 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 bodily discomfort. The questionnaire is scored on a scale of 0 to 100, with lower scores indicating better perceived health status and higher scores indicating a more severe state of disease. The PDQ-39 can be used as a reliable tool to measure quality of life for individuals with PD. Its inclusion in comprehensive assessments is particularly important due to the tendency of treatments to focus on underlying features other than clinical features, including motor impairment, depression, cognitive impairment, and fall risk, which can greatly affect quality of life.

[0177] In one embodiment, the methods described herein further comprise determining an initial PDQ-39 score for the subject prior to administering any of the rAAVs described herein. In one embodiment, the methods described herein further comprise receiving an initial PDQ-39 score for the subject prior to administering any of the rAAVs described herein, i.e., receiving an initial PDQ-39 score previously determined by a clinician / researcher not administering the rAAV.

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

[0179] In one embodiment, the subject shows a decrease in their initial PDQ-39 score for at least 6 months after introduction of any of the rAAVs described herein. In one embodiment, the subject shows a decrease 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 longer after introduction of any of the rAAVs described herein. In one embodiment, the decrease in initial PDQ-39 score is at least 20%. In one embodiment, the reduction in initial PDQ-39 score 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%;8 6%;87%;88%;89%;90%;91%;92%;93%;94%;95%;96%;97%;98%;99% or greater, or 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 their initial PDQ-39 score for at least 6 months after being introduced with any of the rAAVs described herein. In one embodiment, the subject does not show an 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 longer after being introduced with any of the rAAVs described herein.

[0181] In one embodiment, the subject does not show a substantial increase in their initial PDQ-39 score for at least 6 months after being administered any of the rAAVs described herein. As used herein, "substantial increase" refers to an increase of 10% or less of the initial PDQ-39 score. In one embodiment, the subject does not show a substantial 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 longer after being administered any of the rAAVs described herein.

[0182] In one embodiment, the subject shows stabilization of its initial PDQ-39 score for at least 6 months immediately after introduction of any of the rAAVs described herein. As used herein, "stabilization" refers to an initial PDQ-39 score that does not increase or decrease by more than 10%. In one embodiment, the subject shows stabilization of 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 longer immediately after introduction of any of the rAAVs described herein.

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

[0184] The modified Hoehn and Yahr scale is a 5-point scale that measures the overall level of disability due to PD ( Hoehn and Yahr, 1967 ).

[0185] The Stand-Walk-Sit (SWS) is an assessment of postural stability and gait that includes rising from a chair, walking 7 meters (23 feet) in a straight line, turning, and walking back to the chair and sitting down. The timer stops when the subject's back touches the back of the chair. Subjects perform this test in pragmatically defined off- and on-medication states. The SWS test can be video-recorded and combined with the MDS-UPDRS assessment.

[0186] The 9-Hole Pegboard Dexterity Test is a brief test of manual dexterity that records the time required for participants to accurately place and remove nine plastic pegs from a plastic pegboard.

[0187] The standing balance test assesses a person's ability to orient their body in space, maintain an upright posture in both static and dynamic states, and move and walk without falling. It involves participants assuming and maintaining up to five poses for 50 seconds each. The series of poses 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 eyes open in a tandem stance on a solid surface. Detailed stopping rules are in place to ensure participant safety during these increasingly demanding poses. Postural tilt is recorded for each pose using an accelerometer worn by the participant at waist level. The test takes approximately 7 minutes to administer.

[0188] In one embodiment, the subject undergoes any of the standard clinical tests described herein prior to administration / induction and again at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. In one embodiment, the subject demonstrates an improvement in the score on any of the standard clinical tests at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. 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%;91%;92%;93%;94%;95%;96%;97%;98%;99%; or greater.

[0189] In one embodiment, the severity of dyskinesia is measured using the Unified Dyskinesia Rating Scale (UDysRS). This scale assesses involuntary movements often associated with treated PD. It includes two main sections: historical (Part 1 (on-dyskinesia) and Part 2 (off-dystonia)) and objective (Part 3 (impairment) and Part 4 (disability)). On-dyskinesia refers to choreiform and dystonic movements described by the patient as jerking or twisting movements that occur when PD medication is working. Off-dystonia refers to spasms or spasticity that are painful and may occur when PD medication is not being taken or is not working. Throughout the evaluation, the focus is on these two forms of movement, and continuous attention should be placed on excluding parkinsonism itself and the effects of tremor from the evaluation (Goetz 2008).

[0190] In one embodiment, subjects complete a subject-reported PD motor diary. Hauser and colleagues developed a paper motor diary to assess PD motor symptoms over a 24-hour period (Hauser 2004). The diary involves participants recording their time in 30-minute intervals: on-state with no dyskinesias, on-state with non-interfering dyskinesias, on-state with interfering dyskinesias, off-state, or while asleep. Participants are required to record this information at 30-minute intervals throughout the day.

[0191] In one embodiment, the subject completes a UDysRS and / or PD motor diary prior to administration / induction and again at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. In one embodiment, the subject demonstrates improved scores on the UDysRS and / or PD motor at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. 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%;91%;92%;93%;94%;95%;96%;97%;98%;99% or greater.

[0192] In one embodiment, subjects are provided with a wearable activity monitor (e.g., Fitbit®) to assess daily activity immediately after administration / introduction for at least 18 months.

[0193] In one embodiment, the subject undergoes a global disability and quality of life assessment prior to administration / introduction and / or immediately thereafter at least 3, 6, 9, 12 months or thereafter. Exemplary global disability and quality of life assessments include the Comprehensive Global Impression Index (CGI & PGI); Brief Smell Identification Test (BSIT); Parkinson's Disease Sleep Scale-2 (PDSS-2); and the Parkinson's Disease Outcome Assessment Scale-Autonomic Nervous System (SCOPA-AUT).

[0194] The Clinical Global Impression (CGI) provides an overall clinician-determined summary measure that takes into account all available information, including knowledge of the patient's medical history, psychosocial situation, symptoms, behavior, and the impact of symptoms on the patient's ability to function. The CGI actually includes items from two instruments: (a) disease severity on a 1-7 scale, and (b) a scale assessing change from the start of treatment on a similar 7-point scale. The Patient Global Impression (PGI) is identical to the CGI but is completed by the patient. The PGI and CGI are completed separately. The PGI is a self-assessment tool and is completed independently by participants at home by completing a paper assessment or on a sponsor-provided tablet.

[0195] The Brief Smell Identification Test (BSIT) is a 12-item test of olfactory system function using "scratch and sniff" strips. After each scent is released by scratching with a pencil, participants smell it and then answer a four-option multiple-choice question about the scent. This is a self-directed assessment.

[0196] The Parkinson's Disease Sleep Scale-2 (PDSS-2) uses a visual analog scale to address 15 commonly reported symptoms associated with sleep disturbances in PD. Subjects complete 15 questions based on their experiences over the past week. It is a self-administered scale.

[0197] The Scale for Assessment of Outcomes in Parkinson's Disease - Autonomic Nervous System (SCOPA-AUT) is a 26-item self-report questionnaire of autonomic function. Questions cover upper and lower gastrointestinal function, urinary function, cardiovascular function, sexual performance, and various other autonomic problems. It is a self-administered symptom assessment scale.

[0198] In one embodiment, the subject is administered any of the global disability and quality of life assessments described herein prior to administration / induction and again at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. In one embodiment, the subject demonstrates improved scores on the global disability and quality of life assessments at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. 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%;91%;92%;93%;94%;95%;96%;97%;98%;99% or greater.

[0199] In one embodiment, before administration / introduction, and / or immediately after at least 3, 6, 9, 12 months or after, subject undergoes neuropsychological testing.Exemplary neuropsychological testing includes global cognitive assessment via Montreal Cognitive Assessment (MoCA); 30-item Boston Naming Test (BNT); Verbal Fluency Test; Cambridge Neuropsychological Test Automated Battery (CANTAB); Beck Depression Inventory-II (BDI-II); Beck Anxiety Inventory (BAI); and Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease (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, visuo-constructive skills, conceptual thinking, calculation, and orientation. The BNT assesses confrontational naming and language deficits that may be present in PD. Participants are presented with line drawings of objects with increasing naming difficulty and are required to provide a response within 20 seconds. Scoring is based on the number of correct responses spontaneously provided, the number of cues given, and the number of post-cue responses. This brief verbal assessment is completed with remote guidance from the research team via video call.

[0201] In the original word fluency test, subjects are asked to create as many words as possible from preselected letters and categories within 60 seconds, with the instruction not to use proper nouns or words that vary only by suffixes. The number of correct responses is scored for each category.

[0202] The Cambridge Neuropsychological Test Automated Battery (CANTAB) was developed to include sensory and objective measures of cognitive function in the assessment of neurological disorders. Cognitive assessments were developed to detect changes in neuropsychological performance over time as an effect of intervention. Several assessments have been validated in PD, focusing on the domains of working memory, episodic memory, executive function, planning, and information processing. All tasks are completed by the subject using a tablet, including the collection of response times. The CANTAB allows for electronically captured outcome measures validated in various neurodegenerative disorders. The CANTAB can be completed self-directed or remotely guided by a study coordinator or investigator on a sponsor-provided tablet.

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

[0204] The Motor Screening Task (MOT) provides a general assessment of whether sensorimotor deficits or lack of comprehension may limit the ability to collect valid data from participants. This task measures participants' response speed and accuracy in pointing to the center of the screener's target.

[0205] The Cambridge One Touch Stocking (OTS) is an assessment of executive function, assessing the subdomains of spatial planning and working memory. Participants are asked to create a 3D configuration on a screen in a set number of movements. This is measured by the number of problems solved in the first choice, the mean revision of choices, the mean latency to respond, and the mean latency to correct.

[0206] The Paired Associate Learning Test (PAL) is a visual memory and learning task that assesses the ability to learn new things. Participants are asked to select boxes in a pre-specified pattern with increasing levels of difficulty. Results are measured by the number of errors made, the number of attempts needed to correctly identify the pattern, the stages completed, and the memory score. Estimated completion time: 8 minutes.

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

[0208] The Multitasking Test (MTT) assesses an individual's ability to interpret and manage conflicting information and correctly ignore task-irrelevant information. Changing rules between trials places higher cognitive demands on participants to reveal underlying deficits in executive function, a domain frequently affected in PD patients. This is measured by response latency and number of errors.

[0209] The Beck Depression Inventory-II (BDI-II), for example, can be administered at screening as part of eligibility assessment. Participants with a screening score of >20 are excluded from the study and referred to their primary care physician for psychiatric evaluation and treatment. Scoring guidelines for the BDI-II provide recommendations that thresholds be adjusted based on sample characteristics and the purpose for using the BDI-II. Generally, a total BDI-II score of 0–13 indicates minimal depression, a score of 14–19 indicates mild depression, a score of 20–28 indicates moderate depression, and a score of 29–63 indicates severe depression. If the post-treatment BDI-II score is higher than 28, participants remain in the study but are referred to their primary care physician for psychiatric evaluation and treatment. This assessment is a self-administered scale.

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

[0211] The Questionnaire for Obsessive-Compulsive Disorder (QUIP-RS) is a rating scale designed to measure symptom severity and support the diagnosis of impulse control disorders and related disorders in PD. This rating scale covers impulse control behaviors on a 5-point Likert scale to assess the frequency of the following behaviors: gambling, shopping, eating, hypersexuality, simple (repetitive stereotyped behaviors) and / or complex (preoccupation with hobbies), and compulsive overuse of medications (dopamine dysregulation syndrome). This assessment is a self-administered scale.

[0212] In one embodiment, the subject is administered any of the neuropsychological tests described herein before administration / induction and again at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. In one embodiment, the subject shows an improvement in scores on any of the neuropsychological tests at least 3, 6, 9, 12 months or thereafter immediately after administration / induction. 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%;91%;92%;93%;94%;95%;96%;97%;98%;99%; or greater.

[0213] In one embodiment, the subject does not show any serious adverse events for at least 6 months immediately after introduction or administration.In one embodiment, the subject does not show any serious adverse events for at least 12 months immediately after introduction or administration.In one embodiment, the subject does not show any serious adverse events for at least 1 month, 2 months, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, 10 months, 11 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or longer immediately after introduction or administration. Serious adverse events include, but are not limited to, blood and lymphatic system disorders (e.g., anemia, decreased lymphocyte count, and leukocytosis); gastrointestinal disorders (e.g., dyspepsia, dysphagia, and constipation); localized edema; fatigue; falls; neck contusion; increased aspartate aminotransferase; decreased platelet count; prolonged activated partial thromboplastin time; weight loss; blood lactate dehydrogenase; metabolic and nutritional disorders (e.g., hyperglycemia, hypocalcemia, hypoalbuminemia, hypophosphatemia, hypernatremia, hypoglycemia, hyperkalemia, and hyponatremia). musculoskeletal and connective tissue disorders (e.g., pain, neck pain, back pain, chest wall pain, and limb pain); nervous system disorders (e.g., headache, involuntary movements, memory impairment, sleep disorders - 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); surgery and medical procedures (e.g., carpal tunnel release); transient paresthesias; transient tremors; hypotension; and vascular diseases (e.g., hypertension).

[0214] nucleic acid In some aspects, the present disclosure provides isolated nucleic acids useful for 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 of the present disclosure and proteins translated therefrom are isolated. As used herein, the term "isolated" means artificially produced. As used herein with respect to nucleic acids, the term "isolated" means (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, such as by cleavage and gel separation, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is one that can be 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 are disclosed is considered isolated, whereas a nucleic acid sequence present natively in its natural host is not. An isolated nucleic acid may, but need not, be substantially purified. For example, nucleic acid isolated in cloning or expression vector is not pure in that it may comprise only a small percentage of the material in the cell in which it exists.However, as this term is used herein, such nucleic acid is isolated because it can be easily manipulated by standard techniques known to those skilled in the art.When used herein in relation to protein or peptide, the term "isolated" refers to a protein or peptide that is isolated from its natural environment or is artificially produced (for example, by chemical synthesis, by recombinant DNA technology, etc.).

[0215] Those skilled in the art will also recognize that conservative amino acid substitutions may be made to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, the present disclosure encompasses sequence changes that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, methods found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made within the following groups of amino acids: (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 can be made to the amino acid sequences of the proteins and polypeptides disclosed herein.

[0216] The isolated nucleic acid described herein may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid described by the present disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) may 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" typically comprises a minimal transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise, for example, a protein-coding region and / or an expression control sequence (e.g., a polyA tail), as described elsewhere in this disclosure.

[0217] Generally, the ITR sequence is about 145 bp in length. In various embodiments, the left and right ITRs are independently 145 bp or less, or 130 bp or less. "Independently" means that the left and right ITRs can be the same length or different lengths. As a non-limiting example, the left and right ITRs can be independently 145 bp, 130 bp, 128 bp, 124 bp, or 119 bp. Preferably, substantially the entire ITR-encoding sequence is used in the nucleic acid sequence, although some degree of minor modification of these sequences is acceptable. The ability to modify these ITR sequences is within the skill of the art (see, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a nucleic acid molecule is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including the mammalian AAV types identified by the present invention. In some embodiments, the isolated nucleic acid (e.g., rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. 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.) comprising 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 separation site (TRS). The term "lacking a terminal separation site" may refer to an AAV ITR containing a mutation (e.g., a sense mutation, such as a nonsynonymous mutation, or a missense mutation) that abolishes the function of the terminal separation site (TRS) of the ITR, or a truncated AAV ITR (e.g., an ATRS ITR) that lacks a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors containing ITRs that lack functional TRSs generate self-complementary rAAV vectors, for example, as described by McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0219] In addition to the elements identified above for the recombinant AAV vector, the vector also contains conventional control elements operably linked to the transgene elements in a manner that allows its transcription, translation, and / or expression in cells transfected with the vector or infected with the virus produced by the methods described herein. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest (i.e., GDNF) and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing signals 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, if desired, sequences that enhance secretion of the encoded product. Several expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0220] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked 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 linkage 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. Thus, a promoter region is operably linked to a nucleic acid sequence if it can effect transcription of its DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that are translated in frame. In some embodiments, the operably linked coding sequences result in a fusion protein.

[0221] promoter 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 start sequences, stop sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals 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, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0222] A "promoter" refers to a DNA sequence recognized by or introduced into the synthetic machinery of a cell necessary 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 location and orientation with respect to the nucleic acid to control the initiation of RNA polymerase and expression of the gene.

[0223] For nucleic acids encoding proteins, a polyadenylation sequence is generally inserted after the transgene sequence (i.e., downstream of the transgene sequence or 3' of the transgene sequence) and before the 3'AAV ITR sequence. rAAV constructs useful in the present disclosure may also contain an intron, which is preferably located between the promoter / enhancer sequence and the transgene. One exemplary intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. Another vector element that can be used is an internal ribosome entry site (IRES). IRES sequences are used to generate two or more polypeptides from a single gene transcript. IRES sequences will be used to generate proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional, and many such sequences are available (see, e.g., Sambrook et al., and the references cited therein, e.g., pages 3.18 3.26 and 16.17 16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the polyprotein, which is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (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 2A sequences has previously been demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retrovirus) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; pp. 8124-8127; Furler, S et al., Gene Therapy, 2001; pp. 864-873; and Halpin, C et al., The Plant Journal, 1999; pp. 453-459; de Felipe, P et al., Gene Therapy, 1999; pp. 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; pp. 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 phosphoglycerol kinase (PGK) promoter, and the EF1a 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 the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as the presence of an acute phase, a specific differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from various 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 an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, 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-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268: 1766-1769 (1995), and Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1995)). (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0226] In another embodiment, the native promoter of the transgene is used. A native promoter may be preferred when it is desired that the expression of the transgene mimic native expression. A native promoter may be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression. As used herein, "native promoter" refers to the endogenous promoter of the transgene.

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

[0228] Other exemplary promoters include the beta-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 bone 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)), the CD2 promoter (Hansal et al., J. Immunol., 161: 1063-8 (1996)), among others that will be apparent to those skilled in the art. (1998)); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal promoters such as the 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)).

[0229] Nervous system (NS)-specific promoters contemplated for use in the present methods and compositions also include those described in International Patent Applications WO / 2022 / 049385 and WO / 2021 / 214443, which are incorporated herein by reference in their entireties. In some embodiments, the NS-specific promoter is a promoter in Table 1, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter in Table 1. In some embodiments, the NS-specific promoter is a promoter in Table 1, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter in Table 1, and retaining the NS-specific promoter activity of the promoter in Table 1.

[0230] 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 Tables 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter of Tables 2-4. In some embodiments, the CNS-specific promoter is a promoter of Tables 2-4, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter of Tables 2-4 that retains the CNS-specific promoter activity of a promoter of Tables 2-4.

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

[0232] In some embodiments, the CRE may comprise one or more CREs known in the art. For example, in one embodiment, the one or more CREs may be selected from SEQ ID NOs: 19-24, 27, 28, 37, and 38 of International Patent Application No. WO / 2022 / 049385. For example, in one embodiment, the one or more CREs may be selected from SEQ ID NOs: 1-8 of WO2019 / 199867A1, SEQ ID NOs: 1-7 of WO2020 / 076614A1, and SEQ ID NOs: 25-51, 177-178, and 188 of WO2020 / 097121. The foregoing references are incorporated herein by reference in their entirety.

[0233] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Table 1-7

[0234]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0235]

Table 3

[0236]

Table 4

[0237]

Table 5-1

Table 5-2

[0238]

Table 6-1

Table 6-2

[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 at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61. In one aspect, provided herein is a composition comprising a recombinant viral vector comprising a promoter comprising a nucleic acid sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61.

[0240] [ka] [ka]

[0241] In one embodiment, nucleic acid further comprises the enhancer sequence that helps drive the expression to CNS, for example, to specific CNS tissue or cell type.Exemplary enhancer sequence is described in, for example, US Patent Application No. 17 / 283,232; US17 / 291,584; or International Patent Publication No. WO2020168279A2; WO2021195591A2; WO2021248085A2; WO2021216778A2, and 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 useful for treating or preventing 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%, or at least 98% sequence identity to SEQ ID NO:2. In some embodiments, therapeutic proteins and genes encoding such proteins are useful for treating or slowing the progression of PD.

[0243] The nucleic acids described herein may further comprise a reporter sequence (e.g., a nucleic acid sequence encoding a reporter protein). Reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with regulatory elements that drive their expression, reporter sequences provide a signal that can be detected by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorescent assays or other spectroscopic assays, fluorescent-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is a LacZ gene, the presence of a vector carrying the signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-carrying vector may be visually measured by color or light production in a luminescence spectrophotometer. Such reporters may be useful, for example, to verify the tissue-specific targeting ability of nucleic acids and tissue-specific promoter-regulating activity.

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

[0245] Many secreted proteins, including neurotrophic factors, are synthesized in the form of a precursor, a pre-pro-mature protein. The preregion, consisting of an ER signal peptide, is clipped off during translation by signal peptidases, and the pro-mature protein is released into the lumen of the ER immediately after synthesis. Proteolytic cleavage of the mature protein can occur inside the cell, in the extracellular matrix, or both.

[0246] The pro-mature protein may also remain uncleaved and have a different function than the cleaved mature protein. For example, both mature brain-derived neurotrophic factor (BDNF) and pro-BDNF are secreted from neuronal cells. Mature BDNF binds to the TrkB receptor, which induces neuronal survival, differentiation, and synaptic modulation, whereas pro-BDNF binds to the p75 receptor, which induces apoptosis. NTRand binds to the sortilin receptor (for review, see Thomas and Davies, Curr. Biol., 15:262-264 (2005); Teng et al., J. Neurosci., 25:5455-5463 (2005)).

[0247] In scientific textbooks, the terms GDNF mRNA and GDNF protein are used for the full-length pre-(α)pro-GDNF mRNA and for the mature GDNF protein produced by proteolytic cleavage of the (α)pro-GDNF protein. The mature GDNF protein has been extensively studied, with over 2,500 citations available for GDNF in PubMed. GDNF was identified based on its ability to increase neurite length, cell size, and number of dopaminergic neurons, as well as its high-affinity dopamine uptake in culture (Lin et al., Science, 260:1130-1132 (1993)). GDNF is a potent factor for the protection of dopaminergic neurons of the substantia nigra against their toxin-induced degradation in animal models of PD and also in the treatment of patients with PD (reviewed in Airaksinen and Saarma, Nat. Rev. Neurosci. 3:383-394 (2002) and Bespalov and Saarma, Trends Pharmacol. Sci. 28:68-74 (2007)). In addition, GDNF has a therapeutic role in the treatment of animal models of amyotrophic lateral sclerosis (ALS), addiction, alcoholism, and depression (reviewed in Bohn, Exp. Neurol., 190:263-275 (2004); Messer et al., Neuron, 26:247-257 (2000); He et al., J. 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 morphogen in kidney development and regulates spermatogonia differentiation (reviewed in Sariola and Saarma, J. Cell Sci. 116:3855-3862 (2003)).

[0248] In some embodiments, the present disclosure provides a viral vector for slowing or inhibiting the progression of PD, the vector comprising a GDNF-encoding nucleic acid. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector (e.g., 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 that is capable of encoding a particular polypeptide or protein after being transcribed or translated.

[0252] The terms "coding sequence" or "sequence encoding a particular protein" refer 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' (carboxy) terminus. Coding sequences can 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 for GDNF is disclosed in Genbank Accession NM_000514.4 (SEQ ID NO: 1). The amino acid sequence is set forth in SEQ ID NO: 2. Methods of using nucleic acid constructs comprising the sequence of SEQ ID NO: 1 or variants thereof to slow or inhibit the progression of PD are described herein. Variants include, for example, naturally occurring variants resulting from allelic variation (e.g., polymorphisms) between individuals, alternative splice forms, and the like. The term variant also includes GDNF genes from other sources or organisms. Variants are preferably substantially homologous to SEQ ID NO: 1 and / or 2, i.e., typically exhibit at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95% nucleotide sequence identity with SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid construct comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1 and retaining the activity of SEQ ID NO: 1 or 2. Variants of GDNF gene also include nucleic acid sequences that hybridize to the above-defined sequence (or its complementary strand) 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 salt content of less than about 500mM, preferably less than 200mM. Hybridization conditions can be adjusted by those skilled in the art by modifying temperature, salt content, and / or the concentration of other reagents such as SDS, SSC, etc.

[0254] Variants at nucleotides 277, 633, and 1389 of GDNF have been reported. 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 variants are possible, including 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 example, for one or more of the following: (1) enhanced in vivo expression, (2) reduced CpG islands, or (3) reduced innate immune response. Those skilled in the art 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] [ka]

[0258] [ka]

[0259] [ka] [ka]

[0260] [ka]

[0261] vector 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. When used herein with respect to AAV, the term "isolated" refers to an artificially produced or obtained AAV. An isolated AAV may be produced using recombinant methods. Such an AAV is referred to herein as a "recombinant AAV." Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities, so that the nuclease and / or transgene of the rAAV is specifically delivered to one or more predetermined tissues. The AAV capsid is an important factor in determining these tissue-specific targeting capabilities. Therefore, a rAAV having a capsid appropriate for the tissue to be targeted can be selected.

[0262] Methods for obtaining recombinant AAVs with desired capsid proteins are well known in the art (see, e.g., US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing host cells containing a nucleic acid sequence encoding the AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to enable packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the AAV cap gene. AAVs contain three capsid proteins, designated virion proteins 1 to 3 (VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, 72 kDa, and 62 kDa, respectively. In some embodiments, during translation, capsid protein forms a spherical 60-mer protein shell around the AAV genome.In some embodiments, the function of capsid protein is to protect the viral genome, deliver the genome, and interact with the host.In some aspects, 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 of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV 2G9, AAV 2.5G9, AAV9, and AAV10. In some embodiments, the AAV capsid protein is of a serotype derived from a non-human primate, such as the AAVrh10 serotype. In some embodiments, the AAV capsid protein is of the AAV9 serotype. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or 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, AAV rh74, AAV10, or AAV11, or a chimera thereof.

[0264] In one embodiment, the AAV serotypes and / or capsids described herein are selected from Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

[0265] In certain embodiments, 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 the chemically modified capsid disclosed in WO2017 / 212019 is incorporated herein by reference in its entirety. In a further embodiment, the AAV capsid proteins and viral capsids used herein may be polyploid (also referred to as rational monoploid) in that a single AAV capsid may contain different combinations of VP1, VP2, and VP3 AAV serotypes, as described in PCT / US18 / 22725, PCT / US2018 / 044632, or US10,550,405, which are incorporated by reference.

[0266] The components cultured in the host cell to package the rAAV vector into an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components using methods known to those of skill in the art. Most preferably, such a stable host cell contains the required components under the control of an inducible promoter. However, the required components may also be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, the selected stable host cell 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, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by those skilled in the art. In some embodiments, the present disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a protein (e.g., a wild-type huntingtin protein, optionally a "hardened" wild-type huntingtin protein). In some embodiments, the present disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell further comprises a cryopreservation agent.

[0267] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to generate the rAAV of the present disclosure may be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure are known to those skilled in the art of nucleic acid manipulation and include 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 a suitable method is not a limitation on 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 may be produced using a triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper 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 the pHLP19 vector described in U.S. Pat. No. 6,001,650 and the pRep6cap6 vector described in U.S. Pat. No. 6,156,303, both of which are incorporated herein by reference in their entireties. Accessory function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (i.e., "accessory functions") on which AAV replication depends. Accessory functions include those functions required for AAV replication, including, but not limited to, moieties involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0269] In some aspects, the present disclosure provides a transfected host cell. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are generally 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. Using such techniques, one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into a suitable host cell.

[0270] "Host cell" refers to any cell that harbors or is capable of harboring a substance of interest. Host cells are often mammalian cells. Host cells may be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNA involved in the generation of recombinant AAV. The term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" may refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in overall genetic or DNA complement to the original parent 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. In many cases, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during the storage or transfer of such clonal populations. Thus, the cells derived from the cell line referred to may not be exactly identical to the ancestral cell or culture, and the cell line referred to includes such variants.

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

[0273] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which, when associated with the appropriate control elements, is capable of replication and can transfer gene sequences between cells. Thus, the term "vector" includes cloning and expression vehicles as well as viral vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Certain vectors are also capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. 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 one that can replicate autonomously or integrate into the genome of a host cell, and is further characterized by one or more endonuclease restriction sites, at which the vector can be cut in a determinable manner and the desired DNA sequence can be ligated so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within a host cell, such as a host bacterium, or can occur only once per host before the host reproduces by mitosis. In the case of a phage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0275] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in identifying cells transformed or transfected with the vector, or cells that have not been transformed or transfected. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity is detectable 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 certain embodiments, the vectors used herein are capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably linked.

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

[0277] In some aspects of the present invention, recombinant AAV containing a nucleic acid encoding GDNF (AAV2-GDNF) is produced by the method described in PCT / US2022 / 013279, published as WO2022159679, the entire contents of which are incorporated herein by reference.

[0278] In some embodiments, useful vectors are intended to be those vectors in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter.When it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably linked if the induction of the promoter in the 5'regulatory sequence results in the transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frameshift mutations, (2) does not interfere with the promoter region's ability to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein.Therefore, a promoter region will be operably linked to a coding sequence if the promoter region can cause the transcription of that DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide.

[0279] The exact nature of the regulatory sequences necessary for gene expression may vary between species or cell types, but generally may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also optionally include enhancer sequences or upstream activator sequences. The vectors described herein may also optionally include 5' leader or signal sequences. The selection and design of an appropriate vector is within the ability and discretion of one skilled 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. This heterologous DNA (RNA) is placed under the operable control of transcriptional elements that allow the expression of the heterologous DNA in the host cell.

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

[0282] The foregoing methods for packaging a recombinant vector into a desired AAV capsid to generate the rAAV of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to those of skill in the art.

[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, the nucleic acid molecules can be introduced by standard protocols such as chemical transformation and transformation, including electroporation, transduction, particle bombardment, etc. Expression of the nucleic acid molecules encoding the enzymes of the invention described herein can also be achieved by integrating the nucleic acid molecules into the genome.

[0285] In one embodiment, the genome packaged within the AAV2-GDNF comprises the sequence of SEQ ID NO: 64. In one embodiment, the genome packaged within the AAV2-GDNF consists of or consists essentially of the sequence of SEQ ID NO: 64. In one embodiment, the genome packaged within the AAV2-GDNF comprises, consists essentially of, or consists 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 higher identical to the sequence of SEQ ID NO:64.

[0286] In one embodiment, the AAV2-GDNF vector comprises the ITR-ITR portion of the sequence of SEQ ID NO:64 (i.e., base pairs 12 to 2,716 of SEQ ID NO:64). In one embodiment, the AAV2-GDNF vector consists of, or consists essentially of, the ITR-ITR portion of the sequence of SEQ ID NO:64 (i.e., base pairs 12 to 2,716 of SEQ ID NO:64). In one embodiment, the AAV2-GDNF vector comprises, consists of, or consists essentially of the ITR-ITR portion of the sequence of SEQ ID NO:64 (i.e., base pairs 12 to 2,716 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 has a sequence similar to that of the ITR-ITR portion of SEQ ID NO:64 (i.e., base pairs 12 to 2,716 of SEQ ID NO:64) at 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%, 108%, 109%, 1109%, 1110%, 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%, %, 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 higher. In some embodiments, rAAV is produced using plasmid DNA set forth in SEQ ID NO:64, which is depicted in FIG. 26. In some embodiments, rAAV is produced using closed-end linear double-stranded DNA. In various embodiments, AAV2-GDNF comprises a plasmid containing the ITR-ITR portion of the sequence of SEQ ID NO:64 (i.e., base pairs 12 to 2,716 of SEQ ID NO:64).In other embodiments, the AAV2-GDNF comprises a closed-end linear double-stranded DNA comprising the ITR-ITR portion of the sequence of SEQ ID NO: 64 (i.e., base pairs 12 to 2,716 of SEQ ID NO: 64), a non-limiting example of which is Doggybone DNA (dbDNA™) disclosed in U.S. Application No. 2018 / 0037943 and Karbowniczek et al., Bioinsights, 2017, which are incorporated by reference in their entireties. [ka] [ka] [ka] [ka]

[0287] In one embodiment, the plasmid depicted in Figure 26 is used to generate the AAV2-GDNF genome.

[0288] In one embodiment, the genome packaged within AAV2-GDNF is depicted in FIG.

[0289] In one embodiment, AAV2-GDNF is produced using the plasmid depicted in FIG.

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

[0291] Modified capsid In one embodiment, the capsid described herein is further modified to increase its tropism for the CNS. In one embodiment, the tropism of the capsid, and thus the AAV, is 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%, or 27% of the unmodified AAV. ;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-fold, 2-fold, 3-fold, 4-fold; 5-fold; 10-fold; 15-fold; 20-fold; 25-fold; 30-fold; 35-fold; 40-fold; 45-fold; 50-fold; 55-fold; 60-fold; 65-fold; 70-fold; 75-fold; 80-fold; 85-fold; 90-fold; 95-fold; 100-fold; 250-fold; 500-fold; 750-fold or 1,000-fold or more.

[0292] In one embodiment, the capsid is modified to reduce its tropism for non-CNS tissues, for example, a capsid with liver-specific tropism can be modified so that it no longer has such tropism. In one embodiment, the capsid has a tropism for non-CNS tissue that is 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%; or modified to reduce by 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 yet another embodiment, the modified capsid is modified so that its tropism for CNS tissue is increased and its tropism for non-CNS tissue is decreased. For example, a capsid with liver-specific tropism can be modified so that it exhibits CNS-specific tropism and decreased liver-specific tropism. In one embodiment, the CNS tropism of the capsid is 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% or or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 250-fold, 500-fold, 750-fold, or 1,000-fold or greater increase in tropism for non-CNS tissues compared to the unmodified capsid, and ;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% or greater decrease.

[0294] Provided herein is a composition comprising a modified viral capsid containing a payload, wherein the payload comprises a regulatory sequence and a nucleic acid sequence flanked by inverted terminal repeats (ITRs) that target central nervous system disorders, and the modification is a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a transgene (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, modified viral capsid comprises modifications that cause it to be preferentially targeted to CNS.For example, modified viral capsid has increased tropism to CNS, and / or reduced tropism to at least a second location, for example, liver.Preferential targeting of CNS does not exclude targeting to other sites, but rather indicates that it is more highly targeted to CNS than other sites.

[0296] In one embodiment, the modified viral capsid comprises a modification that results in its targeting of the CNS. For example, modifications to a capsid that typically targets a non-CNS site (e.g., the liver) can redirect the capsid to immediately target both CNS and non-CNS sites. In such an embodiment, CNS targeting does not need to be preferential.

[0297] In one embodiment, the modification to the capsid is an amino acid modification, such as an amino acid deletion, insertion, or substitution. 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 comprises, consists of, or consists essentially of a nucleic acid sequence whose contents are 90% identical to SEQ ID NOS: 1-4 of U.S. Patent Application No. 16 / 511,913, the entire contents of which are incorporated herein by reference. This U.S. patent application describes chimeric AAV capsid sequences that exhibit a dominant tropism for oligodendrocytes and can be used to generate AAV vectors that transduce oligodendrocytes in the CNS of a subject.

[0299] In one embodiment, the modified viral capsid is an AAV capsid protein containing one or more amino acid substitutions, wherein the substitutions introduce new glycan binding sites into the AAV capsid protein. In some embodiments, the amino acid substitutions are at amino acid 266, amino acids 463-475, and amino acids 499-502 in AAV2, or at corresponding amino acid positions in AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV10. Such AAV capsid proteins are further described, for example, in 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 comprises, consists of, or essentially consists of an AAV 2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT / US2020 / 029493, the contents of which are incorporated herein by reference in their entirety) containing 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, such as 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, SQAGASDIRDQSR464-476SX1AGX2SX3X4X5X6QX7R (SEQ ID NOs: 153 and 154, respectively) (wherein X1-7 can be any amino acid), and EYSW500-503 (SEQ ID NO: 155)EX8X9W (wherein X8-9 can be any amino acid). In embodiments of the capsid protein, capsid, viral vector, and method described herein, X1 is V or a conservative substitution thereof; X2 is P or a conservative substitution thereof; X3 is N or a conservative substitution thereof; X4 is M or a conservative substitution thereof; X5 is A or a conservative substitution thereof; X6 is V or a conservative substitution thereof; X7 is G or a conservative substitution thereof; X8 is F or a conservative substitution thereof; and / or X9 is A or a conservative substitution thereof. In embodiments of the capsid protein, capsid, viral vector, and method described herein, X1 is V, X2 is P, X3 is N, X4 is M, X5 is A, X6 is V, X7 is G, X8 is F, and X9 is A, wherein the new 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, such as those described in U.S. Patent Application No. 16 / 956,306, 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, the contents of which are incorporated herein by reference in their entirety. Such AAV capsids cross the blood-brain barrier, enabling, for example, delivery of a payload.

[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 has a modification (e.g., replacement of a tyrosine residue with a non-tyrosine residue and / or replacement of a threonine residue with a non-threonine residue) to a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191; the contents of which are incorporated herein by reference in their entirety). The capsid protein may contain one or more of the following residues: Y705, Y731, and T492 of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); one or more of the following residues: Y436, Y693, and Y719 of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); or one or more of the following residues: Y705, Y731, and T492 of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 ...). Such AAV capsids target neurons and astrocytes.

[0303] In one embodiment, the modified viral capsid has a Y to F (tyrosine to phenylalanine) or T to V (threonine to valine) modification in the VP3 region of the capsid replaced with one or more of: Y705F, Y731F, and T492V of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); The AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein) contains one or more of the following residues at positions corresponding to Y436F, Y693F, and Y719F in SEQ ID NO:2 of U.S. Patent Application No. 16 / 565,191; or one or more of the following residues at positions corresponding to Y705F, Y731F, and T492V in 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.

[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 has 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 Y705, Y731, and Y732 of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191). One or more of Y436, Y693, and Y719, or each of them, of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 in U.S. Patent Application No. 16 / 565,191); or one or more of Y705, Y731, and T492, or each of them, of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 in U.S. Patent Application No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0305] In one embodiment, the modified viral capsid replaces a Y to F (tyrosine to phenylalanine) or a T to V (threonine to valine) modification in the VP3 region of the capsid protein with one or more of: Y705F, Y731F, and T492V of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); The AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein) contains one or more of Y436F, Y693F, and Y719F, or each of them, in SEQ ID NO:2 of U.S. Patent Application No. 16 / 565,191; or one or more of Y705F, Y731F, and T492V, or each of them, in a position corresponding to 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.

[0306] In one embodiment, the amino acid modification allows the modified capsid to avoid, for example, neutralizing antibodies raised against, for example, a viral vector of the same serotype, hi one embodiment, the amino acid modification allows the modified capsid to be used for repeated administration, e.g., the modification allows the capsid to have a therapeutic effect upon re-administration.

[0307] In one embodiment, the modified viral capsid is a chimeric capsid. A "chimeric" capsid protein, as used herein, refers to an AAV capsid protein (e.g., any one or more of VP1, VP2, or VP3) that has been modified by the 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 compared to the wild-type, and the insertion and / 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 compared to the wild-type. In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype can be substituted for corresponding wild-type domains, functional regions, epitopes, etc. from a different AAV serotype in any combination to generate a chimeric capsid protein. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a number of chimeric capsid proteins that can be included in capsids are described in the literature and herein.

[0308] In one embodiment, the modified viral capsid is a monoploid capsid. As used herein, the term "monoploid AAV" refers to the AAV described in International Application No. WO2018 / 170310 or US Application No. US2018 / 037149, the entire contents of which are incorporated herein by reference. In some embodiments, the population of virions is a monoploid AAV population capable of assembling virion particles, wherein at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins required to form virion particles capable of encapsulating the AAV genome. For each viral protein present (VP1, VP2, and / or VP3), the protein is of the same type (e.g., all AAV2 VP1). In one example, at least one viral protein 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 not chimeric. For example, only viral particles composed of VP1 / VP2 from chimeric AAV2 / 8 (N-terminus of AAV2 and C-terminus of AAV8) paired with only VP3 from AAV2, or only chimeric VP1 / VP2 28m-2P3 (N-terminus from AAV8 without VP3 start codon mutation and C-terminus from AAV2) paired with only VP3 from AAV2. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are not 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 paired with VP3 from AAV3. In another example, no chimeric protein exists.

[0309] In some embodiments of the technology described herein, the modified viral capsid comprises one or more modifications, such as chemical, non-chemical, or amino acid modifications to the capsid, which can, for example, alter the tissue-type or cell-type tropism of the modified capsid, among other things.

[0310] The modifications can directly alter the properties of the capsid, including biochemical properties such as receptor binding, so that the modifications themselves alter the behavior of the capsid, or can allow for further modifications, such as the attachment of ligands, that in turn alter 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 coding sequence, allows for covalent attachment of a ligand via disulfide bond formation (see, e.g., WO2005 / 106046, the contents of which are incorporated herein by reference).

[0312] A variety of ligands are contemplated, including, but not limited to, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by the target cell (see, e.g., WO2000 / 002654, which is incorporated herein by reference).

[0313] WO2015 / 062516, the contents of which are also incorporated herein by reference, describes genetic modification of capsid genes to insert amino acids containing azide groups, followed by chemical conjugation of ligands via the azide groups.

[0314] Altering AAV capsid tropism by glycosylation, or chemical conjugation of sugar moieties, is described by Horowitz et al., Bioconjugate Chem. 22: 529-532 (2011). This and similar approaches are contemplated for the capsid modifications described herein.

[0315] In other embodiments, coating of viral capsids with polymers 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, e.g., 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, viral capsids can be modified, for example, as described in WO 2017 / 212019 (see also U.S. National Phase Publication No. USSN 16 / 308,740, the contents of which are each incorporated herein by reference). The method described therein couples the viral capsid to a ligand via a bond containing -CSNH- and an aromatic moiety. Genetically modified viral capsids can be further modified using this method, but the modifications described therein do not require genetic modification of the viral capsid. The ligands described therein include, for example, targeting agents, steric shielding agents to avoid neutralizing antibody interactions, labeling agents, or magnetic agents. The targeting ligands described therein include, 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 mimics cell adhesion proteins and can bind to integrins), vitamins, and small molecules.

[0318] In one embodiment, the chemical modifications described herein are modifications described in International Patent Application No. WO / 2017 / 212019, the contents of which are incorporated herein by reference in their entirety.

[0319] In one embodiment, the chemical modifications described herein are modifications described in International Patent Application No. 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 the capsid, wherein said chemically modified tyrosine residue has a structure represented by formula (I): [ka] It is of

[0321] [In the formula,

[0322] - X1 [ka] is selected from the group consisting of

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

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

[0325] [In the formula,

[0326] Xi and Ar are as defined herein above,

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

[0328] - n is 0 or 1,

[0329] - M is a functional moiety including a steric agent, labeling agent, cell type-specific ligand or 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 formula (Ic): [ka] and at least one chemically modified tyrosine having

[0332] [In the formula,

[0333] X2 is -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] X2 is in the para, meta or ortho position of the phenyl group, preferably in the para position;

[0335] - the spacer, n and M are as defined herein above].

[0336] In one embodiment, the "spacer", when present, is selected from the group consisting of an optionally substituted saturated or unsaturated, linear or branched C2-C40 hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-hydroxypropyl) methacrylamide), polylactic-co-glycolic acid (PLGA), polymers of alkyldiamines, and combinations thereof; and / or

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

[0338] In one embodiment, the "spacer" (if present) is selected from the group consisting of linear or branched C2-C20 alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyldiamines, and combinations thereof, said polymers having 2 to 20 monomers, and / or "M" comprises or consists of a cell-type specific ligand derived from a protein selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor 13 (FGF), a mono- or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNac 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 comprises at least one additional chemically modified amino acid residue in the capsid, which is different from a tyrosine residue, said amino acid residue preferably having the formula (V): [ka] having an amino group chemically modified with a group

[0340] [In the formula,

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

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

[0343] In one embodiment, the capsid is incubated with a chemical reagent having a reactive group selected from an aryldiazonium and a 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moiety under conditions conducive to reacting said reactive group with a tyrosine residue present in the capsid to form a covalent bond.

[0344] In one embodiment, the capsid is incubated with a chemical reagent of formula VId to form a compound of formula Ic [ka] At least one chemically modified tyrosine residue in the capsid of the antibody is obtained.

[0345] Pharmaceutical Composition The expression cassettes, vectors or virions of the invention may be formulated into pharmaceutical compositions using 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 may be provided in the form of a kit.

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

[0347] In various embodiments, the pharmaceutical composition comprises a phosphate buffer solution containing about 1 mM to about 50 mM phosphate, e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM phosphate. The phosphate solution is prepared from a combination of a dibasic phosphate (e.g., NaHPO, KHPO) and a monobasic phosphate (e.g., NaHPO, KHPO) in a molar ratio of about 1:10 to about 10:1 dibasic phosphate:monobasic phosphate. For example, in various exemplary embodiments, 10 mM phosphate includes 9.5 mM dibasic phosphate and 0.5 mM monobasic phosphate, 9 mM dibasic phosphate and 1 mM monobasic phosphate, 8.5 mM dibasic phosphate and 1.5 mM monobasic phosphate, 8 mM dibasic phosphate and 2 mM monobasic phosphate, 7.5 mM dibasic phosphate and 2.5 mM monobasic phosphate, or 7 mM dibasic phosphate and 3 mM monobasic phosphate. The pH of the phosphate buffer is about 6.5 to about 7.5, e.g., 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 to 7.3. In one embodiment, the pH of the phosphate buffer is 7.22. The phosphate buffer may also contain NaCl at a concentration of about 50 mM to about 200 mM, for example, 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 contain KCl at a concentration of about 0.5 mM to about 10 mM, for example, 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 contain CaCl2 at a concentration of about 0.20 mM to about 10 mM, e.g., 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, e.g., 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 contain poloxamer 188 (e.g., Pluronic™ F-68 nonionic surfactant) at a concentration of about 0.0001% to about 0.005% by weight, e.g., about 0.0001%, about 0.0002%, about 0.0003%, about 0.0004%, about 0.0005%, about 0.0006%, about 0.0007%, about 0.0008%, about 0.0009%, about 0.001%, about 0.0015%, about 0.002%, about 0.0025%, about 0.003%, about 0.0035%, about 0.004%, about 0.0045%, or about 0.005% by weight. The phosphate buffer may also contain sorbitol at a concentration of about 0.005% to about 10% by weight, e.g., about 0.005%, about 0.075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight.The rAAV comprising the 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) is about 1 x 10. 12 vg / mL ~ approx. 4×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 3×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 2×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 9 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 4 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 5 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 6 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 7 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 8 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 8×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 7×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 6×10 12 vg / mL; 8 × 10 11vg / mL to approximately 5×10 12 vg / mL; 8×10 11 vg / mL to approximately 4×10 12 vg / mL; 8×10 11 vg / mL to approximately 3×10 12 vg / mL; 8×10 11 vg / mL to approximately 2×10 12 vg / mL; 8×10 11 vg / mL to approximately 1×10 12 vg / mL; 8×10 11 vg / mL to approximately 9×10 11 vg / mL; 1×10 12 vg / mL to approximately 7×10 12 vg / mL; 3×10 12 vg / mL to approximately 6×10 12 vg / mL; 4×10 12 vg / mL to approximately 5×10 12 vg / mL; 3.1×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.2×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.3×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.4×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.5×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.6×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.7×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.8×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.9×10 12 vg / mL to approximately 4×10 12 vg / mL; 3×10 12 vg / mL to approximately 3.9×10 12 vg / mL; 3×10 12 vg / mL to approximately 3.8×10 12 vg / mL; 3×10 12 vg / mL to approximately 3.7×10 12 vg / mL; 3×1012 vg / mL ~ approx. 3.6×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.5×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.4×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.3×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.2×10 12 vg / mL; and 3 × 10 12 vg / mL ~ approx. 3.1×10 12 The antibody may have a titer in phosphate buffer of 1000 mg / mL.

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

[0349] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of phosphate (monobasic and dibasic phosphate), 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 (monobasic and dibasic phosphate), 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 NaHPO, about 2 mM NaHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3.

[0353] In some embodiments, the pharmaceutical composition comprises 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 ~ approx. 3.1×10 13 vg / mL AAV2-GDNF.

[0354] In some embodiments, the pharmaceutical composition comprises 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 AAV2-GDNF.

[0355] In some embodiments, the pharmaceutical composition comprises 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 12 vg / mLvg / mL AAV2-GDNF.

[0356] In some embodiments, the pharmaceutical composition comprises 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 AAV2-GDNF.

[0357] In some embodiments, the pharmaceutical composition comprises 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 AAV2-GDNF.

[0358] In some embodiments, the pharmaceutical composition comprises about 8 mM KHPO, about 2 mM KHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and about 1×10 12 vg / mL ~ approx. 3.1×10 13 vg / mL AAV2-GDNF.

[0359] In some embodiments, the pharmaceutical composition comprises about 8 mM KHPO, about 2 mM KHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1×10 12 vg / mL AAV2-GDNF.

[0360] In some embodiments, the pharmaceutical composition comprises about 8 mM KHPO, about 2 mM KHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 5×10 12 vg / mLvg / mL AAV2-GDNF.

[0361] In some embodiments, the pharmaceutical composition comprises about 8 mM KHPO, about 2 mM KHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 1×10 13 vg / mL AAV2-GDNF.

[0362] In some embodiments, the pharmaceutical composition comprises about 8 mM KHPO, about 2 mM KHPO, about 180 mM NaCl, and about 0.001% poloxamer; pH 7.2-7.3; and at least 3×10 13 vg / mL AAV2-GDNF.

[0363] Administration 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, rAAV, preferably suspended in a physiologically compatible carrier (i.e., in a composition), can be administered to a subject, i.e., a host animal, for example, 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 human.

[0364] It may be desirable for the rAAV described herein to be delivered directly to the CNS of a subject. "CNS" refers to all cells and tissues of the brain and spinal cord of a vertebrate.Therefore, this term includes, but is not limited to, nerve cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. Recombinant AAV can be delivered directly to the CNS or brain by injection into the ventricular region, and to the striatum (e.g., the caudate nucleus and / or putamen of the dorsal striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule using a needle, catheter, or related device, using neurosurgical techniques known in the art, such as by stereotactic injection (see, for example, 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). In some embodiments, the rAAV 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, rAAV is delivered by intracranial injection. In some embodiments, rAAV is delivered by cisternal injection. In some embodiments, rAAV is delivered by lateral ventricle injection of the cerebrum.

[0365] In one embodiment, the rAAV or composition thereof is delivered locally to the CNS, e.g., directly to the putamen, via a stepped cannula, as described, e.g., in U.S. 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 entireties.

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

[0367] In one embodiment, a composition described herein is administered topically to the putamen, e.g., 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 / min; 16 μL / min; 17 μL / min; 18 μL / min; 19 μL / min; 20 μL / min; 21 μL / min; 22 μL / min; 23 μL / min; 24 μL / min; 25 μL / min; 26 μL / min; 27 μL / min; 28 μL / min; 29 μL / min; or 30 μL / min.

[0369] Furthermore, in certain instances, it may be desirable to deliver rAAV to a mammalian subject, for example, by intramuscular injection or by administration into the mammalian subject's bloodstream. 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 isolated limb perfusion, a technique well known in the surgical arts; this method essentially allows one skilled in the art to isolate a limb from the systemic circulation before administering rAAV virions. Variants of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 can also be used by those skilled in the art to administer virions into the blood vessels of an isolated limb, potentially enhancing transduction of muscle cells or tissues.

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

[0371] In one embodiment, the rAAV or composition thereof is administered during the subject's "on" periods.

[0372] Aspects of the present disclosure relate to a composition for slowing or inhibiting the progression of PD in a subject, the composition comprising any of the recombinant adeno-associated viruses (rAAVs) comprising a genome including a glial cell line-derived neurotrophic factor (GDNF) gene operably linked to a promoter described herein, and a pharmaceutically acceptable carrier. In one aspect, the composition comprises any of the viral vectors described herein, and optionally a pharmaceutically acceptable carrier. The composition of the present disclosure may comprise an rAAV alone or in combination with one or more other viruses (e.g., a second rAAV carrying and encoding one or more different transgenes). In some embodiments, the composition comprises one, two, three, four, five, six, seven, eight, nine, ten, or more different rAAVs, each carrying 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 therapeutic agent described herein. The compositions of the present disclosure may further comprise any immune modulator described herein. The compositions of the present disclosure may further comprise a second therapeutic agent, such as an anti-Parkinson's therapeutic agent described herein, and any immune modulator described herein.

[0374] Suitable carriers can be easily selected by those skilled in the art, taking into account the indications for which rAAV is intended. For example, one suitable carrier includes saline, which may be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of carriers does not limit the present disclosure.

[0375] If necessary, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0376] The rAAV is administered in an amount sufficient to transfect cells of the desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ (e.g., delivery to the putamen), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Optionally, routes of administration may be combined. In some embodiments, all or at least one of the nucleic acid sequences disclosed herein are delivered via a non-viral DNA construct containing at least one DD-ITR. For example, the non-viral DNA constructs described in WO2019 / 246554 can be used to deliver one or more of the nucleic acids described herein. WO2019 / 246554 is incorporated herein by reference in its entirety.

[0377] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., a dose in genome copies per kilogram of body weight (GC / kg), will vary based on several factors, including, but not limited to, the route of 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. One of ordinary skill in the art can readily determine the dose range of rAAV virions for treating a patient with a particular disease or disorder based on the aforementioned factors and other factors well known in the art.

[0378] In one embodiment, the rAAV is 5×10 12 vg~approx. 1.5×10 13 In another embodiment, the rAAV is administered at a total dose in the range of 1 x 10 12 vg~approx. 6.5×10 13 vg;2×10 12 vg~approx. 6.5×10 13 vg;3×10 12 vg~approx. 6.5×10 13 vg;4×1012 vg~approx. 6.5×10 13 vg;6×10 12 vg~approx. 6.5×10 13 vg;7×10 12 vg~approx. 6.5×10 13 vg;8×10 12 vg~approx. 6.5×10 13 vg;9×10 12 vg~approx. 6.5×10 13 vg;1×10 13 vg~approx. 6.5×10 13 vg;1×10 12 vg~approx. 1.5×10 13 vg;2×10 12 vg~approx. 1.5×10 13 vg;3×10 12 vg~approx. 1.5×10 13 vg;4×10 12 vg~approx. 1.5×10 13 vg;6×10 12 vg~approx. 1.5×10 13 vg;7×10 12 vg~approx. 1.5×10 13 vg;8×10 12 vg~approx. 1.5×10 13 vg;9×10 12 vg~approx. 1.5×10 13 vg;1×10 13 vg~approx. 1.5×10 13 vg;1×10 12 vg~approx. 6.5×10 13 vg;2×10 12 vg~approx. 6.5×10 13 vg;3×10 12 vg~approx. 6.5×10 13 vg;4×10 12 vg~approx. 6.5×10 13 vg;6×10 12 vg~approx. 6.5×10 13 vg;7×10 12 vg~approx. 6.5×10 13 vg;8×10 12 vg~approx. 6.5×10 13 vg;9×10 12 vg~approx. 6.5×10 13 vg;1×10 13vg~approx. 6.5×10 13 vg;1×10 12 vg~approx. 1×10 13 vg;1×10 12 vg~approx. 9×10 12 vg;1×10 12 vg~approx. 8×10 12 vg;1×10 12 vg~approx. 7×10 12 vg;1×10 12 vg~approx. 6×10 12 vg;1×10 12 vg~approx. 5×10 12 vg;1×10 12 vg~approx. 4×10 12 vg;1×10 12 vg ~ approx. 3×10 12 vg;1×10 12 vg~approx. 2×10 12 vg;5×10 12 vg~approx. 1×10 13 vg;5×10 12 vg~approx. 9×10 12 vg;5×10 12 vg~approx. 8×10 12 vg;5×10 12 vg~approx. 7×10 12 vg;5×10 12 vg~approx. 6×10 12 vg;5×10 12 vg~approx. 5.5×10 13 vg;5×10 12 vg~approx. 4.5×10 13 vg;5×10 12 vg~approx.3.5×10 13 vg; and 5 × 10 12 vg~approx. 2.5×10 13 The total dose is administered in the range of vg.

[0379] In one embodiment, the rAAV is at least 5.1 x 10 12vg ;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 12vg;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;6.9×10 12 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;および1.4×1013 In one embodiment, the rAAV is administered at a total dose of at least 1 x 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 13 vg; 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 higher total dose.

[0380] In one embodiment, administration or induction is performed locally to the subject's putamen, with half of the total dose being administered to each of the subject's putamen. In yet another method, the total dose is divided substantially equally between the subject's left and right putamen.

[0381] The effective amount of rAAV is sufficient to target infection in animals or target desired tissue.In some embodiments, the effective amount of rAAV is sufficient to generate stable somatic transgenic animal model.Effective amount mainly depends on factors such as subject species, age, weight, health and tissue to be targeted, and therefore may vary between animals and tissues.For example, the effective amount of rAAV is generally about 10 9 ~10 16 In some cases, the volume of the solution containing the genome copies ranges from about 1 ml to about 100 ml. 11 ~10 13 In certain embodiments, a dosage of between 10 and 100 rAAV genome copies is appropriate. 12 or 10 13rAAV genome copies are effective in targeting CNS tissues (e.g., putamen). In some cases, stable transgenic animals are generated by multiple doses of rAAV.

[0382] In one embodiment, the rAAV is introduced or administered in a liquid composition. In one embodiment, the liquid composition contains about 3 x 10 12 vg / mL ~ approx. 4×101 2 In one embodiment, the liquid composition has an rAAV concentration of about 1 x 10 vg / mL. 12 vg / mL ~ approx. 4×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 3×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 2×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 9 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 4 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 5 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 6 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 7 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 8 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 8×10 12 vg / mL; 8 × 1011 vg / mL to approximately 7×10 12 vg / mL; 8×10 11 vg / mL to approximately 6×10 12 vg / mL; 8×10 11 vg / mL to approximately 5×10 12 vg / mL; 8×10 11 vg / mL to approximately 4×10 12 vg / mL; 8×10 11 vg / mL to approximately 3×10 12 vg / mL; 8×10 11 vg / mL to approximately 2×10 12 vg / mL; 8×10 11 vg / mL to approximately 1×10 12 vg / mL; 8×10 11 S vg / mL to approximately 9×10 11 vg / mL; 1×10 12vg / mL to approximately 7×10 12 vg / mL; 3×10 12vg / mL to approximately 6×10 12 vg / mL; 4×10 12vg / mL to approximately 5×10 12 vg / mL; 3.1×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.2×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.3×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.4×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.5×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.6×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.7×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.8×10 12 vg / mL to approximately 4×10 12 vg / mL; 3.9×10 12 vg / mL to approximately 4×10 12 vg / mL; 3×10 12 vg / mL to approximately 3.9×10 12 vg / mL; 3×1012 vg / mL ~ approx. 3.8×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.7×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.6×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.5×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.4×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.3×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.2×10 12 vg / mL; and 3 × 10 12 vg / mL ~ approx. 3.1×10 12 with an rAAV concentration of 1000 mg / mL.

[0383] In one embodiment, the liquid composition contains about 8×10 11 vg / mL; 9 × 10 11 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 with an rAAV concentration of 1000 mg / mL.

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

[0385] In one embodiment, the rAAV or a composition thereof is administered as an injection via a transfrontal (e.g., bilateral) trajectory. A transfrontal trajectory is at least one trajectory that is substantially perpendicular to the AP axis of each putamen and accessed through the frontal bone of the skull. Transfrontal administration can include injecting the rAAV through two or more trajectories that are substantially perpendicular to the AP axis of each putamen. Each trajectory can be accessed through a single burr hole through the frontal bone of the skull or through separate individual burr holes. Therefore, at least one burr hole in the frontal bone is required for each of the left and right putamen. For example, administration can be performed using a transfrontal trajectory, e.g., a bilateral frontal trajectory, in which a cannula is guided from the frontal bone through a first trajectory that is substantially perpendicular to the AP axis of the first putamen, and the rAAV is injected using the first trajectory. The cannula is then placed in a second trajectory (different from the first trajectory) that is substantially perpendicular to the AP axis of the first putamen, and the rAAV is injected using the second trajectory. The process is then repeated in the second putamen. It is understood that each trajectory results in the cannula contacting the putamen at a different location. Figure 13A shows an exemplary transfrontal trajectory.

[0386] In one embodiment, rAAV or a composition thereof is administered as an injection via a bilateral occipital trajectory. The occipital trajectory is a single posterior trajectory substantially parallel to the AP axis of the putamen and is accessed through the occipital bone of the skull. Therefore, a single burr hole in the occipital bone is required for each putamen. For example, administration can be performed using a bilateral occipital trajectory, in which a cannula is guided from the occipital bone using a trajectory substantially parallel to the AP axis of the first putamen, and rAAV is injected while advancing the cannula toward the rostral end of the first putamen. The process is then repeated with the second putamen. In a variation, rAAV is injected into each putamen without advancing the cannula to the rostral end of each putamen, with the cannula fixed. Figure 13B shows an exemplary occipital trajectory.

[0387] In one embodiment, each putamen is injected using the same orbital technique, i.e., each putamen is injected via bilateral frontal orbits. In one embodiment, each putamen is injected using different orbital techniques, i.e., one putamen is injected via bilateral frontal orbits and the other putamen is injected via bilateral occipital orbits.

[0388] In one embodiment, the injection volume of a single putamen is less than or equal to 2000 μL and greater than 1,800 μL.For example, a single putamen can be injected with a total volume of 1,800 μL; 1,810 μL; 1,820 μL; 1,830 μL; 1,840 μL; 1,850 μL; 1,860 μL; 1,870 μL; 1,880 μL; 1,890 μL; 1,900 μL; 1,910 μL; 1,920 μL; 1,930 μL; 1,940 μL; 1,950 μL; 1,960 μL; 1,970 μL; 1,980 μL; 1,990 μL; or 2,000 μL.In one embodiment, the injection volume of each putamen is the same. In one embodiment, the injection volume for each putamen is different (eg, one putamen is injected with a volume of 1,800 μl and the other putamen is injected with a volume of 2,000 μl).

[0389] In some embodiments, the rAAV composition may be particularly useful in cases where a high concentration of rAAV is present (e.g., -10 13 The composition is formulated to reduce aggregation of AAV particles in the composition when the concentration of rAAV is less than 100 ng / ml (GC / ml or more). Methods for reducing aggregation of rAAV are well known in the art and include, for example, adding detergents, adjusting pH, adjusting salt concentration, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0390] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for use with the particular compositions described herein in various treatment regimens.

[0391] One embodiment described herein is a formulation for slowing or inhibiting the progression of PD in a subject, comprising 3 x 10 hydroxybenzoates per mL of a pharmaceutically acceptable carrier. 12 vg~4×10 12 In one embodiment, the concentration of the viral vector is about 1 x 10 vg. 12 vg / mL ~ approx. 4×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 3×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 2×10 12 vg / mL; 2 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 8 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 9 × 10 11 vg / mL ~ approx. 9×10 12 vg / mL; 1 × 10 12 vg / mL ~ approx. 9×10 12 vg / mL; 2 × 10 12vg / mL to approximately 9×10 12 vg / mL; 3×10 12 vg / mL to approximately 9×10 12 vg / mL; 4×10 12 vg / mL to approximately 9×10 12 vg / mL; 5×10 12 vg / mL to approximately 9×10 12 vg / mL; 6×10 12 vg / mL to approximately 9×10 12 vg / mL; 7×10 12 vg / mL to approximately 9×10 12 vg / mL; 8×10 12 vg / mL to approximately 9×10 12 vg / mL; 8×10 11 vg / mL to approximately 8×10 12 vg / mL; 8×10 11 vg / mL to approximately 7×10 12 vg / mL; 8×10 11 vg / mL to approximately 6×10 12 vg / mL; 8×10 11 vg / mL to approximately 5×10 12 vg / mL; 8×10 11 vg / mL to approximately 4×10 12 vg / mL; 8×10 11 vg / mL to approximately 3×10 12 vg / mL; 8×10 11 vg / mL to approximately 2×10 12 vg / mL; 8×10 11 vg / mL to approximately 1×10 12 vg / mL;​​​​​​​​​​​​​​​​​​​​​​​​12 vg / mL; 3.4 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3.5 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3.6 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3.7 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3.8 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3.9 × 10 12 vg / mL ~ approx. 4×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.9×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.8×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.7×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.6×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.5×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.4×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.3×10 12 vg / mL; 3 × 10 12 vg / mL ~ approx. 3.2×10 12 vg / mL; and 3 × 10 12 vg / mL ~ approx. 3.1×10 12 In one embodiment, the concentration of the viral vector is about 8 x 10 11 vg / mL; 9 × 10 11 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 × 1012 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 formulations may contain at least about 0.1% or more of the active compound, although the percentage of active ingredient may, of course, vary and may conveniently be between about 1% or 2% and about 70% or 80% or more of the total formulation weight or volume. Essentially, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations; as such, various dosage and treatment regimens may be desirable.

[0393] In certain circumstances, it may be desirable to deliver the rAAV-based therapeutic construct in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the rAAV may be delivered using the administration modalities described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety). In some embodiments, the preferred mode of administration is via portal vein injection.

[0394] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, this form is sterile and fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), a suitable mixture thereof, and / or vegetable oils. For example, proper fluidity can be maintained by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

[0395] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermic fluid or injected at the planned site of infusion (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0396] Sterile injectable solutions are prepared by incorporating the active rAAV with various other ingredients listed herein in the required amount into a suitable solvent, and then optionally sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0397] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), such as inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution is administered in a manner compatible with the dosage formulation and in such an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as 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, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to a host.

[0399] Delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, vesicles, etc., may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the transgene delivered by the rAAV vector may be formulated for delivery by being encapsulated in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, etc.

[0400] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the 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). Furthermore, various methods for liposomes and liposome-like preparations 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 in several cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

[0402] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain aqueous solution in their cores.

[0403] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overloading, such ultrafine particles (approximately 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 contemplated for use.

[0404] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used as a device to enhance the rate and efficiency of drug penetration into and through the circulatory system and is described in U.S. Patent No. 5,656,016. Other contemplated drug delivery alternatives are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (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 method described herein relates to treating the subject who has PD or has been diagnosed with PD with the nucleic acid described herein.The subject who has PD can be identified by clinicians using the current method of diagnosing such diseases and disorders, for example, the method described herein above.The symptoms and / or complications of PD that characterize this condition and help diagnose it, and the clinical tests that carry it out are well known in the art and are described herein above.Family history of PD can also help determine whether a subject may have PD or make a diagnosis of PD.

[0406] In one embodiment, the subject has been diagnosed with PD before undergoing the treatment described herein. In one embodiment, the subject has been 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 before undergoing the treatment described herein.

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

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

[0409] In one embodiment, the subject is diagnosed with PD before undergoing a treatment described herein. In one embodiment, the subject is diagnosed as being at risk for having PD before undergoing a treatment described herein.

[0410] In one embodiment, before administering the treatment, the person administering the treatment receives the results of a diagnostic assay diagnosing the subject as having PD. In one embodiment, before administering the treatment, the person administering the treatment receives the results of an assay diagnosing the subject as at risk for having PD.

[0411] The compositions and methods described herein can be administered to subjects who have or have been diagnosed with PD. In some embodiments, the methods described herein include administering an effective amount of the compositions described herein, such as the nucleic acids 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. Compared with comparable untreated controls, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique.

[0412] Effective dose, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine the minimum effective dose and / or maximum tolerated dose. Dosages can vary depending on the dosage form used and the route of administration utilized. The therapeutically effective dose can be initially estimated from cell culture assays. Dosages can also be formulated in animal models to achieve a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dosage can be monitored by suitable bioassays, for example, assays for neuronal degradation or functionality, among others. Dosages are determined by a physician and can be adjusted, if necessary, to match the observed effects of treatment.

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

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

[0415] In one embodiment, the at least one anti-PD therapeutic agent excludes 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 drugs and therapies for PD. "Administered in combination," as used herein, means that two (or more) different treatments are delivered to a subject during the course of the subject's illness, for example, two or more treatments are delivered after the subject is diagnosed with PD and before the illness is cured or eliminated, or before the treatments are stopped for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, resulting in an overlap in the administration period. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, e.g., a comparable effect may be seen with less of the second treatment, or the second treatment may reduce symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation may be seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disease is greater than that observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. The rAAV described herein and at least one additional therapy may be administered simultaneously or sequentially in the same or separate compositions. For sequential administration, the agent described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed. The drugs and / or other therapeutic agents, procedures or modalities can be administered during periods of active disease or during periods of remission or less active disease.The agent can be administered prior to another treatment, concurrently with a treatment, after a treatment, or during remission of the disorder.

[0417] Exemplary therapeutic agents used to treat PD are described herein above.

[0418] When administered in combination with rAAV, an additional therapeutic agent (e.g., a second or third anti-PD therapeutic agent) can be administered in an amount or dosage that is higher, lower, or the same as the amount or dosage of each therapeutic agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the additional therapeutic agent is lower (e.g., at least 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95% or more lower) than the amount or dosage of each additional therapeutic agent used individually.

[0419] In other embodiments, the amount or dosage of the additional therapeutic agent that produces the 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 more lower) than the amount or dosage of each additional therapeutic agent individually required to achieve the same therapeutic effect.

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

[0421] In one embodiment, a subject reduces the amount or dosage of at least one anti-PD therapeutic agent after introduction or administration of any of the rAAVs described herein. 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 introduction or administration of any of the rAAVs.

[0422] In one embodiment, the subject is no longer receiving anti-PD therapy after the introduction or administration of any of the rAAVs described herein.

[0423] immune modulators In some embodiments, the compositions described herein include an immune modulator, and the methods further include administering the immune modulator. The immune modulator can be administered at the time of, before, or after administration. In cases where the subject is re-administered at least a second composition, the immune modulator can be administered before, with, or after at least the second administration.

[0424] In preferred embodiments, the immune modulator is administered prior to administration of the recombinant viral vector. In various embodiments, the immune modulator is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more prior to administration of the recombinant viral vector. In one embodiment, the immune modulator is administered 24 hours or less prior to administration of the recombinant viral vector.

[0425] In one embodiment, the immune modulator is administered at substantially the same time as the recombinant viral vector, for example, shortly before (i.e., within 6 hours, or 5 hours, or 4 hours, or 3 hours, or 2 hours, or 1 hour) administration of a recombinant viral vector disclosed herein. In some embodiments, the immune modulator is administered simultaneously with 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 immune modulator allows for the administration of a recombinant viral vector to a subject who would not otherwise be a good candidate for receiving such a vector. Subjects who would not otherwise be good candidates for receiving such a vector include, for example, subjects who have previously received and / or been exposed to a recombinant viral vector and subsequently develop an antibody response to the vector. Typically, subjects are considered to be candidates, i.e., good candidates, for administration of a recombinant viral vector if they have a titer for viral vector-binding antibodies of less than 1:5 (e.g., 1:1, 1:2, 1:3, or 1:4). In contrast, subjects are considered not to be good candidates for administration of a recombinant viral vector if they have a titer for viral vector-binding antibodies of 1:5 or greater (e.g., 1:6, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:50, 1:100, 1:1,000, or greater). Those skilled in the art can use standard techniques in the art to assess a subject's antibody titer, for example, by obtaining a biological sample from the subject, such as the subject's blood, challenging the biological sample with a known antigen, and detecting the presence of virus-bound antibodies against 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 the sample. Antibody titer is usually expressed as a ratio, for example, 1:100, meaning that 1 part serum to 100 parts saline solution (i.e., diluent) results in 50% antibody neutralization in the sample. That is, the reciprocal of the serum dilution required to inhibit viral infection by 50% can be designated as the neutralizing antibody titer at 50% inhibition. A viral vector antibody titer of 1:10 is therefore indicative of a lower level of viral vector antibody than a titer of 1:100.

[0427] Thus, in one embodiment, the subject is evaluated for the presence of anti-AAV antibodies against the gene therapy AAV vector before administering the gene therapy. In one embodiment, the subject is evaluated for the presence of neutralizing anti-AAV antibodies against the gene therapy AAV vector before administering 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. Jan 6, 2022, the contents of which are incorporated herein by reference in their entirety.

[0428] In one embodiment, an immune modulator 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), where 1 part biological sample diluted in 10,000 parts buffer results in 50% viral vector neutralization.

[0429] In one embodiment, an immune modulator is administered to a subject with a titer of viral vector-binding antibodies present in a biological sample or blood product from the subject that is greater than or equal to 1.5 and less than about 1:10 (e.g., 1:6, 1:7, 1:8, or 1:9), in order to expand the pool of subjects that can be effectively treated with AAV gene therapy, where 1 part biological sample or blood product diluted in 10,000 parts buffer results in 50% viral vector neutralization. Currently, prospective patients with virus-neutralizing antibody levels of 1:5 or higher are excluded from such treatment, i.e., they are not good candidates. Administration of an immune modulator to a subject with an antibody titer greater than or equal to 1:5 but less than 1:10 is expected to reduce the antibody titer present 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, an immune modulator is administered to a subject 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), where 1 part biological sample or blood product diluted in 10,000 parts buffer results in 50% viral vector neutralization. For example, administration of an immune modulator to a subject with a titer greater than or equal to 1:5 but less than 1:15 is expected to reduce the antibody titer present 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 immune modulator 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, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1

[0013] The immunomodulator may be administered to a subject having an antibody titer of viral vector-binding antibodies present in a biological sample from the subject of 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, and 1:99), where 1 part biological sample or blood product diluted in 10,000 parts buffer results in 50% viral vector neutralization. Administration of an immune modulator to a subject having an antibody titer of greater than or equal to 1:5, but less than 1:25, is expected to reduce the antibody titer present 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 immune modulator is capable of repeated dosing or administration of the AAV vector disclosed herein. For example, administration of a viral vector, e.g., an AAV vector, and the immune modulator (at substantially the same time as, or before or after, administration of the AAV vector) disclosed herein can be administered multiple times (i.e., more than once) over a defined period of time. By way of example, the AAV vector can be administered several times, i.e., more than once over a period of several weeks (e.g., 2 weeks) to several months (e.g., 2 months). Without wishing to be limited by theory, administration of the AAV vector and the immune modulator according to the methods disclosed herein can be, by way of non-limiting example, monthly for a 6-month period, 3-4 times over a 6-week period, weekly for a 1-month (or about 4 weeks) or 2-month (or about 8 weeks) period. In some embodiments, when an AAV vector disclosed herein is administered multiple times (e.g., multiple doses) with an immune modulator (either at substantially the same time as, before, or after administration of the AAV vector), the dose of the viral vector, e.g., AAV vector, is lower than typically used in single dose regimens, e.g., at a lower dose than in the single dose regimens described herein. For example, the dose of the AAV vector is about 10 12 Less than or equal to, or about 10 12 For example, the dose may be less than about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 , or 10 7 ~10 12In some embodiments, repeated doses of the AAV vectors disclosed herein are administered to a subject according to the methods disclosed herein together with an immune modulator, and the immune modulator can be varied between doses, i.e., the same or different immune modulators can be used in the repeated doses. For example, if a first dose of AAV is co-administered with immune modulator A, the second immune modulator administered with the second or third dose of AAV is different from immune modulator A, e.g., the second immune modulator is immune modulator B. For illustrative purposes, a dosing regimen according to the disclosed methods can be 10 12 or 10 12The dosing regimen may involve administration of an AAV vector at a concentration of less than 100 mg / mL, where the immune modulator is administered as ABCD, or AABC, or ABAC, where A, B, C, and D are different immune modulators disclosed herein. In other words, the dosing regimen may include multiple doses of the immune modulator over a period of time, where each dose of the plurality includes an immune modulator independently selected from immune modulator A, immune modulator B, immune modulator C, immune modulator D, and combinations thereof (i.e., each dose may include two or more immune modulators). Such different immune modulators allow for repeated dosing of the AAV vectors disclosed herein. In some embodiments, for example, immune modulator "A" may be, for example, IdeS, and immune modulator "B" may be ImmTOR™, as disclosed herein. In some embodiments, an AAV vector disclosed herein is administered at a first time point with an IdeS immune modulator, and an AAV vector disclosed herein is administered at a second time point with a different immune modulator, e.g., an immunoglobulin-degrading protein or small molecule, e.g., ImmTOR™, or vice versa. For example, an AAV vector disclosed herein can be administered at a first time point with an ImmTOR™ immune modulator, and an AAV vector disclosed herein can be administered at a second time point with IdeS.

[0432]

[0013] One aspect of the present disclosure provides a method for administering a recombinant viral vector (e.g., a gene therapy vector) to a subject who has previously received a recombinant viral vector, e.g., the same recombinant viral vector or another viral vector having a similar serotype, wherein the method comprises administering an immune modulator to the subject prior to administering the recombinant viral vector. In one embodiment, the previously received recombinant viral vector elicits an immune response that results in anti-AAV antibodies that target (i.e., recognize and bind to) the administered recombinant viral vector.

[0433] Another aspect herein provides a method for administering a recombinant viral vector (e.g., a gene therapy vector) to a subject who has previously been exposed to a viral vector, wherein the exposure elicits an immune response that results in anti-AAV antibodies that target the administered recombinant viral vector, and the subject has an anti-AAV antibody titer of at least 1:5 to 1:15, at least 1:5 to 1:25, at least 1:5 to 1:50, or at least 1:5 to 1:100, and the method comprises administering an immune modulator to the subject prior to administering the recombinant viral vector.

[0434] In one embodiment, the immune modulator is administered systemically.

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

[0436] In one embodiment, the immune modulator is administered locally. For example, if a recombinant viral vector is administered locally to brain tissue and the immune modulator does not cross the blood-brain barrier, it is preferred to administer the immune modulator locally to brain tissue, either directly to the brain tissue, e.g., via a suitable catheter, or indirectly to the brain tissue through cerebrospinal fluid circulating in the spinal cord (i.e., spinal tap).

[0437] In one embodiment, the immune modulator is administered locally to central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF)). CNS tissue also includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Any of the compositions described herein can be delivered directly to the CNS or brain, for example, by injection into the ventricular region, as well as the 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 immune modulator is administered locally to any of the following: neural pathways, the somatosensory system, the visual system, the auditory system, nerves, the neuroendocrine system, the neurovascular system, the brain neurotransmitter system, the dural meningeal system, or a combination thereof.

[0439] In one embodiment, the immune modulator is administered locally to the eye, for example, to the vitreous, retina, or sclera.

[0440] In one embodiment, the immune modulator is administered systemically.

[0441] In some embodiments, the immune modulator 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 herein in its entirety.

[0442] In some embodiments, the immune modulators disclosed herein can be administered to a subject at any suitable dose, e.g., a suitable dose determined by a medical professional. For example, a suitable dosage can be from about 0.05 mg / kg to about 5 mg / kg of the subject's body weight, or from about 0.1 mg / kg to about 4 mg / kg of the subject's body weight.

[0443] In some embodiments, an immune modulator disclosed herein, such as IdeZ, is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg of the subject's body weight. For example, a suitable dosage may be about 0.05 mg / kg to about 5 mg / kg of the subject's body weight, or about 0.1 mg / kg to about 4 mg / kg of the subject's body weight.

[0444] In some embodiments, an immune modulator disclosed herein, such as IdeS, is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg of the subject's body weight. For example, a suitable dosage may be about 0.05 mg / kg to about 5 mg / kg of the subject's body weight, or about 0.1 mg / kg to about 4 mg / kg of the subject's body weight.

[0445] In some embodiments, an immune modulator disclosed herein, such as EndoS, is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg of the subject's body weight. For example, a suitable dosage may be about 0.05 mg / kg to about 5 mg / kg of the subject's body weight, or about 0.1 mg / kg to about 4 mg / kg of the subject's body weight.

[0446] Additionally, reference to numerical ranges, e.g., "0.01 to 10," includes 0.011, 0.012, 0.013, etc., and 9.5, 9.6, 9.7, 9.8, 9.9, 10, etc. For example, a dosage of about "0.01 mg to about 10 mg" per kg of subject body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., and 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

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

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

[0449] In certain embodiments, the protease and / or glycosidase is administered to the 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 the heterologous polynucleotide, protein, or peptide encoded by the heterologous polynucleotide encapsulated by the 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; or within 1 day, 2 days, 3 days, 4 days, 5 days, or less after administration of the recombinant viral vector. This also happens over a long period of time.

[0450] In some embodiments, the immunomodulator is a proteasome inhibitor. In some embodiments, the immunomodulator is a protease or glycosidase. In certain aspects, the proteasome inhibitor is bortezomib. In some aspects of the embodiment, the immunomodulator comprises bortezomib and an anti-CD20 antibody, such as rituximab. In other aspects of the embodiment, the immunomodulator comprises bortezomib, rituximab, methotrexate, and intravenous gamma globulin. Non-limiting examples of proteasome inhibitors and their combinations with rituximab, methotrexate, and intravenous gamma globulin are described in US 10,028,993, US 9,592,247, and US 8,809,282, each of which is incorporated by reference in its entirety.

[0451] In alternative embodiments, the immunomodulator is an inhibitor of the NF-kB pathway. In certain aspects of the embodiment, the immunomodulator is rapamycin or a functional variant thereof. Non-limiting examples of the use of rapamycin are described in US10,071,114, US20160067228, US20160074531, US20160074532, US20190076458, and US10,046,064, each of which is incorporated herein by reference in its entirety. In other aspects of the embodiment, 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, administration and uses are described in US20150320728, US20180193482, US20190142974, US20150328333, US20160243253, US10,039,822, US20190076522, US20160022650, US10,441,651, US10,420, Nos. 835, 10,434,088, 10,335,395, 10,357,483, 10,357,482 ...

[0452] In some embodiments, the immune modulator comprises a synthetic nanocarrier comprising rapamycin (i.e., ImmTOR™ 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 and US Patent No. 9,006,254, each of which is incorporated by reference in its entirety. In some embodiments, the immune modulator is an engineered cell, for example, an immune cell modified using SQZ technology as described in WO2017192786, which is incorporated by reference in its entirety.

[0453] In some embodiments, the immune modulator is poly-ICLC, 1018 ISS, 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 IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide In another further embodiment, the immunomodulator is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, and combinations thereof.

[0454] In some embodiments, the immune modulator is a small molecule that inhibits the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and / or 2-aminopurine (a PKR inhibitor), which can also be administered in combination with a composition comprising at least one rAAV 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) (involved in innate immune signaling), such as 2-aminopurine, BX795, chloroquine, and H-89, can also be used in the compositions and methods comprising at least one rAAV vector disclosed herein for in vivo protein expression.

[0455] In some embodiments, the immune modulator is photopheresis, also known as extracorporeal photochemotherapy, or ECP. Photopheresis treatment is performed on the subject's blood. Using either an IV or catheter, blood from the subject is passed through a device that separates a portion of the white blood cells (leukocytes). The separated white blood cells are treated with a naturally occurring photosensitizing chemical called 8-methoxypsoralen (8-MOP) and then exposed to a specific wavelength of ultraviolet (UVA) light. After exposure to UVA light, the blood is administered back to the subject. Photopheresis can be performed at least once daily. In one embodiment, photopheresis is performed at least 1, 2, 3, 4, 5, 6, or 7 times per week prior to administration of the recombinant viral vector. In one embodiment, photopheresis is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of the recombinant viral vector. Thus, administering an immune modulator to a subject can include performing photopheresis on the subject. It is understood that photopheresis can be performed in conjunction with administration of a second immune modulator selected from the enzymes, nanoparticles, and chemical compositions described herein, and / or a portion of multiple dosing regimens.

[0456] In some embodiments, rAAV vectors having modified viral capsids can also encode negative regulators of innate immunity, such as NLRX1. Thus, in some embodiments, the rAAV vector can also optionally encode one or more of NLRX1, NS1, NS3 / 4A, or A46R. In addition, in some embodiments, compositions comprising at least one rAAV vector disclosed herein can also include synthetic, modified RNA encoding an inhibitor of the innate immune system to avoid an innate immune response generated by a tissue or subject.

[0457] In some embodiments, the immunomodulator for use in the administration methods disclosed herein is an immunosuppressant or immunosuppressant agent. As used herein, the term "immunosuppressant or immunosuppressant agent" refers to a pharmaceutical agent that inhibits or interferes with normal immune function. Examples of immunosuppressants or immunosuppressants suitable for the methods disclosed herein include agents that inhibit the T cell / B cell costimulatory pathway, such as agents that interfere with the coupling of T cells and B cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Publication No. 2002 / 0182211, which is incorporated herein by reference in its entirety. In one embodiment, the immunosuppressant is cyclosporin A. Other examples of immunosuppressants include myophenylate mofetil, rapamycin, and antithymocyte globulin. In various embodiments, the immunosuppressant is administered in a composition comprising at least one rAAV vector disclosed herein, or in a separate composition, but simultaneously with, prior to, or after administration of a composition comprising at least one rAAV vector according to the methods of administration disclosed herein. The immunosuppressant is administered in a formulation compatible with the route of administration and in a dosage sufficient to achieve the desired therapeutic effect. In some embodiments, the immunosuppressant is administered transiently for a time sufficient to induce tolerance to the rAAV vectors disclosed herein.

[0458] In any embodiment of the methods and compositions disclosed herein, the subject receiving the composition disclosed herein is also administered an immunosuppressant. Various methods are known for achieving immunosuppression of the immune response in a patient receiving AAV. Methods known in the art include administering an immunosuppressant, such as a proteasome inhibitor, to the patient. For example, one such proteasome inhibitor known in the art is bortezomib, as disclosed in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15 / 796,137, both of which are incorporated herein by reference in their entireties. In some embodiments, the immunosuppressant is an antibody, including polyclonal antibodies, monoclonal antibodies, SCFV or other antibody-derived molecules, which can suppress the immune response, for example, by eliminating or suppressing antibody-producing cells. In a further embodiment, the immunosuppressive element is a short hairpin RNA (shRNA). In this embodiment, the coding region of the shRNA is included in the rAAV cassette and is generally located downstream, i.e., 3', of the polyA tail. shRNAs can be targeted to reduce or eliminate the expression of immune stimulators such as cytokines, growth factors (including transforming growth factors β1 and β2, TNF, and others known in the art).

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

[0460] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to help describe particular embodiments, and are not intended to limit the claimed technology described herein, since the claimed technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein 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 is 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 context of rAAV-mediated delivery) a sufficient number of target cells in a target tissue of a subject. 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., one that can be delivered via rAAV) can be an amount sufficient to have a therapeutic benefit in a subject, e.g., reduce or stabilize the subject's MDS-UPDRS score, extend the subject's lifespan, ameliorate one or more symptoms of a disease (e.g., PD symptoms) in a subject, etc. The effective amount depends on various factors, such as the subject's species, age, weight, health, and tissue being targeted, and as such, may vary between subjects and tissues, as described elsewhere in this disclosure.

[0463] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce," "reduction," or "reducing" or "inhibiting" typically refers to a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or agent), and can 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 greater. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be to a level that is accepted as within the normal range for individuals without the given disorder.

[0464] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase compared to a reference level, or any increase between 10 and 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 greater, compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0465] As used herein, "subject" refers to a human or non-human animal. Typically, non-human animals are vertebrates such as primates, rodents, livestock animals, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0466] Preferably, the subject is a mammal.Mammal can be, but not limited to, human, non-human primate, mouse, rat, dog, cat, horse or cow.Non-human mammals can be advantageously used as subjects representing the animal model of PD.Subject can be male or female.

[0467] The subject may be a subject who has been previously diagnosed with, or has been identified as having, a condition requiring treatment (e.g., PD) or one or more complications associated with such a condition, and, if necessary, has already received 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 a 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 risk factors.

[0468] A "subject in need" of treatment for a particular condition (e.g., PD) can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0469] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, although the use of these terms overlaps in the art. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0470] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn using default settings).

[0471] Alterations to the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered by the required substitution, deletion, or insertion. Techniques for making such modifications are very well established and include, for example, those 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 entireties. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.

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

[0473] In some embodiments of any of the aspects, the polypeptides, nucleic acids, or cells described herein can be engineered. As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" if at least one aspect of the polypeptide, such as its sequence, has been manipulated by the hand of man so that it differs from its naturally occurring form. As is common practice and understood by those skilled in the art, the progeny of an engineered cell are typically still referred to as "engineered," even if actual manipulation was previously performed on the entity.

[0474] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn using default settings).

[0475] The term "exogenous" refers to a substance present in a cell other than its native origin. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide in which it is not normally found and which has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, in an effort to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide in which it is found in relatively low amounts and which has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, in an effort to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell. As used herein, "ectopic" refers to a substance found in an unusual location and / or amount. An ectopic substance can be a substance that is normally found in a given cell, but found in very low amounts and / or at a different time. Ectopic also includes substances, such as polypeptides or nucleic acids, that are not found in nature or are not expressed in a given cell in its natural environment.

[0476] The term "vector," as used herein, refers to a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replication and can transfer gene sequences into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

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

[0478] In some embodiments of any of the aspects, the vectors or nucleic acids described herein are codon-optimized, e.g., the native or wild-type sequence of a nucleic acid sequence has been modified or engineered to include alternative codons such that the modified or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism (or cells derived from suc...

Claims

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

2. The rAAV is introduced via local delivery, and optionally the local introduction is directly into the putamen of said subject; and / or the local introduction comprises directly introducing the rAAV into each of the subject's putamen; and / or The local introduction is performed simultaneously with non-invasive imaging, and optionally and / or the non-invasive imaging is selected from the group consisting of intraoperative magnetic resonance imaging (iMRI)-guided convection-enhanced delivery (CED), ultrasound, computed tomography (CT); functional magnetic resonance imaging (fMRI); positron emission tomography (PET); electroencephalography (EEG); magnetoencephalography (MEG); functional near-infrared spectroscopy (fNIRS); and combinations thereof; and / or the local introduction comprises introducing approximately half of the rAAV vector into each putamen via intraoperative magnetic resonance imaging (iMRI)-guided convection-enhanced delivery (CED); and / or the local introduction further comprises introducing an MRI contrast agent substantially simultaneously with said AAV vector; or the MRI contrast agent is gadoteridol, and / or the MRI contrast agent is introduced into the subject in the same composition as the rAAV; or 10. The composition of claim 1, wherein the MRI contrast agent is introduced into the subject in a different composition than the rAAV.

3. The composition of claim 1, wherein the rAAV is introduced via systemic introduction.

4. Transduction and / or coverage of the putamen is assessed via magnetic resonance imaging; and / or at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the volume of the subject's putamen is transduced with the GDNF transgene; and / or the subject does not exhibit a substantial increase in PD-related symptoms for at least 12 months immediately following said administration compared to before said administration; or the subject exhibits a reduction in PD-related symptoms for at least six months or longer immediately after the administration compared to before the administration; or 10. The composition of claim 1, wherein the subject exhibits a reduction in PD-related symptoms immediately after administration for at least 12 months or longer compared to before administration.

5. the subject has an early Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) score of less than 32 prior to induction, and optionally the slowing or inhibiting of the progression of Parkinson's disease in the subject is characterized by a second MDS-UPDRS score six months immediately following the induction that is not substantially higher than the initial MDS-UPDRS score; and / or or said slowing or inhibiting the progression of Parkinson's disease in said subject is characterized by a second MDS-UPDRS score about 12 months immediately following said administration that is not substantially higher than said initial MDS-UPDRS score; or the subject has an initial MDS-UPDRS score of greater than or equal to 32 prior to initiation, and optionally the subject exhibits a decrease in the initial MDS-UPDRS score for at least six months immediately following the administration compared to before administration; and / or or 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 immediately following the administration that is at least about 20% lower than the initial MDS-UPDRS score; or or 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 immediately following the initiation that is at least about 30% lower than the initial MDS-UPDRS score; or the method further comprises, prior to administering, determining an initial MDS-UPDRS score for the subject; or the method further comprises, prior to the introducing, receiving the results of an assay that provides an initial MDS-UPDRS score for the subject; or and optionally, slowing or inhibiting the progression of PD in the subject is characterized by a reduction in an initial MDS-UPDRS score after induction. the reduction is at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater reduction in the initial MDS-UPDRS score 6 months after induction; or the reduction is at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater reduction in the initial MDS-UPDRS score 12 months after initiation; or and optionally, slowing or inhibiting the progression of PD in the subject is characterized by a stabilization of an initial MDS-UPDRS score after induction. the stabilization is characterized by an increase or decrease of no more than 10% in the initial MDS-UPDRS score; and / or Stabilization occurs for at least 6 months or longer, or The subject is mildly affected by PD, and optionally The subject mildly affected by PD had an initial MDS-UPDRS score of less than 32 prior to the administration of rAAV and was diagnosed with PD less than 5 years prior to the administration; or the method further comprises, prior to said introducing, diagnosing said subject as being mildly affected by PD; or the method further comprises, prior to said introducing, receiving results of an assay diagnosing said subject as being mildly affected by PD; or The subject is moderately affected by PD, and optionally The subject moderately affected by PD had an initial MDS-UPDRS score equal to or greater than 32 prior to the administration of rAAV, and was diagnosed with PD less than 4 years prior to the administration; or the method further comprises, prior to said introducing, diagnosing said subject as being moderately affected by PD; or the method further comprises, prior to the introducing, receiving results of an assay diagnosing the subject as being moderately affected by PD; or the promoter is a cytomegalovirus (CMV) promoter, or The promoter is a nervous system (NS) or central nervous system (CNS) specific promoter, and optionally the NS-specific promoter is selected from the NS-specific promoters of Table 1; or the CNS-specific promoter is selected from the CNS-specific promoters of Table 2; or 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; or the rAAV is AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or a rational singlet thereof, and optionally the rAAV is AAV2; or the rAAV exhibits brain-specific tropism; or The rAAV comprises modifications that increase its brain-specific tropism, and optionally or has at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater increase in brain-specific tropism compared to unmodified AAV; The rAAV is introduced at a total dose ranging from 5×10 12 vg to about 1.5×10 13 vg, and optionally about one-half of the total dose is administered to each putamen of the subject; or The rAAV is introduced as a liquid composition comprising the rAAV and a pharmaceutically acceptable carrier, and optionally the liquid composition has an rAAV concentration of about 3×10 12 vg / mL to about 4×10 12 vg / mL; or the subject is administered at least one anti-PD therapeutic agent prior to the introduction of the rAAV; or the subject is administered at least one anti-PD therapeutic agent before and after the introduction of the rAAV; and / or the at least one anti-PD therapeutic agent is selected from the group consisting of levodopa, Sinemet, Rytary, Stalevo, amantadine, pramipexole, rotigotine, ropinirole, apomorphine, entacapone; and / or The subject maintains or reduces the dose of the at least one anti-PD therapeutic agent after induction, and as needed.

10. The composition of claim 1, wherein 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.

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

7. A composition for use in a method of slowing or inhibiting the progression of PD in a subject in need thereof, comprising a glial cell line-derived neurotrophic factor (GDNF) transgene, said method comprising: transducing greater than or equal to about 30% of the volume of the putamen of said subject with said glial cell line-derived neurotrophic factor (GDNF) transgene. Including, the subject does not show a substantial increase in PD-related symptoms for at least six months after the introduction, and optionally wherein said transducing is effected by administering an rAAV containing said GDNF transgene to each of said subject's putamen.

8. 1. A composition for use in a method for reducing or stabilizing an Early Movement Disorder Society Unified Parkinson's Disease Rating Scale-Part (MDS-UPDRS) score in a subject with Parkinson's disease (PD), the composition comprising a recombinant adeno-associated virus (rAAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, the method comprising: administering the recombinant adeno-associated virus (rAAV) containing a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter to the putamen of the subject. Including, the subject has a second MDS-UPDRS score at 6 months after administration that is reduced or stabilized compared to the subject's initial MDS-UPDRS score before administration, and optionally the method further comprises, prior to administering, obtaining or receiving an initial MDS-UPDRS score from the subject; or 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 12 months after administration; or The composition, wherein stabilization is an increase or decrease of no more than 10% in said initial MDS-UPDRS score.

9. 1. A composition for use in a method of treating a subject mildly affected by Parkinson's disease (PD), comprising a recombinant adeno-associated virus (rAAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, said method comprising: administering the recombinant adeno-associated virus (rAAV) containing a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter to each of the subject's putamen. Including, at least 30% of the putamen of said subject is transduced with said GDNF transgene; the subject has a second MDS-UPDRS score at 6 months after administration that is stabilized compared to the initial MDS-UPDRS score, and optionally the subject has a stabilized MDS-UPDRS score at 12 months after administration compared to the initial MDS-UPDRS score before administration; and / or The composition, wherein stabilization is an increase or decrease of no more than 10% in said initial MDS-UPDRS score.

10. 1. A composition for use in a method of treating a subject moderately affected by Parkinson's disease (PD), comprising a recombinant adeno-associated virus (AAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter, said method comprising: administering the recombinant adeno-associated virus (AAV) containing a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter to each of the subject's putamen. Including, at least 30% of the putamen of said subject is transduced with said GDNF transgene; the subject has a second MDS-UPDRS score at 6 months after administration that is at least about 20% lower than the initial MDS-UPDRS score, and optionally The composition, wherein the reduction is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the initial MDS-UPDRS score.

11. A composition for use in a method for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject in need thereof, the composition comprising a recombinant adeno-associated virus (rAAV) vector comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter; The method comprises: locally introducing into each of the subject's putamen said recombinant adeno-associated virus (rAAV) vectors comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter; and and locally introducing an MRI contrast agent into each of the subject's putamen substantially simultaneously with said rAAV. Including, at least 30% of the volume of the putamen of said subject is transduced with said transgene; the subject does not exhibit a substantial increase in PD-related symptoms for at least six months immediately following the administration compared to before the administration; or The method comprises: introducing into the subject the recombinant adeno-associated virus (rAAV) comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter. Including, at least 30% of the volume of the putamen of said subject is transduced with said GDNF transgene; the subject does not exhibit a substantial increase in PD-related symptoms for at least six months immediately following the administration compared to before the administration; composition.

12. 1. A composition for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, said 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 Including, as needed the composition has an rAAV concentration of 3×10 12 vg to 4×10 12 vg per mL; or A composition, wherein the composition comprises an rAAV concentration of 3.3 x 1012 vg per mL.

13. 1. A formulation for slowing or inhibiting the progression of Parkinson's disease (PD) in a subject, said formulation comprising 3 x 10 hydroxybenzoates per mL of a pharmaceutically acceptable carrier. １２ vg ~ 4 x 10 １２ vg of adeno-associated virus (AAV), The rAAV comprises a genome comprising a glial cell line-derived neurotrophic factor (GDNF) transgene operably linked to a promoter. formulation.

14. 3. The composition of claim 2, wherein the introducing is performed at a flow rate of about 1 μL / min to about 30 μL / min.

15. The composition of any one of claims 1 to 12 and 14 and the formulation of claim 13, wherein the subject does not exhibit any serious adverse events for at least six months immediately following the introduction or administration.

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