High-dose GAD gene therapy for treating parkinson's disease

Abstract

Provided herein are compositions and methods for treating or preventing neurodegenerative disease and for treating or preventing symptoms associated with neurodegenerative disease, including Parkinson's disease. For example, provided are methods of treating a patient with Parkinson's disease, the method comprising administering to the patient vectors comprising a nucleic acid encoding glutamic acid decarboxylase (GAD).

Description

162027.49676HIGH-DOSE GAD GENE THERAPY FOR TREATING PARKINSON’S DISEASECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This International Patent Application claims priority to U.S. Provisional Application No. 63 / 707,508, filed on October 15, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on September 19, 2025, is named SeqList- 162027-49676. xml and is 17,394 bytes in size.FIELD

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating disease, particularly Parkinson’s disease.BACKGROUND

[0004] Parkinson’s disease (PD) is a neurodegenerative disorder with characteristic impairments of motor function such as slowed movement, impaired dexterity, gait disturbance, tremors, and rigidity of muscles. More than 10 million people worldwide and nearly one million patients in the U.S. are living with PD, which makes it one of the most common central nervous system disorders. Approximately 90,000 new patients are diagnosed annually in the U.S. The cause of PD is unknown for a majority of patients, while a much smaller percentage have a known genetic cause. However, in all cases, there is dysfunction of the key circuits that control movement.

[0005] The clinical features of PD are associated with progressive loss of neurons in the midbrain, particularly the substantia nigra pars compacta (SNpc). The net effect is a disruption of basal ganglia activity and downstream brain networks that control movement and possibly other neurological functions, including cognitive function and autonomic regulation.

[0006] The progression of PD can be divided into five stages. Stage 1 of PD is characterized by disturbances in facial expression, speech and / or locomotion. The symptoms are initiallyonly seen on one side of the body (unilateral involvement), and there is usually minimal or no functional impairment. During stage 2 of PD, both hemispheres of the brain become affected by the disease. As a result, tremors gradually become bilateral and can affect the patient’s midline. Additional symptoms of PD in stage 2 may include the loss of facial expression on both sides of the face, decreased blinking, speech abnormalities, soft voice, monotone voice, slurring speech, stiffness or rigidity of the muscles in the trunk that may result in neck or back pain, stooped posture, and general slowness in activities of daily living. Stage 3 is characterized by loss of balance and slowness of movement. Balance is compromised by the inability to make the rapid, automatic, and involuntary adjustments necessary to prevent falling, and falls are common at this stage. At stage 4, patients show severe and limiting symptoms. The patient may be able to stand without assistance, but movement may require a walker. Stage 5 is the most advanced and debilitating stage of PD. Stiffness in the legs may make it impossible to stand or walk. The patient requires a wheelchair or is bedridden. Around-the-clock nursing care is usually required for all activities for patients at this stage of PD.

[0007] Histologically, PD is characterized by neuronal inclusions of a-synuclein in neuronal cell bodies (Lewy bodies) and within neuronal cell processes (Lewy neurites). Accumulated a-synuclein causes degeneration of dopaminergic neurons in the SNpc, either directly due to the toxic impact of a-synuclein or indirectly through induction of mitochondrial dysfunction and proteasomal / lysosomal dysfunctions.

[0008] Dopamine functions as an inhibitory neurotransmitter that regulates the excitability of neurons, which are involved in controlling balance and body movement. PD symptoms are further correlated with the disturbance of various other neurotransmitters, such as acetylcholine (ACh) and y-aminobutyric acid (GABA). The GABA pathway is essential in regulating the inhibitory tone to prevent excessive stimulation of the cerebral cortex. Additionally, decreasing GABA activity promotes mitochondrial dysfunction and oxidative stress, which are highly related to PD neuropathology. The loss of dopamine signaling in PD leads to dysregulation of the subthalamic nucleus (STN) due to a reduction in GABA inputs to this structure, resulting in excessive STN mediated activation of globus pallidus internus (GPi) / Substantia Nigra pars reticulata (SNpr) outflow, which increases the inhibitory brake on the thalamus. Loss of dopamine also leads to a decrease in GABA-mediated inhibition of GPi / Substantia nigra pars reticulata (SNr), further exacerbating the excessive basal ganglia outflow inhibition of the thalamus. This inhibition of the thalamus underlies the motor symptoms characteristic of PD.

[0009] The standard treatment of PD involves oral administration of the dopamine precursorL-3,4-dihydroxyphenylalanine (levodopa or L-Dopa), which eliminates symptoms associated2178092112.1with PD, but does not ultimately prevent the degeneration of dopaminergic cells. Thus, currently used treatments for PD merely reduce PD symptoms without slowing or halting disease progression.

[0010] Administration of L-Dopa allows a PD patient to have so called “on” time, a period of time in which a PD patient has adequate control of PD symptoms. When the effect of L- Dopa wears off, the symptoms of PD reemerge. This time period is referred to as “off’ time. The measurement of “on” time and “off’ time are typically calculated by asking the patients to keep a medication diary. In the early stages of PD, a patient’s “on” time is about 16 hours with the administration of L-Dopa. However, as the disease progresses, the amount of “on” time gradually decreases even with larger doses of medication. Additionally, the side effects associated with long-term administration of L-dopa can be quite severe, and include mental changes such as depression, hallucination, mania, delusions, agitations, and excessive sleeping. Administration of L-dopa can also have a detrimental effect in patients with cardiovascular or pulmonary, renal, hepatic or endocrinal diseases. Some of the side effects associated with longterm administration of L-dopa can be mitigated by co-administration of N-amino-a-methyl-3- hydroxy-L-tyrosine monohydrate, an inhibitor of aromatic amino acid decarboxylase (AADC), an enzyme that decarboxylates L-Dopa to dopamine. However, this drug combination still may cause nausea, dyskinesia, psychosis, and hypotension.

[0011] Early in the course of PD, dopaminergic replacement therapy and other medications are sufficient for marked symptomatic improvement of the motor deficits, which are the major source of disability for these patients. Facilitation of dopaminergic neurotransmission benefits most patients early in the disease, but generally requires both increasing drug doses and a move to more complex polytherapy, while still targeting the dopaminergic pathway. As the disorder advances and brain physiology changes in response to chronic medication exposure, drug- related complications develop and often cause significant disability, such as disabling dyskinesias and motor fluctuations. Thus, almost all PD patients reach a stage sometime after the initiation of treatment where their symptoms cannot be adequately controlled with current therapy. The development of these complications, among other factors, are among the primary reasons why patients with moderate to advanced PD and their physicians consider surgical intervention to reduce symptoms and improve function.

[0012] In view of the current limitations in the treatment of PD, new methods for treating PD are urgently needed.3178092112.1SUMMARY

[0013] Provided is a method of treating or preventing Parkinson’s disease (PD) or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding glutamic acid decarboxylase (GAD), wherein the pharmaceutical composition is delivered bilaterally into each of the two subthalamic nuclei (STN) of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

[0014] Provided is a method of treating or preventing a neurodegenerative disease or disorder associated with y-aminobutyric acid (GABA) deficiency or treating or preventing symptoms associated with said neurodegenerative disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding GAD, wherein the pharmaceutical composition is delivered bilaterally into each of the two STN of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

[0015] In one embodiment, the pharmaceutical composition is administered at a total dose of at least about 2.09 x io11vg. In one embodiment, the pharmaceutical composition is administered at a total dose of about 2.09 x io11vg.

[0016] In one embodiment, the pharmaceutical composition is administered at a dose of at least about 1 x io11vg / STN. In one embodiment, the pharmaceutical composition is administered at a dose of at least about 1.045 x io11vg / STN. In one embodiment, the pharmaceutical composition is administered at a dose of about 1.045 x io11vg / STN.

[0017] In one embodiment, the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-65. In some embodiments, the GAD-65 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In one embodiment, the GAD- 65 comprises SEQ ID NO: 1. In one embodiment, the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-67. In some embodiments, the GAD- 67 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 3 or 4. In one embodiment, the GAD-67 comprises SEQ ID NOs: 3 or 4. In a preferred embodiment, the GAD-65 comprises SEQ ID NO: 1 and the GAD-67 comprises SEQ ID NO: 3. In one4178092112.1embodiment, the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-65 and a nucleic acid encoding GAD-67.

[0018] In one embodiment, the pharmaceutical composition comprises vectors comprising a nucleic acid encoding GAD-65 and vectors comprising a nucleic acid encoding GAD-67. In one embodiment, the pharmaceutical composition comprises vectors comprising a nucleic acid encoding GAD-65 and vectors comprising a nucleic acid encoding GAD-67 at a ratio of about 1 : 1 (ratio of viral genomes).

[0019] In one embodiment, the vectors comprising the nucleic acid encoding GAD are viral vectors. In one embodiment, the viral vectors are adeno-associated virus (AAV) vectors. In one embodiment, the AAV vectors comprise a viral genome derived from an AAV-2 serotype. In one embodiment, the AAV vectors comprise capsid proteins derived from an AAV-2 serotype.

[0020] In one embodiment, the subject is a human.

[0021] In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part 3 score during “medication off’ time by at least 2 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 5 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 10 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 15 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 score during “medication on” time by at least 2 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication on” time by at least 5 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication on” time by at least 10 points as compared to a control. In some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s Parkinson’s Disease Questionnaire (PDQ)-39 score by at least 2 points as compared to a control. In some embodiments, administration of the pharmaceutical composition results in an improvement of the subject’s PDQ-39 score by at least 5 points as compared to a control. In5178092112.1some embodiments, the administration of the pharmaceutical composition results in an improvement of the subject’s PDQ-39 score by at least 8 points as compared to a control.BRIEF DESCRIPTION OF THE FIGURES

[0022] Fig. 1. Treatment with a high dose of AAV-GAD leads to larger reduction (compared to baseline) in the UPDRS Part 3 (motor examination) score during the “medication off’ state after 26 weeks as compared to treatment with the low dose of AAV- GAD or no treatment. The three experimental groups were: (1) AAV-GAD at a dose of 3.5 * IO10vg / STN (low dose), (2) AAV-GAD at a dose of 10.45 x io10vg / STN (high dose); (3) sham surgery. Patients were injected bilaterally. The total dose per participant were 7.0 IO10vg or 20.9x 1010vg, in the low and high dose groups, respectively.

[0023] Fig. 2. Treatment with a high dose of AAV-GAD leads to larger reduction (compared to baseline) in the UPDRS Part 3 (motor examination) score during the “medication on” state after 26 weeks as compared to treatment with the low dose of AAV- GAD. The three experimental groups were: (1) AAV-GAD at a dose of 3.5 x io10vg / STN (low dose), (2) AAV-GAD at a dose of 10.45 x 1O10vg / STN (high dose); (3) sham surgery. Patients were injected bilaterally. The total dose per participant were 7.Ox lO10vg or 2O.9x lO10vg, in the low and high dose groups, respectively.

[0024] Fig. 3. Treatment with a high dose of AAV-GAD leads to larger reduction (compared to baseline) in the PDQ-39 score during the “medication on” state after 26 weeks as compared to treatment with the low dose of AAV-GAD. The three experimental groups were: (1) AAV-GAD at a dose of 3.5 x io10vg / STN (low dose), (2) AAV-GAD at a dose of 10.45 x io10vg / STN (high dose); (3) sham surgery. Patients were injected bilaterally. The total dose per participant were 7.Ox lO10vg or 2O.9x lO10vg, in the low and high dose groups, respectively.DETAILED DESCRIPTION

[0025] Provided herein are methods for treating neurodegenerative diseases or disorders including PD by administering to a subject in need thereof vectors for the expression of glutamic acid decarboxylase (GAD) or a pharmaceutical composition comprising such vectors. The vectors or the pharmaceutical composition may be delivered to the intrinsic neurons of the STN, where GAD expression can increase GABA production. As shown herein, GAD gene therapy can restore normal outflow of the basal ganglia to improve motor function.6178092112.1

[0026] Benefits of GAD gene therapy

[0027] Early in the course of PD, medications enhancing dopaminergic neurotransmission are often sufficient for achieving symptomatic improvement of most clinical features. Facilitation of dopaminergic signaling continues to benefit most patients, but as the disorder advances, higher and / or more frequent doses of dopaminergic drugs are required, as well as increased numbers of medications. Further, there is an increasing chance for patients to develop drug-related complications such as involuntary movements (dyskinesias), increased “off’ times, and motor fluctuations. As a result, PD usually progresses, and patients typically worsen and become less responsive to dopaminergic medication, all while developing adverse effects of medical therapy. When patients have disabling motor symptoms that no longer respond adequately to medical therapy and / or have complications of medical therapy (such as dyskinesias or motor fluctuations), medical options are limited.

[0028] Surgical treatment options for these more advanced patients include destructive lesioning of the STN ( / .< ., subthalamotomy) or deep brain stimulation (DBS) of the STN to reduce excessive excitatory glutamatergic drive from STN projection neurons. However, subthalamotomy is permanent and can adversely affect critical nearby white matter connections. It is often not well tolerated when administered bilaterally, which is usually required for most patients. Deep brain stimulation is better tolerated than subthalamotomy, since it modulates STN activity rather than destroying STN neurons. However, electrical stimulation from DBS is not specific to STN neurons and can also influence nearby white matter connections, leading to dose-limiting adverse effects such as speech and swallowing problems, sensory changes, and gait dysfunction.

[0029] Although DBS is effective and the current standard of care for patients who do not respond adequately to medications, the procedure has many limitations, which complicate the patient’s experience and limit access to therapy. Since DBS requires installation of electrodes into the brain and an implanted electrical stimulator, there is risk of inflammation, hardware- related infections, wound breakdown or skin erosion and pain from scarring, or skin sensitivity from the subcutaneous hardware. Patients must undergo two surgeries, the first to implant the brain electrode and a second to implant the pulse generator, with the second procedure usually performed under general anesthesia to facilitate tunneling of lead extensions. Patients must live in reasonable proximity to a DBS center, since most require frequent visits to reprogram and optimize DBS therapy for several months after surgery and periodic follow-ups indefinitely after the initial programming period. The DBS pulse generators also have a limited life span and must be replaced after some time. Finally, DBS is a local therapy that is designed to7178092112.1influence only the target structure and does not directly influence the basal ganglia circuitry, which controls movement beyond local effects on the STN.

[0030] The AAV-GAD gene therapy described herein offers a method to increase GAD activity in the STN and to enhance GABA production within the STN to reduce abnormal activity and release GABA downstream through STN projections to GPi / SNr. This improves the neurochemistry and neuronal activity of all these structures, thus normalizing basal ganglia outflow and restoring motor function. Furthermore, AAV-GAD gene therapy has the potential to overcome many of the limitations of DBS. Unlike DBS, AAV-GAD treatment requires only one routine stereotactic surgery, carried out under local anesthesia with no need for general anesthesia. No equipment is required to be permanently implanted in the brain, and there is no need for burdensome and lengthy post-surgical optimization of therapy. AAV-GAD treatment therefore avoids the potential risk of indwelling hardware-related infections. As shown herein, AAV-GAD gene therapy led to clinical benefits in motor function without the need for ongoing management or adjustment of the therapy. Further, it was confirmed that AAV-GAD gene therapy was both well tolerated and safe, with no significant adverse events related to the gene transfer. AAV-GAD was also infused into the STN, which is the most common target for PD surgery, so the procedure to identify and insert an infusion catheter into the STN is standard and has been utilized for decades in surgery for PD.

[0031] GAD vectors

[0032] GAD catalyzes the decarboxylation of glutamate to GABA and carbon dioxide, using pyridoxal-phosphate (PLP) as a cofactor. Indeed, GAD is the rate-limiting enzyme for GABA production.

[0033] Mammals, including humans, express two isoforms of GAD with molecular weights of 67 and 65 kDa, respectively ( / .< ., GAD-67 and GAD-65). These isoforms are encoded by two different genes (GAD1 and GAD2) on different chromosomes (chromosomes 2 and 10 in humans, respectively). Inhibitory neurons express both GAD-65 and GAD-67 isoforms, which serve complementary and synergistic roles in regulation of brain excitability.

[0034] GAD-65 and GAD-67 show significant differences in their levels of expression in different brain regions. GAD-67 is expressed uniformly throughout the brain, whereas GAD- 65 expression is concentrated primarily in the axon terminals. Together, these two enzymes maintain most of the physiological supply of GABA in mammals.

[0035] GAD gene therapy in PD patients can drive expression of GAD in the transduced cells of the STN, which catalyzes the enzymatic conversion of glutamate to GABA. This restores a more normal glutamate / GABA balance in both the STN and its efferent targets, the8178092112.1GPi / SNr. Increased STN activity (more abnormal activity) leads to increased GABA release to regulate this circuitry. As a result, the activity of downstream thalamocortical networks is normalized in an autoregulatory fashion, leading to improved motor function in PD patients.

[0036] Provided is a vector for the expression of GAD-65. Human GAD-65 cDNA encodes a polypeptide of 585 amino acid residues (Genbank Accession No. NM000818, see SEQ ID NOs: l and 5). See Tables 1-3. A person skilled in the art will be aware of the variety of naturally occurring variants of GAD-65 (e.g., isoforms) that exist in humans and that are suitable for use in the methods disclosed herein. Alternatively, artificial variants of human GAD-65 may be created for use in the methods disclosed herein.

[0037] In some embodiments, the GAD-65 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In a preferred embodiment, the GAD-65 protein comprises SEQ ID NO: 1.

[0038] In one embodiment, the GAD-65 protein is fused to an affinity tag, such as a His- tag. In some embodiments, the GAD-65 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In one embodiment, the GAD-65 protein comprises SEQ ID NO:2.

[0039] In some embodiments, the sequence encoding the GAD-65 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 5 or 6. In one embodiment, the sequence encoding the GAD-65 protein comprises SEQ ID NOs: 5 or 6.

[0040] Provided is a vector for the expression of GAD-67. Human GAD-67 encodes a polypeptide of 594 amino acid residues (Genbank Accession Nos. M81883, NP_000808.2, and BC026349.1, see SEQ ID NOs:3, 4, 7, and 8). See Tables 1-3. A person skilled in the art will be aware of the variety of naturally occurring variants of GAD-67 (e.g., isoforms) that exist in humans and that are suitable for use in the methods disclosed herein. Alternatively, artificial variants of human GAD-67 may be created for use in the methods disclosed herein.

[0041] In some embodiments, the GAD-67 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In a preferred embodiment, the GAD-67 protein comprises SEQ ID NO:3.

[0042] In some embodiments, the GAD-67 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at9178092112.1least 99% identical to SEQ ID NO: 4. In one embodiment, the GAD-67 protein comprises SEQ ID NO:4.

[0043] In some embodiments, the sequence encoding the GAD-67 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 7 or 8. In one embodiment, the sequence encoding the GAD-67 protein comprises SEQ ID NOs:7 or 8.

[0044] In a preferred embodiment, the GAD-65 comprises SEQ ID NO: 1 and the GAD-67 comprises SEQ ID NO: 3.

[0045] As used herein, the term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package, BLASTP, BLASTN, FASTA, and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources. In comparing sequences, these methods account for various substitutions, deletions, and other modifications.

[0046] In embodiments, provided is a variant of a GAD-65 or GAD-67 protein disclosed herein. In some embodiments, the GAD-65 variant has one or more amino acid substitutions as compared to a GAD-65 protein disclosed herein. In some embodiments, the GAD-67 variant has one or more amino acid substitutions as compared to a GAD-67 protein disclosed herein. In some embodiments, the GAD-65 variant has one or more conservative amino acid substitutions as compared to a GAD-65 protein disclosed herein. In some embodiments, the GAD-67 variant has one or more conservative amino acid substitutions as compared to a GAD- 67 protein disclosed herein.

[0047] As used herein, the terms “conservative amino acid substitutions” and “conservative modifications” refer to amino acid substitutions or modifications that do not significantly affect or alter the function and / or activity of the presently disclosed proteins comprising the amino10178092112.1acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0048] Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.Table 1. Overview of sequencesTable 2. Amino acid sequences11178092112.112178092112.1Table 3. Nucleic acid sequences. Any stop codon(s) can be used for the expression of GAD- 65 and / or GAD-67.13178092112.114178092112.115178092112.116178092112.1

[0049] In some embodiments, the sequence encoding GAD-65 and / or the sequence encoding GAD-67 are codon-optimized.

[0050] GAD vectors

[0051] Provided herein is a method of treating PD in a subject in need thereof, the method comprising administering to the subject a vector encoding GAD-65 and / or a vector encoding GAD-67.

[0052] “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA minicircle, or a virus (including virus derived sequences). A vector may also refer to a virion comprising a nucleic acid to be delivered into a host cell, either in vitro or in vivo. In some embodiments, a vector refers to a virion comprising a recombinant viral genome, optionally wherein the viral genome comprises one or more ITRs and a transgene.

[0053] In one embodiment, the genes for the expression of GAD-65 and GAD-67 are delivered in a single vector. Alternatively, the genes for the expression of GAD-65 and GAD- 67 are delivered in two separate vectors (e.g., a pair of vectors).

[0054] In some embodiments, the nucleic acid encoding GAD-65 and / or the nucleic acid encoding GAD-67 can be delivered using a non-viral delivery system, for example, using a colloidal dispersion system such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, or liposomes.17178092112.1

[0055] In one embodiment, the vector for the expression of GAD-65 and / or the vector for the expression of GAD-67 is a viral vector. Examples of viral vectors include, but are not limited to, retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses (AAV)), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses may include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus.

[0056] In one embodiment, the vector for the expression of GAD-65 and / or the vector for the expression of GAD-67 is an AAV vector. AAV are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function mainly in regulating AAV replication and rescue and integration of the AAV into a host cell's chromosomes. The Cap reading frame encodes three structural proteins of molecular weight 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. More than 80% of total proteins in AAV virion comprise VP3. Flanking the rep and cap open reading frames at the 5' and 3' ends are about 145 bp long inverted terminal repeats (ITRs). The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene.

[0057] Recombinant adeno-associated virus “rAAV” vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and / or Cap genes, but retain functional flanking ITR sequences.

[0058] In some embodiments, the viral vector is an rAAV virion, which comprises an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises an expression construct disclosed herein. The AAV-GAD-65 and AAV-GAD67 vectors disclosed herein are rAAV vectors.18178092112.1

[0059] In some embodiments, the viral vector disclosed herein comprises a nucleic acid comprising an AAV 5' ITR and 3' ITR located 5' and 3' to sequence encoding GAD-65 and / or GAD-67. However, in certain embodiments, it may be desirable for the nucleic acid to contain the 5' ITR and 3' ITR sequences arranged in tandem, e.g., 5' to 3' or a head-to-tail, or in another alternative configuration. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have 5' ITRs (or conversely, 3' ITRs) located both 5' and 3' to the sequence encoding GAD-65 and / or GAD-67. The ITRs sequences may be located immediately upstream and / or downstream of the heterologous molecule, or there may be intervening sequences. The ITRs need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion, or substitution of nucleotides) so long as the sequences provide for functional rescue, replication, and packaging. The ITRs may be selected from AAV2, or from among the other AAV serotypes, as described herein.

[0060] In some embodiments, the viral vector is an AAV vector, such as an AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV containing AAV9 ITRs and AAV9 capsid proteins), AAVrh74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid proteins), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid proteins), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid proteins).

[0061] In one embodiment, the vector for the expression of GAD-65 and / or GAD-67 is an AAV2 vector (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins).

[0062] In some embodiments, the viral vector is a pseudotyped AAV vector, containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., an AAV containing AAV2 ITRs and AAV8 capsid proteins).

[0100] The nucleic acid encoding GAD-65 and / or GAD-67 may comprise additional expression control elements that are operably linked to the GAD transgene(s) in a manner19178092112.1which permits its transcription, translation and / or expression in a target cell. As used herein, “operably linked” refers to a relationship between two or more nucleic acid sequences where certain nucleic acid sequences (e.g., control elements) influence characteristics of another nucleotide sequence (e.g, influencing expression of a transgene). Operably linked sequences include both expression control elements that are included in or are contiguous with the GAD transgene, and expression control elements that act in trans or at a distance to control expression of the transgene. As used herein, an “expression construct” is a nucleic acid that allows for the expression of a transgene (e.g, GAD).

[0063] Expression control elements as used herein include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequence); sequences that enhance protein stability; a post transcriptional regulatory sequence, and when desired, sequences that enhance secretion of the encoded product. For example, as used herein, a nucleic acid sequence (e.g., a GAD coding sequence) and regulatory sequences are considered to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. In an embodiment, the human GAD-65 and / or GAD-67 are under regulation of the cytomegalovirus (CMV) enhancer-chicken P-actin (CBA) promoter, a woodchuck post-transcriptional regulatory (WPRE) element, and / or a bovine growth hormone polyadenylation (bgh-PolyA) signal.

[0064] The promoter operably linked to a GAD transgene can either be inducible or constitutive. Inducible promoters allow regulation of gene expression and can be regulated by exogenously circumstances or compounds. Examples of inducible promoters regulated by exogenously supplied promoters include a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system; a ecdysone insect, a tetracycline-repressible system. Constitutive promoters are unregulated promoter that allows for continual transcription of its associated gene. Examples of constitutive promoters include, without limitation, a chicken beta actin promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), a SV40 promoter, a dihydrofolate reductase promoter and a P-actin promoter.

[0065] In embodiments, a native promoter or fragment thereof for the GAD transgene may be used if the expression of the transgene to mimic the native expression is preferred. In another20178092112.1embodiment, a tissue specific promoter is used to allow expression in specific tissues for targeted gene therapy. Tissue specific promoters such as neuron-specific and glial-specific promoters allow the protein to express in the specific tissue desired. In an embodiment, the promoter is tissue specific and is essentially active only within the central nervous system or has a higher activity within the central nervous system. In an embodiment, the promoter can be specific for a particular cell type or neurons. In another embodiment, the promoter is specific for cells located in a particular region of the brain, for example, the cortex, subthalamic nucleus, striatum, nigra and / or hippocampus.

[0066] Some suitable neuronal specific promoters include, but are not limited to, neuron specific enolase (NSE) (GenBank Accession No: X51956), and human neurofilament light chain promoter (NEFL) (GenBank Accession No: L04147). Glial specific promoters include, but are not limited to, glial fibrillary acidic protein (GFAP) promoter (GenBank Accession No:M65210), SI 00 promoter (GenBank Accession No: M65210) and glutamine synthase promoter (GenBank Accession No: X59834).

[0067] Provided are methods of treating or preventing of PD and of treating or preventing the symptoms associated with PD by administering to a subject in need thereof a first vector comprising a nucleic acid encoding GAD-65 and a second vector comprising a nucleic acid encoding GAD-67. Provided are methods of treating or preventing of PD and of treating or preventing the symptoms associated with PD by administering to a subject in need thereof a first vector comprising a nucleic acid encoding GAD-67 and a second vector comprising a nucleic acid encoding GAD-65. In one embodiment, the method comprises administering a pharmaceutical composition comprising a vector comprising a nucleic acid encoding GAD-65 and a pharmaceutical composition comprising a vector comprising a nucleic acid encoding GAD-67.

[0068] In one embodiment, the GAD-65 vector and the GAD-67 vector are administered at the same time. In one embodiment, the GAD-65 vector (or the pharmaceutical composition comprising the GAD-65 vector) is administered prior to the GAD-67 vector (or the pharmaceutical composition comprising the GAD-67 vector). In one embodiment, the GAD- 67 vector (or the pharmaceutical composition comprising the GAD-67 vector) is administered prior to the GAD-65 vector (or the pharmaceutical composition comprising the GAD-65 vector).

[0069] Provided herein is a pharmaceutical composition that comprises two recombinant GAD vectors, namely a GAD-65 vector and a GAD-67-vector.21178092112.1

[0070] Provided herein is a pharmaceutical composition that comprises two recombinant GAD viral vectors, namely a viral GAD-65 vector and a viral GAD-67-vector.

[0071] Provided herein is a pharmaceutical composition that comprises two rAAV-GAD vectors, namely an rAAV-GAD-65 vector and an rAAV-GAD-67-vector.

[0072] In some embodiments, the ratio for the two vectors (one encoding GAD-65 and the other encoding GAD-67) in the composition is from about 5: 1 to about 1 :5. In some embodiments, the ratio for the two vectors (one encoding GAD-65 and the other encoding GAD-67) in the composition is from about 4: 1 to about 1 :4. In some embodiments, the ratio for the two vectors (one encoding GAD-65 and the other encoding GAD-67) in the composition is from about 3: 1 to about 1 :3. In some embodiments, the ratio for the two vectors (one encoding GAD-65 and the other encoding GAD-67) in the composition is from about 2: 1 to about 1 :2. In one embodiment, the ratio for the two vectors (one encoding GAD-65 and the other encoding GAD-67) in the composition is about 1 : 1.

[0073] Methods of determining vector ratios are known in the art. In one embodiment, the two vectors are provided in a 1 : 1 ratio of viral particles. In a preferred embodiment, the two vectors are provided in a 1 : 1 ratio of viral genomes.

[0074] rAAV virion production

[0075] The rAAV virions disclosed herein may be constructed and produced using the materials and methods described herein, as well as those known to those of skill in the art. Such engineering methods used to construct embodiments of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989), both of which are incorporated herein in their entireties. Further, methods suitable for producing a rAAV cassette in an adenoviral capsid have been described in U.S. Pat. No. 5,856,152 (entitled Hybrid adenovirus-AAV vector and methods of use therefor) and U.S. Pat. No. 5,871,982 (entitled Hybrid adenovirus-AAV virus and methods of use thereof), both of which are incorporated herein in their entireties. See also Asaad W et al., AAV genome modification for efficient AAV production, Heliyon. 2023 Apr l;9(4):el5071.

[0076] Briefly, in order to package the rAAV genome into a rAAV virion, a host cell is used that contains sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production. The AAV rep and cap sequences22178092112.1are obtained from an AAV source as identified herein. The AAV rep and cap sequences, as well as the transgene, may be introduced into the host cell in any manner known to one in the art, including, without limitation, transfection, infection, calcium phosphate precipitation, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences may be transfected into the host cell by one or more nucleic acid molecules and exist stably in the cell as an episome. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has the rep and cap sequences transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence. Alternatively, infection of proviral cell lines with adenovirus or herpes simplex virus vector carrying a Rep and Cap expression cassette can be used. Further, baculovirus expression vector systems for rAAV vector production in insect SF9 cells have been developed.

[0077] The rep and cap sequences, along with their expression control sequences, may be supplied on a single vector, or each sequence may be supplied on its own vector. Preferably, the rep and cap sequences are supplied on the same vector. Alternatively, the rep and cap sequences may be supplied on a vector that contains other DNA sequences that are to be introduced into the host cells. Preferably, the promoter used in this construct may be any suitable constitutive, inducible or native promoters known to one of skill in the art. The molecule providing the rep and cap proteins may be in any form which transfers these components to the host cell. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as those for marker genes. This molecule does not contain the AAV ITRs and generally does not contain the AAV packaging sequences. To avoid the occurrence of homologous recombination, other virus sequences, particularly those of adenovirus, are avoided in this plasmid. This plasmid is desirably constructed so that it may be stably transfected into a cell.

[0078] Although the molecule providing rep and cap may be transiently transfected into the host cell, it is preferred that the host cell be stably transformed with sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into the chromosome of the host cell. Depending upon the promoter controlling expression of such stably transfected host cell, the rep / cap proteins may be transiently expressed (e.g., through use of an inducible promoter).23178092112.1

[0079] The methods employed for constructing embodiments of this disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above. For example, the rAAV may be produced utilizing a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.), according to manufacturer’s instructions. See, also, Herzog et al., Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector, Nat Med. 1999 Jan;5(l):56-63, employing the plasmid with the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying adenovirus helper functions of E2A, E40rf6 and VA. While this specification provides illustrative examples of specific constructs, using the information provided herein, one of skill in the art may select and design other suitable constructs, using a choice of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of polyadenylation sites.

[0080] The rAAV virions are then produced by culturing a host cell containing a rAAV virus as described herein which contains a rAAV genome to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. Suitable viral helper genes, e.g., adenovirus E2A, E40rf6 and VA, among other possible helper genes, may be provided to the culture in a variety of ways known to the art, preferably on a separate plasmid. Thereafter, the recombinant AAV virion which directs expression of the GAD-65 and / or GAD-67 transgene is isolated from the cell or cell culture in the absence of contaminating helper virus or wildtype AAV.

[0081] rAAV purification processes can include one or more of the following phases: (i) harvest of the producer cells, occasionally with the supernatant, (ii) chemical (e.g, detergent and ion containing lysis buffers) and / or mechanical (e.g, freeze-thaw, microfluidization) cell lysis to liberate AAV particles, (iii) cellular and viral nucleic acid removal, e.g., by enzymatic digestion (Benzonase), (iv) one to three step particle separation by chromatography and optionally density gradients, and (v) concentration, formulation, and sterile filtration.

[0082] Purification methods for rAAV virions are known in the art and include density gradient ultracentrifugation, gradient sedimentation, nonionic iodixanol gradients followed by ion-exchange or heparin-affinity column chromatography, ion-exchange chromatography, mucin columns, tangential flow filtration, etc.

[0083] Expression of the GAD-65 and / or GAD-67 transgene may be measured in ways known in the art. For example, a target cell may be infected in vitro, and the number of copies of the transgene in the cell monitored by Southern blotting or quantitative polymerase chain24178092112.1reaction (PCR). The level of RNA expression may be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the level of protein expression may be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specific methods detailed below in the Examples.

[0084] Pharmaceutical compositions

[0085] Provided herein are pharmaceutical compositions comprising any of the vectors disclosed herein and a pharmaceutically acceptable excipient.

[0086] In some embodiments, the rAAV comprising the gene encoding GAD is assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0087] Formulations of the vectors disclosed herein involve the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels.

[0088] The vector of the disclosure can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically and / or physiologically acceptable excipient, carrier, buffer, stabilizer, antioxidants, preservative, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of an active agent. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0089] In some embodiments, the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant may be selected from an anionic surfactant, nonionic surfactants, and mixtures thereof. Anionic surfactants include, but are not limited to sodium lauryl sulfate, potassium laurate, sodium dodecyl sulfate, alkyl polyoxyethylene sulfates, sodium alginate, dioctyl sodium sulfosuccinate, glyceryl esters, sodium carboxymethylcellulose, cholic acid and other bile acids (e.g., cholic acid, deoxy cholic acid, glycocholic acid, taurocholic acid, glycodeoxy cholic acid) and salts thereof (e.g., sodium deoxycholate, etc.). Suitable cationic surfactants include but are not limited to quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide,25178092112.1lauryldimethylbenzylammonium chloride, acyl carnitine hydrochlorides, and alkyl pyridinium halides, and mixtures thereof. Nonionic surfactants include, but are not limited to Vitamin E TPGS, a-Hydro-o-hydroxypoly(oxyethylene)poly(oxypropylene) poly-(oxyethylene) block copolymer (Pol oxamer 188), Oxirane, methyl-, polymer with oxirane (Pol oxamer 407), PEG 4000, Tween-80, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene-derivatized lipids such as mPEG- PSPC (palmitoyl-stearoyl-phophatidylcholine), mPEG-PSPE (palmitoyl-stearoyl- phophatidylethanolamine), sorbitan esters, glycerol monostearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers, poloxamines, methylcellulose, hydroxycellulose, hydroxy propylcellulose, hydroxy propylmethylcellulose, noncrystalline cellulose, polysaccharides, starch, starch derivatives, hydroxy ethyl starch, polyvinyl alcohol, and polyvinylpyrrolidone, and mixtures thereof.

[0090] In some cases, Ringer's Injection, Lactated Ringer's Injection, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required. For delayed release, the vector may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0091] In one embodiment, the vectors are provided in KH2PO4, Na2HPC>4, NaCl, and MgCh, with pH 7.4.

[0092] If the vector is to be stored long-term, it may be frozen in the presence of a cryoprotectant such as glycerol.

[0093] Methods of treatment

[0094] Provided herein are methods of treating or preventing PD and / or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or an AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s). Provided are method of reducing the frequency, severity, and / or duration of PD symptoms in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or a AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s). In one embodiment, the AAV-GAD-65 and the AAV-GAD-67 vectors are administered in a single pharmaceutical26178092112.1composition. In one embodiment, the AAV-GAD-65 and the AAV-GAD-67 vectors are administered in separate pharmaceutical compositions.

[0095] In some embodiments, the subject is a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals, include, but are not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline, etc. In a preferred embodiment, the subject is human. Individuals and patients are also subjects herein.

[0096] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization ( / .< ., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of one or more symptoms of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0097] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0098] The medical uses contemplated by the specification may be formulated as a vector for use as a medicament for prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), but could equally be formulated as (i) a method of prevention or treatment of the disease(s) and / or disorder(s) defined herein (or prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), as (ii) a vector for use in the preparation of a medicament for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), or as (iii) use of a vector according for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein). Such medical uses are all envisaged by the present disclosure.27178092112.1

[0099] Provided herein is a pharmaceutical composition, wherein the pharmaceutical composition is delivered bilaterally into each of the STN of a subject.

[0100] Proof of concept of STN administration of AAV-GAD in the nonclinical models has been performed successfully. Further, a Phase 1, first-in-human, unilateral treatment, dose escalation study was conducted. See Kaplitt et al., Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial, Lancet. 2007 Jun 23;369(9579):2097-105. In this open-label study, 12 participants with advanced Parkinson’s disease had AAV-GAD vectors (i.e., a 1 : 1 mixture of AAV-GAD- 65 and AAV-GAD-67 vectors) in a final volume of 50 pL delivered to one STN. This allowed the untreated side to serve as a control. Participants were divided into 3 equal groups, with the low dose group receiving a total of 3.5 x 109vg, the middle dose group receiving 1.05 x 1010vg and the high dose group receiving 3.5 x 1010vg. The respective vector concentrations before dilution and injection were I x lO11viral genomes (vg) / mL (low dose), 3x l0nvg / mL (medium dose), and I x lO12vg / mL (high dose) with l x phosphate-buffered saline solution.

[0101] Following completion of the Phase 1 study, a randomized, double-blinded, sham surgery, controlled, multicenter, Phase 2 study was conducted. See LeWitt et al., AAV2-GAD gene therapy for advanced Parkinson's disease: a double-blind, sham-surgery controlled, randomised trial, Lancet Neurol. 2011 Apr; 10(4): 309- 19; Niethammer et al., Long-term follow-up of a randomized AAV2-GAD gene therapy trial for Parkinson's disease, JCI Insight. 2017 Apr 6;2(7):e90133. In this study, 22 participants were randomized to treatment with 35 pL AAV-GAD (I x lO12vg / mL concentration) per hemisphere for a dose of 3.5x l010vg, corresponding to the high dose from the phase 1 clinical trial (7.Ox lO10vg total dose to the participant), and 23 participants were randomized to a sham surgery procedure. Of the 23 sham participants, 14 participants subsequently received AAV-GAD in the open-label phase of the study.

[0102] The Phase 2 study further identified a unique18FDG-PET brain network pattern associated with response to AAV-GAD therapy, involving the generation of new polysynaptic functional pathways linking the STN to the motor cortical regions (Niethammer et al., Gene therapy reduces Parkinson's disease symptoms by reorganizing functional brain connectivity, Sci Transl Med. 2018 Nov 28;10(469):eaau0713). This new18FDG-PET metabolic network is termed the GAD-related pattern (GADRP) and is quantified using a z-scale unit score. This network was defined in an unbiased fashion while the data were blinded, using mathematical modeling to identify patterns which might distinguish AAV-GAD from sham.28178092112.1

[0103] Efficacy in these two clinical trials was determined by a reduction in the UPDRS Part 3 (motor examination) score during the “medication off’ state. In addition, treatment with AAV-GAD therapy was associated with a unique brain network defined by18FDG-PET (Niethammer et al. 2018).

[0104] A third clinical trial is described in the Examples.

[0105] Provided herein is a method of treating or preventing PD or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding GAD, wherein the pharmaceutical composition is delivered bilaterally into each of the two STN of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

[0106] Provided is a method of treating or preventing a neurodegenerative disease or disorder associated with GABA deficiency or treating or preventing symptoms associated with said neurodegenerative disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding GAD, wherein the pharmaceutical composition is delivered bilaterally into each of the two STN of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

[0107] Provided herein are methods for treating diseases or disorders of the central nervous system associated with dopaminergic hypoactivity, disease, injury or chemical lesioning. In one embodiment, the neurodegenerative disease or disorder is cognitive impairment. In one embodiment, the neurodegenerative disease or disorder is PD.

[0108] In some embodiments, the pharmaceutical composition is administered at a total dose of at least about 2.09 x io11vg. In one embodiment, the pharmaceutical composition is administered at a total dose of about 2.09 x io11vg.

[0109] In some embodiments, the pharmaceutical composition is administered at a dose of at least about 1 x io11vg / STN. In some embodiments, the pharmaceutical composition is administered at a dose of at least about 1.045 x io11vg / STN. In one embodiment, the pharmaceutical composition is administered at a dose of about 1.045 x io11vg / STN.

[0110] In some embodiments, the pharmaceutical composition is administered in a volume of about 10 pL, about 15 pL, about 20 pL, about 25 pL, about 30 pL, about 35 pL, about 40 pL, 45 pL, about 50 pL, about 55 pL, about 60 pL, 65 pL, about 70 pL, about 75 pL, about 80 pL, about 85 pL, about 90 pL, about 95 pL, or about 100 pL. In some embodiments, the pharmaceutical composition is administered in a volume of about 35 pL. In some29178092112.1embodiments, the pharmaceutical composition is administered in a volume of about 50 pL. In some embodiments, the pharmaceutical composition is administered at a concentration of about 1 x IO10vg / mL, about 1 x 1011vg / mL, or about 1 x 1013vg / mL. In some embodiments, the pharmaceutical composition is administered at a concentration of about 1-5 x IO10vg / mL, about 1-5 x 1011vg / mL, or about 1-5 x 1013vg / mL. In one embodiment, the pharmaceutical composition is administered at a concentration of about 2 / 1012vg / mL AAV-GAD. In one embodiment, the pharmaceutical composition is administered at a concentration of about 3 / 1012vg / mL. In one embodiment, the pharmaceutical composition is administered at a concentration of about 2.09* 1012vg / mL.[OHl] In some embodiments, the methods disclosed herein improve the symptoms of PD in frequency, extent, and / or duration. PD symptoms include, but are not limited to, generalized slowing of movements (bradykinesia), resting tremor (e.g., tremor in hands, arms, legs, jaw, or head), rigidity, loss of smell, sleep dysfunction, mood disorders, excess salivation and drooling, constipation, excessive periodic limb movements in sleep, difficulty with balance and coordination, difficulty walking and talking, depression and other emotional changes, memory difficulties, dementia, fatigue, micrographia, orthostatic hypotension, difficulty swallowing, urinary retention, erectile dysfunction are common, muscle stiffness (where muscle remains contracted for a long time), and any of the other symptoms described in this disclosure. In some embodiments, the methods disclosed herein increased “on” time and / or reduce “off’ time.

[0112] Numerous methods are known in the art for assessing the efficacy and safety of a treatment for PD, including, but not limited to, the methods described herein.

[0113] I8FDG-PET

[0114] 18FDG-PET can be used in the screening process to exclude participants with atypical forms of PD most commonly misdiagnosed as idiopathic PD, / .< ., those with multiple system atrophy (MSA) or progressive supranuclear palsy (PSP).18FDG-PET measures the uptake of radiolabeled, peripherally administered glucose in brain tissue, providing an index of the relative activity of neurons within a region or in a network of regions. Therefore, the abnormalities in brain network activity can be evaluated with this imaging technique, and biological changes in brain function can be objectively measured following interventions.

[0115] Baseline18FDG-PET scans can be assessed using an automated differential diagnosis algorithm based on previously identified disease-specific brain networks diagnostic of idiopathic PD to exclude participants with “lookalike” disorders such as multiple system atrophy and progressive supranuclear palsy.30178092112.1

[0116] Scans can be evaluated for PD-related pattern (PDRP) and GAD-related pattern (GADRP) at baseline and at 6 months after study drug administration as an exploratory evaluation for potential correlates of activity. The PDRP score is a z-scale unit score of18FDG- PET brain network pattern. The GADRP score is a z-scale unit score of18FDG-PET brain network pattern associated with response to AAV-GAD therapy, involving the generation of new polysynaptic functional pathways linking the STN to the motor cortical regions.

[0117] Questionnaires and Scales

[0118] Columbia Suicide severity rating scale: used to identify and assess individuals at risk for suicide (assessed by a clinician or individual with medical training).

[0119] Beck Depression Inventory-II: a 21-item self-report inventory measuring the severity of depression in adolescents and adults.

[0120] Beck Anxiety Inventory: a 21 -item self-report inventory measuring the severity of anxiety in adolescents and adults.

[0121] Clinical Global Impression-Severity: a 7-point scale to rate the severity of the participant’s illness at the time of assessment, relative to her / his baseline state (prior to the surgery) (assessed by a clinician or individual with medical training).

[0122] Clinical Global Impression-Improvement: a 7-point scale to assess how much the participant’s illness has improved or worsened relative to her / his baseline state (prior to the surgery) (assessed by a clinician or individual with medical training).

[0123] EuroQol-5 Dimensions-5 Levels Survey: this survey comprises five dimensions: mobility, self-care, usual activities, pain / discomfort and anxiety / depression. Each dimension has 5 levels: no problems, slight problems, moderate problems, severe problems and extreme problems. The participant is asked to indicate his / her health state by ticking the box next to the most appropriate statement in each of the five dimensions. This decision results in a 1 -digit number that expresses the level selected for that dimension. The digits for the five dimensions can be combined into a 5-digit number that describes the patient’s health state.

[0124] Montreal Cognitive Assessment: a brief cognitive screening task assessment of different cognitive abilities such as visuospatial skills, executive function, memory, language and attention (assessed by a clinician or individual with medical training).

[0125] Parkinson’s Disease Questionnaire (PDQ)-39 Scale: a 39-item questionnaire completed by participants that measures health status and quality of life. See, e.g., Peto et al., The development and validation of a short measure of functioning and well being for individuals with Parkinson's disease, Qual Life Res. 1995 Jun;4(3):241-8. The 39-point PDQ provides scores for each of the 8 scales: mobility, activities of daily living, emotional well-31178092112.1being, stigma, social support, cognitions, communications and bodily discomfort. PDQ-39 data can be presented either in profile form or as a single index. A higher score on the PDQ-39 scale indicates more severe PD symptoms and a lower quality of life.

[0126] Parkinson’s Disease Sleep Scale-2: a 15-item scale completed by participants to selfrate and quantify the level of sleep disruption being experienced.

[0127] Unified Dyskinesia Rating Scale: a scale formatted as a questionnaire for the participant used to evaluate the involuntary movements that can be associated with long-term treatment with dopaminergic medication.

[0128] Movement Disorder Society -Unified Parkinson’s Disease Rating Scale (MDS- UPDRS): Part 1 (Non-Motor Experiences of Daily Living), Part 2 (motor experiences of daily living), Part 3 (motor examination) and Part 4 (motor complications). See, e.g., Ebersbach G, Baas H, Csoti I, Miingersdorf M, Deuschi G. Scales in Parkinson’s disease. J Neurol. 2006;253(Suppl 4):IV32-35; Poewe W. Clinical measures of progression in Parkinson’s disease. Mov Disord. 2009;24(S2):S671-76; Goetz et al., Movement Disorder Society- sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS): Process, format, and clinimetric testing plan, Mov Disord. 2007 Jan;22(l):41-7; Goetz et al., Movement Disorder Society UPDRS Revision Task Force. Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS): scale presentation and clinimetric testing results, Mov Disord. 2008 Nov 15;23(15):2129-70.

[0129] Part 1 has two components: 1 A concerns a number of behaviors that are assessed by a clinician or individual with medical training with all pertinent information from participants and caregivers, and IB is completed by the participant with or without the aid of the caregiver, but independently of the clinician or individual with medical training. Part 1 assesses behavioral problems such as intellectual decline, hallucinations, and depression.

[0130] Part 2 is designed to be a self-administered questionnaire like Part IB. Part 2 assesses patients' perceptions of their ability to carry out activities of daily living, including dressing, walking, and eating.

[0131] Part 3 is completed by a clinician or individual with medical training and has instructions for the clinician or individual with medical training to give or demonstrate to the participant. Part 3 covers the evaluation of motor disability and includes ratings for tremor, slowness (bradykinesia), stiffness (rigidity), and balance. Part 3 of 14 items that can have scores from zero (normal) to four (severe). The scores are added to give an overall score of the involuntary movements.32178092112.1

[0132] Part 4 integrates patient-derived information with clinical observations and judgments by a clinician or individual with medical training and is completed by the clinician or individual with medical training. Part 4 covers a number of treatment complications including ratings of involuntary movements (dyskinesias), painful cramps (dystonia), and irregular medication responses (motor fluctuations).

[0133] The MDS-UPDRS Part 3 may be completed while in the “medication off’ state (during medication “off’ time). “Medication off’ state scores can be obtained after at least 12 hours of holding dopaminergic medication. Once this assessment is completed, participants take their usual medications for PD. “Medication on” state evaluations can be assessed when participants and their clinician agree that the participant is in a typical “on” state (as a guidance, the “on” state is expected to be achieved approximately 2 hours after taking medication(s) for PD). Participants may take additional levodopa (in addition to their usual dose) if needed to achieve the “on” state.

[0134] Revised Hopkins Verbal Learning Test (HVLT-R): a test administered by a clinician or individual with medical training, assessing verbal memory. Participants are presented with a list of words over repeated learning trials and are required to recall words from the list immediately and after a delay of 20-25 minutes and complete a yes-no delayed recognition task. The HVLT-R also provides 6 alternate forms in order to reduce the effects of practice and will be administered during each session.

[0135] Stroop Color and Word Test (Delis-Kaplan Executive Function System (DKEFS) version): a test administered by a clinician or individual with medical training, measuring processing speed and executive functioning. There are 4 parts. Participants are required to read printed color names as fast as possible (Part 1), and name color patches as fast as possible (Part 2). To assess executive functioning, and specifically cognitive inhibition, participants are then required to name the ink color of printed color names, avoiding the ‘read’ the printed work. In the 4th part, participants are required to do the same as in Part 3 unless the word is presented in a box, and in these instances, they must read the word instead of naming the color.

[0136] Trail Making Test (TMT) (DKEFS version): a paper-and-pencil test administered by a clinician or individual with medical training, measuring processing speed and executive functioning. There are 4 trials to complete including a cancellation task ( / .< ., cross-out targets as fast as possible), simple tracking tasks requiring participants to connect numbers with a line as fast as possible, connect letters in order, and lastly, a more complex task requiring them to alternate between numbers and letters in sequences.33178092112.1

[0137] Verbal Fluency Test (DKEFS version): a verbal fluency test administered by a clinician or individual with medical training, measuring executive function and language abilities. Participants are required to generate as many words as possible aloud within a one- minute period following letter cues (3 trials) and category cues (3 trials).

[0138] Digit Span test (part of Weschler Memory and Weschler scales): a test administered by a clinician or individual with medical training, measuring attention and working memory span. Participants are required to repeat strings of digits of increasing length in the same order as well as in reverse order.

[0139] In some embodiments, all questionnaires used in repeated assessments are administered by the same rater.

[0140] Hauser Patient Diary

[0141] The Hauser Patient Diary can be used to collect information on how much time a participant spends in the different PD states:(a) ON - Good or practically normal mobility.(b) ON with Troubling Dyskinesia - participant is troubled by involuntary twisting, turning movements. These movements are different from the rhythmic “tremor” which is a symptom of PD itself.(c) OFF - stiffness, marked decrease in mobility, and / or immobility.(d) ASLEEP - time spent sleeping.

[0142] The diary is divided into 30-minute sections starting at midnight and ending at 11 :30 PM. Participation in the study requires that participants demonstrate full understanding of the rating criteria and proficiency in completing the diary. In some embodiments, diary concordance of at least 66% will be required for a participant to qualify for a clinical study. Participants may complete this 24-hour diary of their PD symptoms, for example, twice per week (on 2 nonsequential days) for 3 consecutive weeks prior to the second screening visit ( / .< ., the Day -7 to Day -3 visit) (baseline entries), and on 2 nonsequential days during the week prior to Day 15, and Weeks 12 and 26. The change from baseline in absolute number of daily hours in the “on without troublesome dyskinesias” state to the weeks prior to Day 15, Week 12 and Week 26 may be compared.

[0143] The methods disclosed herein may improve a patient’s “on” time. The term “on” time as used herein refers to the period of time during which a patient is relatively free of symptoms of PD or Parkinsonism. During this time, the PD patient usually has adequate control of PD symptoms. “On” time is usually associated with good or practically normal mobility. An improvement in a patient’s “on” time might refer to an improvement of the severity, duration,34178092112.1and / or frequency of the patient’s PD symptoms during “on” time. An improvement in a patient’s “on” time might refer to an improvement in the duration (z.e., longer) or frequency (z.e., more often) of the time periods in which the patient experiences “on” time.

[0144] The methods disclosed herein may increase a patient’s “on” time by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%. The methods disclosed herein may decrease a patient’s “off’ time by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%.

[0145] For the avoidance of doubt, the methods disclosed herein may improve (e.g., increase) patient reported “on” time or improve clinical scores obtained during clinician reported “medication on” time. The methods disclosed herein may be improve (e.g., reduce) patient reported “off’ time or improve clinical scores obtained during clinician reported “medication off’ time.

[0146] Provided herein is a method of reducing symptoms associated with PD during “medication on” time, wherein the reduction of symptoms is reflected in a reduction in the patient’s UPDRS score as compared to a control. Provided herein is a method of reducing symptoms associated with PD during “medication on” time, wherein the reduction of symptoms is reflected in a reduction in the patient’s UPDRS (Part 3) score as compared to a control. Provided herein is a method of reducing symptoms associated with PD during “on” time, wherein the reduction of symptoms is reflected in a reduction in the patient’s Parkinson’s Disease Questionnaire-39 Scale score as compared to a control.35178092112.1

[0147] The methods disclosed herein may improve a patient’s “off’ time. The term “off’ time as used herein refers to the period of time during which a patient is not substantially free of symptoms of PD or Parkinsonism. Often, a patient will experience a state of decreased mobility and / or a state in which symptoms of Parkinson's disease or Parkinsonism have re- emerged. An improvement in a patient’s “off’ time might refer to an improvement of the severity, duration, and / or frequency of the patient’s PD symptoms during “off’ time. An improvement in a patient’s “off’ time might refer to an improvement in the duration (z.e., shorter) or frequency (z.e., less often) of the time periods in which the patient experiences “off’ time.

[0148] Provided herein is a method of reducing symptoms associated with PD during “off’ time, wherein the reduction of symptoms is reflected in a reduction in the patient’s UPDRS score as compared to a control. Provided herein is a method of reducing symptoms associated with PD during “off’ time, wherein the reduction of symptoms is reflected in a reduction in the patient’s UPDRS (Part 3) score as compared to a control. Provided herein is a method of reducing symptoms associated with PD during “off’ time, wherein the reduction of symptoms is reflected in a reduction in the patient’s Parkinson’s Disease Questionnaire-39 Scale score as compared to a control.

[0149] The methods disclosed herein may improve a patient’s general health and quality of life as assessed by the Parkinson’s Disease Questionnaire-39 Scale.

[0150] Provided herein are methods of treating or preventing PD and / or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or an AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s UPDRS (Part 3) score during “medication off’ time as compared to a control by at least 2 points. Provided are method of reducing the frequency, severity, and / or duration of PD symptoms in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or a AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s UPDRS (Part 3) score during “medication off’ time as compared to a control by at least 2 points. Provided herein are methods of treating or preventing PD and / or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or an AAV- GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s UPDRS (Part 3) score during36178092112.1“medication on” time as compared to a control by at least 2 points. Provided are method of reducing the frequency, severity, and / or duration of PD symptoms in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or a AAV- GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s UPDRS (Part 3) score during “medication on” time as compared to a control by at least 2 points. In some embodiments, the UPDRS (Part 3) score is improved by at least 3 points, by at least 4 points, by at least 5 points, by at least 6 points, by at least 7 points, by at least 8 points, by at least 9 points, by at least 10 points, by at least 11 points, by at least 12 points, by at least 13 points, by at least 14 points, by at least 15 points, by at least 16 points, by at least 17 points, by at least 18 points, by at least 19 points, by at least 20 points, by at least 21 points, by at least 22 points, by at least 23 points, by at least 24 points, or by at least 25 points as compared to a control. In some embodiments, the score is improved by 10-25 points, by 10-20 points, by 15-25 points, or by 15-20 points.

[0151] “ On” time and “off time” generally relate to patient-reported time periods during the day when medication benefit has waned and symptoms return, usually quantified in hours per day. This measure integrates the heterogeneity of fluctuations, including partial wearing off, delayed “on”, dose failures, and early-morning “off’ times. “On” and “off’ times capture real- world fluctuations in symptom control during the patient’s usual medication regimen.

[0152] In contrast, the UPDRS (Part 3) motor examination score is a clinician-rated snapshot of motor severity obtained during a standardized, practically defined OFF condition (typically after overnight withdrawal of dopaminergic therapy). The UPDRS (Part 3) motor examination score for the “medication on” state is frequently measured approximately two hours after taking medication(s) for Parkinson’s disease. The UPDRS (Part 3) motor examination score for the “medication off’ state is frequently measured at least 12 hours of holding dopaminergic medication.

[0153] Provided herein are methods of treating or preventing PD and / or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject an AAV-GAD-65 vector and / or an AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s PDQ-39 score by at least 2 points as compared to a control. Provided are method of reducing the frequency, severity, and / or duration of PD symptoms in a subject in need thereof, the method comprising administering to the subject an AAV-GAD- 65 vector and / or a AAV-GAD-67 vector, or a pharmaceutical composition comprising such vector(s), wherein the administration results in an improvement of the patient’s PDQ-39 score37178092112.1by at least 2 points as compared to a control. In some embodiments, the PDQ-39 score is improved by at least 3 points, by at least 4 points, by at least 5 points, by at least 6 points, by at least 7 points, by at least 8 points, by at least 9 points, by at least 10 points, by at least 11 points, by at least 12 points, by at least 13 points, by at least 14 points, by at least 15 points, by at least 16 points, by at least 17 points, by at least 18 points, by at least 19 points, by at least 20 points, or by at least 25 points as compared to a control. In some embodiments, the PDQ- 39 score is improved by, for example, 2 to 10 points, by 3 to 9 points, by 4 to 8 points, by 5 to 8 points.

[0154] An improvement may be assessed, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 9 weeks, after 10 weeks, after 11 weeks, or after 12 weeks. An improvement may be assessed, for example, after 1 week, after 2 months, after 3 months, after 4 months, after 5 months, after 6 months, after 7 months, after 8 months, after 9 months, after 10 months, after 11 months, after 12 months, after 13 months, after 14 months, after 15 months, after 16 months, after 17 months, after 18 months, after 19 months, after 20 months, after 21 months, after 22 months, after 23 months, or after 24 months.

[0155] A “control” may be one or more individuals in a control group (e.g., one or more PD patients that have not received the AAV-GAD therapy). The control may be the same patient at an earlier time (e.g., before receiving the AAV-GAD therapy or at an earlier time during the treatment course). A control value can be a single value or an average of values.

[0156] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.

[0157] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0158] It is to be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or38178092112.1embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0159] All referenced patents, patent applications, book chapters, scientific publications, etc. are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0160] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention.EXAMPLES

[0161] Example 1: Study design - randomized, double-blind, sham-controlled phase 1 / 2 study

[0162] This was a Phase 1 / 2, randomized, double-blind, sham-controlled study designed to assess the safety and tolerability of AAV-GAD administered bilaterally into the STN in participants with PD not adequately controlled with appropriate anti-Parkinsonian medications. Participants had idiopathic Parkinson's disease, a history of levodopa responsiveness for at least 12 months, and a UPDRS Part 3 score of >25 points in the “medication off’ state. “AAV-GAD” in this study was a 1 : 1 mixture (as assessed by viral genomes) of AAV-GAD-65 and AAV- GAD-67 vectors.

[0163] Ob jectives and endpoints for this study

[0164] Primary objective: To evaluate the safety and tolerability of AAV-GAD delivered to the STN in participants with PD. Endpoint: Number of participants with adverse events and serious adverse events.

[0165] Exploratory objective 1 : To evaluate the effect of AAV-GAD on the MDS-UPDRS Part 3 (motor examination) score in the “medication off state”. Endpoint: The mean change from baseline to Weeks 12 and 26 for the AAV-GAD groups compared to the SHAM group in MDS-UPDRS Part 3 (motor examination) score in the “medication off’ state.

[0166] Exploratory objective 2: To evaluate the effect of AAV-GAD on the GAD-related pattern (GADRP) score and PD-related pattern (PDRP) score. Endpoints: (1) the mean change from baseline to Week 26 for the AAV-GAD groups compared to the SHAM group in GADRP39178092112.1score; (2) the mean change from baseline to Week 26 for the AAV-GAD groups compared to the SHAM group in PDRP score.

[0167] Exploratory objective 3: To evaluate the effect of AAV-GAD on the percentage of substantially clinically meaningful responders and clinically meaningful responders. Endpoints: (1) The percentage of substantially clinically meaningful responders. A substantially clinically meaningful responder is defined as a participant who meets both of the following criteria: (a) demonstrates a reduction from baseline to Week 26 in MDS-UPDRS Part 3 (motor examination) score of >10; and (b) demonstrates an increase from baseline to Week 26 in GADRP score of >1.1 z-scale units; (2) The percentage of clinically meaningful responders. A clinically meaningful responder is defined as a participant who meets both of the following criteria: (a) demonstrates a reduction from baseline to Weeks 12 and 26 in MDS- UPDRS Part 3 (motor examination) score of >6; and (b) demonstrates an increase from baseline to Week 26 in GADRP score of >1.1 z-scale units.

[0168] Exploratory objective 4: To evaluate the effect of AAV-GAD on activities of daily living (ADL), dyskinesias, cognition, sleep, depression, dopaminergic medication dose, neuropsychological effects, and quality of life assessments. Endpoint: The mean change from baseline to Weeks 12 and 26 for the AAV-GAD groups compared to the SHAM group in: MDS-UPDRS Part 1 (Non-Motor Experiences of Daily Living), MDS-UPDRS Part 2 (ADL) score, MDS-UPDRS Part 4 (Motor Complications) score, Unified Dyskinesia Rating Scale, Montreal Cognitive Assessment, Parkinson’s Disease Sleep Scale-2, Beck Depression Inventory-II, Beck Anxiety Inventory, Columbia suicide severity rating scale, Levodopa dose and levodopa equivalent daily dose, “on without troublesome dyskinesias” time using the Hauser Patient Diary, Parkinson’s Disease Questionnaire-39 Scale, EuroQol-5 Dimensions-5 Levels Survey, Clinical Global Impression-Severity, Clinical Global Impression- Improvement, Revised Hopkins Verbal Learning Test (HVLT-R), Stroop Color and Word Test (DKEFS version), Trail Making Test (TMT), Verbal Fluency Test (DKEFS version), and / or Digit Span Test.

[0169] Inclusion criteria

[0170] Participants who met all inclusion criteria and none of the exclusion criteria were eligible to participate in the study. Inclusion criteria were:

[0171] Male or female.

[0172] Age 25 to 85 years, inclusive.40178092112.1

[0173] Must sign an informed consent form (ICF) (or their legally acceptable representative must sign) indicating that he or she understands the purpose of, and procedures required for the study and is willing to participate in the study.

[0174] Diagnosis of probable idiopathic PD by United Kingdom Parkinson’s Disease Society Brain Bank criteria and no evidence of another Parkinsonian disorder.

[0175] Completed cranial MRI and18FDG-PET during screening. MRI and18FDG-PET scans obtained during screening will be used as baseline tests and will be reviewed to verify the diagnosis of PD.

[0176] Presence of typical features of PD and history of levodopa responsiveness demonstrated for at least 12 months prior to screening.

[0177] Progressive Parkinsonian disability with demonstrated response from continued use of levodopa (or from other PD medications if part of the treatment regimen), but with insufficient response to medication (e.g., uncontrolled tremors, increased “off’ time, decreased “on” time, motor fluctuations, dyskinesias, gait disturbance) or with intolerable side effects.

[0178] Severe disability, which includes a practically defined “medication off’ state MDS- UPDRS Part 3 (motor examination) score of > or =25 points after overnight omission of all PD medications.

[0179] Continued use of an unchanged stable anti-Parkinsonian drug regimen for > or = 4 weeks prior to screening.

[0180] Willingness to not add any new treatments for PD for the duration of the 6-month study period. Adjustments in the dose of dopaminergic medications are permitted when medically indicated.

[0181] Agrees not to participate in another interventional study until completion of this study.

[0182] Willingness to not receive DBS or any other approved or experimental surgical therapy for PD during the 6-month study period.

[0183] Receiving stable doses of other medications for any other underlying medical conditions for > or = 4 weeks prior to screening.

[0184] Must demonstrate a full understanding of the Hauser Patient Diary rating criteria and demonstrate proficiency in completing the diary by reaching at least 66% of diary concordance with a site rater.

[0185] A woman must be: (1) not of childbearing potential or (2) of childbearing potential and practicing a highly effective method of contraception (failure rate of <1% per year when used consistently and correctly) and agrees to remain on a highly effective method for at least41178092112.16 months after receiving the last dose of study intervention. Examples of highly effective methods of contraception are hormonal or barrier methods of contraceptive, or sexual abstinence.

[0186] If male, willing to use barrier and spermicide form of contraception or maintain sexual abstinence for at least 6 months following surgery. Participants who were randomly assigned to sham surgery were also required to adhere to this restriction to receive treatment in the follow-up study.

[0187] Ability to discontinue use of aspirin, other antiplatelet drugs, or anti-coagulants at least 1 week prior to surgery and for 1 week following surgery.

[0188] Exclusion criteria

[0189] The exclusion criteria were as follows:

[0190] Women who are pregnant or breastfeeding. Women of childbearing potential are required to have a negative serum pregnancy test at a screening visit and within approximately 1 week prior to surgery on Day 1. Participants are considered not of childbearing potential if they are surgically sterile (z. e. , they have undergone a hysterectomy or bilateral oophorectomy), or are postmenopausal.

[0191] Any experimental therapy (drug or biologic) within 3 months prior to screening.

[0192] Have a known allergy to any of the non-investigational drugs (e.g., antibiotics and local anesthesia) to be used in the study.

[0193] Known allergies, hypersensitivity, or intolerance to AAV-GAD excipients.

[0194] Any prior history of brain surgery for PD.

[0195] Ongoing treatment with dopamine receptor-blocking drugs.

[0196] History of any serious cerebral insult, such as head injury, or central nervous system infection, or uncontrolled seizures.

[0197] Mental retardation or impaired cognitive abilities, as judged by patient history or cognitive impairment as defined by Montreal Cognitive Assessment (MoCA) <20.

[0198] Focal or lateralized central nervous system (CNS) neurological deficits other than mild memory disturbance.

[0199] Neurological features suggestive of the diagnosis of a “Parkinson-plus” disorder (such as progressive supranuclear palsy, multiple system atrophy, corticobasal ganglionic degeneration, or “lower body” Parkinsonism) or normal pressure hydrocephalus.

[0200] Evidence of lesions or other significant abnormalities on cranial neuroimaging (either CT, MRI, or18FDG-PET) suggesting findings compatible with a probable diagnosis other than PD.42178092112.1

[0201] Alanine aminotransferase and / or aspartate aminotransferase >1.5 upper limit of normal (ULN) or alkaline phosphatase >1.5 ULN.

[0202] Clinically relevant abnormality on 12-lead ECG.

[0203] Administration of any live virus vaccine within 4 weeks of surgery.

[0204] Evidence of other significant medical or psychiatric disorders, such as psychosis, frequent occurrence of hallucinations, severe depression, significant cognitive decline in recent months, systemic organ failure, or bleeding diathesis, not attributable to adverse effects of antiParkinsonian medications.

[0205] Evidence of active impulse control disorder, or substance or alcohol abuse unrelated to use of dopaminergic medication.

[0206] Significant concurrent or recently diagnosed (<2 months prior to the screening visit) medical condition that, in the opinion of the clinician or individual with medical training, could affect the participant’s ability to tolerate or complete the study.

[0207] Current immunosuppressive therapy or underlying disorder associated with diminished immunocompetence.

[0208] Serum platelet count of <80,000 / mm3, international normalized ratio (INR) >1.3, or partial thromboplastin time (PTT) >40 seconds.

[0209] Score of >20 on the Beck Depression Inventory -II.

[0210] Acute or chronic systemic and / or local skin infections involving the scalp.

[0211] Has any other condition that, at the consideration of a clinician, makes them inappropriate for entry into the study.

[0212] Withdrawal criteria

[0213] Participation in this clinical study was discontinued for any of the following reasons:

[0214] The participant withdraws consent or requests discontinuation from the study for any reason.

[0215] Occurrence of any medical condition or circumstance that exposes the participant to substantial risk and / or does not allow the participant to adhere to the requirements of the protocol.

[0216] Any serious adverse event (SAE), clinically significant adverse event (AE), severe laboratory abnormality, intercurrent illness, or other medical condition that indicates that continued participation is not in the best interest of the participant.

[0217] Requirement of prohibited concomitant medication.

[0218] Participant failure to comply with protocol requirements or study-related procedures.43178092112.1

[0219] Termination of the study by the sponsor or the regulatory authority.

[0220] Any reason for participant withdrawal was documented in the electronic case report form (eCRF).

[0221] Participants who withdrew prior to surgery were replaced. Participants who withdrew after surgery were replaced.

[0222] End of study

[0223] The end of the study (“study completion”) was defined as the date of the last protocol-specified visit / assessment (including telephone contact) for the last participant in the study.

[0224] Treatment groups

[0225] Participants were randomized to one of the three following groups: (1) AAV-GAD at a dose of 3.5 * 1010vg / STN, low dose group, (2) AAV-GAD at a dose of 10.45* 1010vg / STN, high dose group; (3) sham surgery.

[0226] AAV-GAD was administered bilaterally via stereotactic infusion into each STN (1 infusion per hemisphere: 2 infusions in total). Each STN was infused with (1) 35 pL of 1 x 1012vg / mL AAV-GAD, for a dose of 3.5 * 1010vg / STN (low dose group) or (2) 50 pL of 2.09 * 1012vg / mL AAV-GAD, for a dose of 10.45 * 1010vg / STN (high dose group). The total dose per participant were 7.0* 1010vg or 20.9* 1010vg, in the low and high dose groups, respectively. Also see Table 7.

[0227] In the previous Phase 2 study (LeWitt et al. 2011), the same dose was delivered to each STN as the low dose in this current study.

[0228] The “AAV-GAD” study drug comprised two rAAV serotype 2-GAD vectors for the expression of GAD-65 and GAD-67, respectively, mixed in a 1 : 1 ratio (ratio calculated by comparing number of viral genomes). Both vector preparations contain a transgene expression cassette consisting of the CMV enhancer, CBA promoter, and WPRE to facilitate production of the human GAD enzyme from the GAD-65 and the GAD-67 genes. The vectors also contained a bgh-PolyA signal.

[0229] Formulation and packaging

[0230] AAV-GAD was formulated in a buffered solution of KH2PO4, Na2HPC>4, NaCl, and MgCh, with pH 7.4. The AAV-GAD formulation was diluted to a concentration of 1 x 1012vg / mL (low dose) or used at the undiluted concentration of 2.09 * 1012vg / mL (high dose).

[0231] Randomization and blinding

[0232] Participants were randomized to receive 1 of 2 doses of AAV-GAD (5 participants per dose group) or sham surgery (4 participants).44178092112.1

[0233] Randomization occurred after all inclusion and exclusion criteria had been met and surgery is scheduled.

[0234] Breakins the blind

[0235] After a participant completes the Week 26 Visit and that participant’s data were considered clean, that participant’s treatment assignment was unblinded.

[0236] Acute delivery system description

[0237] The following devices were used as part of the procedure:

[0238] Infusion Catheter Kit: Sterile, disposable, single-patient, single-use infusion catheter system consisting of a parenchymal catheter and extension set (including a breakaway connector).

[0239] Cranial Anchor Kit: Sterile, disposable, single-patient, single-use Cranial Anchor kit.

[0240] Transfer Guard and Syringe Kit: Sterile, disposable, single-patient, single-use syringe and sterile, disposable, single-patient, single-use syringe transfer guard.

[0241] Accessory kit.

[0242] Reusable pump.

[0243] These components facilitated infusion by connecting the syringe containing the therapeutic agent(s) to the parenchymal catheter via the extension set. This system included a breakaway connector to preclude accidental movement of the parenchymal catheter during infusion.

[0244] Device packaging

[0245] The single-use disposable infusion catheter kit, anchor kit, and transfer guard and syringe kit (1 per hemisphere for each kit) were packaged and distributed in sterile packaging. The reusable accessory kit was packaged in sterile packaging. The pump was distributed in the packaging provided by the manufacturer.

[0246] AAV-GAD administration and surgery procedure

[0247] On the day of AAV-GAD administration, participants were taken to the operating room. Intravenous antibiotics was given prior to skin incision. The neurosurgeon made a frontal burr hole at a standard entry site normally used for the placement of STN DBS electrodes. Local anesthesia was used unless general anesthesia was necessary for participant safety. This entry site was usually at or just anterior to the coronal suture in the mid-pupillary line; however, the entry site could be adjusted for optimal safety and accuracy depending upon the assessment of the participant’s presurgical CT or MRI. The brain parenchymal infusion catheters were placed in each STN bilaterally using standard stereotactic procedures.45178092112.1

[0248] Briefly, standard stereotactic systems for the insertion of STN DBS electrodes were used to target each STN. After a plan was created for trajectories from the frontal burr hole to the STN target, a burr hole was generated centered over the planned entry site. The base of a specially designed burr hole cover used to secure the catheter was then fixed to the skull. The trajectory plan and / or coordinates were confirmed prior to insertion of the cannula guide for each catheter placement. The surgeon read the settings of the stereotactic frame or the plan in a frameless system while an assistant confirmed and documents in the source documents that these matched the plan written prior to the start of surgery. Standard methods for localizing the STN at the site were then performed to confirm the proper location of the center of the STN. Once the dorsal and ventral limits of the STN were mapped along an optimal trajectory, the rigid cannula guide was inserted to a depth of 10 mm above the intended target. The intended target was the middle of the STN (based upon the intraoperative mapping).

[0249] Prior to thawing the study drug vial, the code was confirmed to match the code provided by the sponsor. Prior to infusion, the vial was thawed at room temperature and the AAV-GAD formulation was prepared as specified herein. An excess of 100 pL AAV-GAD was drawn into the infusion syringe under sterile conditions. The neurosurgeon then connected the syringe to the infusion catheter. Prior to inserting the catheter, the neurosurgeon took the sterile syringe filled with the AAV-GAD solution and primed the catheter system with the solution to flush air and confirm flow.

[0250] The catheter was then disconnected from the infusion system and was inserted to a predetermined depth through the cannula guide to place the tip of the infusion catheter within the center of the STN. Confirmation of the location of the catheter tip was obtained using standard procedures for localizing DBS electrodes at the site. The cannula guide was partially withdrawn to expose the catheter, and then the second stage of the locking mechanism (cranial anchor disk) was inserted securely into the cranial anchor base that was previously fixed to the skull. The ancillary surgical supplies, forceps and standard Rhoton dissector was used to close the locking mechanism and secure the catheter. The catheter and the release ripcord for the locking cap were both externalized through the skin flap by using an IV needle and the incision was then sutured. The catheter was then reattached to the infusion system.

[0251] Prior to inserting the contralateral catheter, the surgeon verbally confirmed the coordinates or localization plan for the catheter tip in the contralateral hemisphere and this was recorded in the source documents to document that they completed this prior to insertion of the contralateral cannula guide. The contralateral catheter was then inserted in the same manner as the first catheter. Following completion of second catheter insertion, the infusion commenced,46178092112.1and the frame or fiducials were removed. Confirmation of the location of this second catheter tip was obtained using standard procedures for localizing DBS electrodes at the site.

[0252] In preparation for the infusion, it was ensured that the pump (pump head and controller) was at approximately the same level as the infusion site. It was ensured that the pump was maintained at the same height for the duration of the infusion. To begin infusion, the syringe was placed into the pump that was programmed to infuse either 35 pL or 50 pL of AAV-GAD, depending upon the dose assignment, at a rate of approximately 0.23 pL / min. The infusion lasted about 2.5 hours or 150 minutes for the low dose group. The infusion latest about 3.5 hours or 210 minutes for the high dose group. After completion of AAV-GAD infusion, a non-contrast CT of the head was performed to document the catheter position upon completion of the infusion. The catheter was then removed in its entirety by removing the release ripcord to open the locking mechanism followed by withdrawal of the catheter. A non-contrast CT of the head was performed within 24 hours following removal of the catheter to evaluate any new bleeding or retained catheter fragment.

[0253] Saline administration and sham surgery procedure

[0254] The same surgical procedure described for AAV-GAD administration occurred for the participants randomized to sham surgery; however, the burr holes were of partial-thickness and neither the inner table of the skull nor the dura were penetrated, thereby minimizing the risk of intracranial injury, hemorrhage, or infection. A sham procedure to simulate mapping of the STN was performed per the site’s standard practice, with electrophysiological recordings made from a routine case played as part of a mock mapping procedure for sites using this method to target the STN. The distal ~10 cm of the catheter was cut, unbeknownst to the participant, and the truncated catheter was inserted into the partial-thickness burr hole and fixed in place with the locking burr hole cover in identical fashion to the penetrating catheters for the AAV-GAD groups. The release ripcord and catheter were externalized in the same fashion as for AAV-GAD participants and then saline infusion was performed through catheters inserted into each partial -thickness burr hole (no brain infusion). The volume of saline was 35 pL on each side (combined total of 70 pL). A sham non-contrast CT of the head was performed within 24 hours following removal of the catheter.

[0255] Excluded medications and / or procedures

[0256] The following medications and procedures were prohibited:

[0257] Deep brain stimulation.

[0258] Other brain surgery for PD.47178092112.1

[0259] Other experimental therapy (drug or biologic) within 3 months prior to screening and throughout participation in the study.

[0260] Dopamine receptor-blocking drugs.

[0261] Live virus vaccine within 4 weeks of surgery.

[0262] Immunosuppressive therapy.

[0263] New treatment for PD.

[0264] Adjustments in the dose of dopaminergic medications were allowed when medically indicated.

[0265] Restricted medications and / or procedures

[0266] The following medications and procedures were restricted: Aspirin, other antiplatelet drugs, or anticoagulants from 1 week prior to surgery through 1 week following surgery. Participants receiving anti coagulation therapy had to be able to maintain, with acceptable risk, a normal international normalized ratio (INR) and partial thromboplastin time (PTT) for at least 1 week prior to surgery and 1 week following surgery as determined by the participant’s primary care physician.

[0267] Allowed medications and / or procedures

[0268] Participants continued to receive any medications that they were receiving at study entry for underlying medical conditions. Participants were to be on an unchanged, stable antiParkinsonian drug regimen and on stable doses of medications for any other underlying medical conditions for > or =4 weeks prior to screening.

[0269] Study procedures

[0270] The diagnosis of idiopathic PD were made before any other screening procedure took place. During screening, assessments that are not part of the study entry criteria were performed after the participant has qualified to enroll in the study.

[0271] Participants randomly assigned to immediate treatment with AAV-GAD were scheduled for surgery and complete the full battery of visits through the Week 26 visit.

[0272] Participants randomly assigned to sham surgery followed the same visit schedule during the first 6 months.

[0273] Any medical condition already present at screening was recorded as medical history and not reported as an AE unless the medical condition or signs or symptoms present at screening change in severity, frequency, or seriousness at any time during the study.

[0274] The Week 19 visit was a telephone visit for assessment of AEs, changes in control of Parkinsonism, and other neurological events.48178092112.1

[0275] During visits that include the UPDRS assessment, the MDS-UPDRS Part 3 “medication off’ state assessment was preferably performed before any other assessments or questionnaires. Site staff were to confirm that the last dose of dopaminergic medication(s) was taken by the participant at least 12 hours before performing the MDS-UPDRS Part 3 “medication off’ state assessment.

[0276] For the other assessment tools, the following order was recommended:1. HVLT-R (10 min + 20-25 min delay*)2. Trail Making Test (10 min)3. Col or- word interference (10 min)4. Digit Span Test (5 min)5. *HVLT-R delayed memory (continued from 1, above, after planned delay)6. Verbal fluency test (10 min)7. All other questionnaires

[0277] Additional serum or urine pregnancy tests were performed, as determined necessary by the clinician or required by local regulation, to establish the absence of pregnancy at any time during the participation in the study. Similarly, unscheduled clinical chemistry examinations were permitted as required to manage emerging issues.

[0278] Efficacy tests

[0279] The efficacy tests are described in detail in the detailed description of the disclosure.The following tests were applied:

[0280] 18FDG-PET.

[0281] Questionnaires and Scales: Columbia Suicide severity rating scale, Beck Depression Inventory-II, Beck Anxiety Inventory, Clinical Global Impression-Severity, Clinical Global Impression-Improvement, EuroQol-5 Dimensions-5 Levels Survey, Montreal Cognitive Assessment, Parkinson’s Disease Questionnaire-39 Scale, Parkinson’s Disease Sleep Scale-2, Unified Dyskinesia Rating Scale, Movement Disorder Society -Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) , Revised Hopkins Verbal Learning Test (HVLT-R), Stroop Color and Word Test (Delis-Kaplan Executive Function System (DKEFS) version) , Trail Making Test (TMT) (DKEFS version), Verbal Fluency Test (DKEFS version), Digit Span test (part of Weschler Memory and Weschler scales), and / or Hauser Patient Diary.

[0282] Safety

[0283] Safety was evaluated by vital signs, neurological examinations, physical examinations, ECGs, radiographic imaging (CT and MRI), clinical laboratory evaluations, descriptive analysis of AEs (including incidence, severity, seriousness, and relatedness),49178092112.1immune response, and the Columbia-Suicide Severity Rating Scale over the 6-month duration of the study.

[0284] Adverse events

[0285] Adverse event definitions are provided in Table 4.Table 4. Adverse event definitions, a. Life threatening refers to an event in which the participant is at risk of death at the time of the event; it does not refer to an event that might hypothetically cause death if it was more severe (e.g., a silent myocardial infarction), b. Hospitalization is defined as an inpatient admission, regardless of length of stay. Hospitalization for an elective procedure for a pre-existing condition does not constitute an SAE. c. Medical judgment was exercised in deciding whether an AE or ADR is serious in other situations. Important AEs or ARs that may not be immediately life threatening or result in death or hospitalization but may seriously jeopardize the participant by requiring intervention to prevent one of the other outcomes listed in the table (e.g., a secondary malignancy, an allergic bronchospasm requiring intensive emergency treatment, seizures or blood dyscrasias that do not require hospitalization or development of drug dependency).

[0286] Adverse events included:

[0287] An exacerbation of a pre-existing illness.

[0288] An increase in the frequency or intensity of a pre-existing episodic event or condition.

[0289] A condition (regardless of whether present prior to the start of the study) that is detected after study drug administration (or sham surgery). (This did not include pre-existing conditions recorded as such at screening.).

[0290] Continuous persistent disease or a symptom present at baseline that worsens following administration of study drug (or sham surgery).50178092112.1

[0291] Overdose of study drug.

[0292] Adverse events did not include:

[0293] Medical or surgical procedures: the condition that leads to the procedure was an AE.

[0294] Pre-existing disease or a condition present before treatment that did not worsen.

[0295] Hospitalization where no untoward or unintended response had occurred, e.g., elective cosmetic surgery.

[0296] Seriousness Assessment: When an AE or adverse drug / device reaction occurred, the clinician responsible for the care of the participant first assessed whether the event was serious using the definition given in Table 4.

[0297] Assessment of Severity: The severity of all AEs and / or adverse drug / device reactions (serious and nonserious) in this study were assessed as mild, moderate, or severe.

[0298] Assessment of Causality: For all AEs, the relationship of the AE to the administration of the study drug, the surgical procedure, and the devices was assessed and captured in the study-specific electronic case report form (eCRF). Relation to study drug was assessed according to the definitions in Table 5.Table 5. Adverse event causality definitions. AE = adverse event; SAE = serious adverse event; AR= adverse reaction; SAR = serious adverse reaction.

[0299] Assessment of Expectedness: If there was at least a reasonable possibility of a causal relationship between the event and the study drug, the expectedness of the event was assessed. An unexpected serious adverse reaction (SAR) was one that is not reported in the current investigator brochure, or one that is more frequently reported or more severe than previously reported.51178092112.1

[0300] Clinical Laboratory Abnormalities: The clinical significance of all abnormal laboratory values as defined by the appropriate reference range(s) was assessed. All abnormal values assessed to be of clinical concern and at least possibly related to study drug or of uncertain causality were repeated. Clinically significant abnormal laboratory values occurring during the clinical study were followed until repeat tests return to normal, stabilize, or are no longer clinically significant.

[0301] Clinical safety laboratory evaluations

[0302] See Table 6 for clinical and safety related laboratory tests.Table 6. Clinical and safety related laboratory tests

[0303] Vital Sign Measurements

[0304] Vital sign measurements included oral or tympanic temperature (Celsius), heart rate (beats per minute), respiratory rate (breaths per minute), and blood pressure (mmHg). In addition, height was measured at screening, whereas weight was measured at both screening and Week 26 visits.52178092112.1

[0305] On the day of surgery, temperature, heart rate, respiratory rate, and blood pressure were measured before surgery, approximately every 30 minutes for at least the first 4 hours after start of infusion (this was prolonged in 30-minute intervals if the infusion took longer than 4 hours), and then at approximately 8 and 12 hours after the start of infusion.

[0306] Electrocardiogram

[0307] Standard 12-lead ECGs (ventricular rate, PR interval, QRS duration, and QT interval) were performed with the participant in a supine position having rested in this position for at least 5 minutes before each reading.

[0308] Immune Response

[0309] Immune responses against AAV2 viral capsids and / or the AAV-GAD product were assessed. Blood samples were obtained to evaluate for anti-GAD antibodies and anti-AAV2 antibodies.

[0310] Radiographic Imaging

[0311] Radiographic imaging of the head (CT or MRI) was performed per site standard for assessments pre-, intra-, and post-operatively.

[0312] Due to the retention of the nylon / titanium cranial anchor base above the skull as a burr hole cover following removal of the infusion system, the specific MRI conditions were taken into account for all post-surgical MRI scans.

[0313] Blood Volume

[0314] The total blood volume collected for the study was approximately 58.5 mL.

[0315] Analysis of Efficacy

[0316] Exploratory Efficacy Endpoints: The exploratory efficacy analyses was performed at Weeks 12 and 26. Descriptive statistics were presented for efficacy endpoints. Differences between the individual and pooled (when appropriate) AAV-GAD groups and the sham group in meant change from baseline to Weeks 12 and 26 were compared for each of the endpoints.

[0317] The change from baseline to Week 12 and Week 26 in MDS-UPDRS Part 3 (motor examination) score in the “medication off’ state was analyzed using a mixed model for repeated measures (MMRM). The model included treatment group, visit, and treatment by visit interaction as factors and the baseline MDS-UPDRS Part 3 (motor examination) score in the “medication off’ state as a covariate.

[0318] The change from baseline to Week 26 in GADRP score was analyzed using an analysis of variance model with change from baseline as the response variable, treatment group as a factor, and baseline GADRP score as a covariate.53178092112.1

[0319] Example 2: Results - randomized, double-blind, sham-controlled phase 1 / 2 study

[0320] As shown herein, AAV-GAD was safe and well tolerated, with no SAEs related to AAV-GAD treatment.

[0321] One way of determining efficacy of AAV-GAD therapy is by determining whether treatment results in a reduction in the UPDRS Part 3 (motor examination) score in the “medication off’ state. Part 3 covers the evaluation of motor disability and includes ratings for tremor, slowness (bradykinesia), stiffness (rigidity), and balance. Higher UPDRS scores indicate more severe symptoms. As such, the goal of treatment is the reduction of UPDRS scores.

[0322] The three experimental groups were as shown in Table 7.Table 7. Treatment groups. AAV-GAD = 1 : 1 mixture of AAV-GAD-65 and AAV-GAD-67 vectors.

[0323] Fig. 1 shows the mean change from baseline to Week 26 in the UPDRS Part 3 (motor examination) score in the “medication off’ state. More negative values indicate a larger improvement. Patients treated with the high dose of AAV-GAD therapy showed a larger improvement in their motor score as compared to patients treated with the low dose and the control (sham). A statistically significant 18-point average improvement from baseline in UPDRS Part 3 “medication off’ medication score was demonstrated in the high dose group. A change of 5 to 10 points is considered clinically meaningful.

[0324] Another way of determining efficacy of AAV-GAD therapy is by determining whether treatment results in a reduction in the UPDRS Part 3 (motor examination) score in the “medication on” state. Most PD medications focus on reducing the severity of symptoms in the “off’ state. As such, there is a need for medications that also improve PD symptoms during the “on” state.

[0325] Fig. 2 shows the mean change from baseline to Week 26 in the UPDRS Part 3 (motor examination) score in the “on” state. More negative values indicate a larger improvement. No improvement of symptoms was seen for the sham group. Further, patients treated with the high dose of AAV-GAD therapy (1: 1 mixture of AAV-GAD-65 and AAV-GAD-67 vectors)54178092112.1showed a larger improvement in their motor score as compared to patients treated with the low dose.

[0326] A third way of determining efficacy of AAV-GAD therapy is by determining whether treatment results in a reduction in the PDQ-39 Quality of Life score, a key patient- reported quality of life measure in PD. A higher score on the PDQ-39 scale indicates more severe PD symptoms and a lower quality of life. As such, the goal of treatment is the reduction of PDQ-39 scores.

[0327] Fig. 3. shows the mean change from baseline to Week 26 in the PDQ-39 score. Patients treated with the high dose of AAV-GAD therapy showed a larger improvement in their PDQ-39 score as compared to patients treated with the low dose and the control (sham). In the high dose AAV-GAD group, the PDQ-39 score improved by 8 points from baseline. A 2 to 4- point change is considered clinically meaningful. A dose response in PDQ-39 score was observed, with 100% of participants in the high dose group, 60% of participants in the low dose group, and 25% of participants in the sham surgery group reporting an improvement.

[0328] In sum, treatment with the high dose of AAV-GAD therapy (1 : 1 mixture of AAV- GAD-65 and AAV-GAD-67 vectors) resulted in enhanced improvements in PD symptoms and quality of life as compared to treatment with the low dose of AAV-GAD therapy or no treatment. The data herein demonstrate the impact of using targeted local delivery of genebased therapy to correct the aberrant circuitry that results from the depletion of dopamine in the brain of idiopathic PD patients as the disease progresses. AAV-GAD treatment is designed to normalize circuit function in all forms of PD with its potential benefit not limited to any single type of PD.55178092112.1

Claims

162027.49676CLAIMSWe claim:

1. A method of treating or preventing Parkinson’s disease (PD) or treating or preventing symptoms associated with PD in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding glutamic acid decarboxylase (GAD), wherein the pharmaceutical composition is delivered bilaterally into each of the two subthalamic nuclei (STN) of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

2. A method of treating or preventing a neurodegenerative disease or disorder associated with y-aminobutyric acid (GABA) deficiency or treating or preventing symptoms associated with said neurodegenerative disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising vectors comprising a nucleic acid encoding GAD, wherein the pharmaceutical composition is delivered bilaterally into each of the two STN of the subject, and wherein the pharmaceutical composition is administered at a total dose of at least about 2 x io11viral genomes (vg).

3. The method of claim 1 or 2, wherein the pharmaceutical composition is administered at a total dose of at least about 2.09 x io11vg.

4. The method of claim 3, wherein the pharmaceutical composition is administered at a total dose of about 2.09 x io11vg.

5. The method of any one of claims 1-4, wherein the pharmaceutical composition is administered at a dose of at least about 1 x io11vg / STN.

6. The method of claim 5, wherein the pharmaceutical composition is administered at a dose of at least about 1.045 x io11vg / STN.

7. The method of claim 6, wherein the pharmaceutical composition is administered at a dose of about 1.045 x 1011vg / STN.

8. The method of any one of claims 1-7, wherein the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-65.

9. The method of claim 8, wherein the GAD-65 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

10. The method of claim 9, wherein the GAD-65 comprises SEQ ID NO: 1.

11. The method of any one of claims 1-10, wherein the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-67.

12. The method of claim 11, wherein the GAD-67 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 3 or 4.

13. The method of claim 12, wherein the GAD-67 comprises SEQ ID NOs: 3 or 4.

14. The method of any one of claims 1-7, wherein the vectors comprising the nucleic acid encoding GAD comprise a nucleic acid encoding GAD-65 and a nucleic acid encoding GAD-67, wherein the GAD-65 comprises SEQ ID NO: 1 and the GAD-67 comprises SEQ ID NO: 3.

15. The method of any one of claims 11-14, wherein the pharmaceutical composition comprises vectors comprising a nucleic acid encoding GAD-65 and vectors comprising a nucleic acid encoding GAD-67 at a ratio of about 1 : 1 (ratio of viral genomes).

16. The method of any one of claims 1-15, wherein the vectors comprising the nucleic acid encoding GAD are viral vectors.

17. The method of claim 16, wherein the viral vectors are adeno-associated virus (AAV) vectors.

18. The method of claim 17, wherein the AAV vectors comprise a viral genome derived from an AAV-2 serotype.

19. The method of claim 17 or 18, wherein the AAV vectors comprise capsid proteins derived from an AAV-2 serotype.

20. The method of any one of claims 1-19, wherein the subject is a human.

21. The method of any one of claims 1-20, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s Movement Disorder Society- Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part 3 score during “medication off’ time by at least 2 points as compared to a control.

22. The method of claim 21, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 5 points as compared to a control.

23. The method of claim 22, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 10 points as compared to a control.57178092112.

124. The method of claim 23, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication off’ time by at least 15 points as compared to a control.

25. The method of any one of claims 1-24, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 score during “medication on” time by at least 2 points as compared to a control.

26. The method of claim 25, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication on” time by at least 5 points as compared to a control.

27. The method of claim 26, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s MDS-UPDRS Part 3 during “medication on” time by at least 10 points as compared to a control.

28. The method of any one of claims 1-27, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s Parkinson’s Disease Questionnaire (PDQj-39 score by at least 2 points as compared to a control.

29. The method of claim 28, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s PDQ-39 score by at least 5 points as compared to a control.

30. The method of claim 29, wherein the administration of the pharmaceutical composition results in an improvement of the subject’s PDQ-39 score by at least 8 points as compared to a control.58178092112.1

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