Methods for treating parkinson's disease

Abstract

To provide methods and compositions for treating Parkinson's disease (PD) and other neurodegenerative disorders.SOLUTION: Disclosed are methods for treating neurological disorders such as Parkinson's disease (PD) using glutamic acid decarboxylase (GAD) and identifying PD patients that will be most receptive to the method of treating PD. In one aspect, the disclosure provides a method of treating PD in a subject in need thereof, the method comprising: (a) identifying a subject having less than about 10 hours, and preferably less than about 8 hours, of on-time per day; and (b) administering to the subject a composition comprising a therapeutically effective amount of one or more vectors to the subthalamic nucleus of the patient, where each vector comprises a nucleic acid sequence encoding glutamic acid decarboxylase (GAD) and the subject's on-time is increased.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 947,418, filed December 12, 2019, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to methods and compositions for treating Parkinson's disease (PD) and other neurodegenerative disorders. More particularly, described herein are methods that include administering to a subject one or more vectors containing nucleic acid sequences encoding isoforms of glutamic acid decarboxylase (GAD), and identifying a target patient population that may be most receptive to a treatment method for PD. [Background technology]

[0003] Disorders of the central nervous system, particularly those involving the dopamine neurotransmitter, affect millions of people worldwide each year. More than 10 million people worldwide and nearly 1 million patients in the United States live with Parkinson's disease (PD), one of the most common central nervous system disorders.

[0004] PD is a multifactorial disease involving both genetic and non-genetic factors. Some mechanisms that may contribute to the development of PD include the accumulation of misfolded protein aggregates, impaired protein clearance pathways, mitochondrial damage, oxidative stress, excitotoxicity, neuroinflammation, and genetic mutations. PD affects the basal ganglia and the substantia nigra, a neuronal region of the brain. The substantia nigra is a structure of the basal ganglia located in the midbrain and plays an important role in reward and movement. This region is primarily composed of dopaminergic neurons (DA), which produce the neurotransmitter dopamine. In the brain, dopamine functions as an inhibitory neurotransmitter, controlling the excitability of neurons involved in the control of balance and body movement. In a normal brain, DA neurons cross synapses and release dopamine, which matches receptors on receiving cells. The cells are stimulated to transmit a message. After the message is transmitted, the receptor releases dopamine molecules into the synapse, where excess dopamine is "taken up" or recycled within the releasing neuron.

[0005] Gamma-aminobutyric acid, or γ-aminobutyric acid (GABA), is the major inhibitory neurotransmitter in the developmentally mature mammalian central nervous system. Most nigral DA neurons express both gamma-aminobutyric acid (A) (GABA(A)) and gamma-aminobutyric acid (B) (GABA(B)) receptors. DA neurons regulate monosynaptic GABA output in the substantia nigra. Dopamine recycling is regulated by the gamma-aminobutyric acid (GABA) pathway. It is well known in the art that activation of the GABA pathway causes an increase in dopamine, which in turn reduces neuronal firing rate. The firing pattern of dopaminergic neurons is also effectively regulated by GABAergic input in vivo. The unavailability of GABA(A) receptors causes dopaminergic neurons to shift to a burst firing pattern, regardless of their original firing pattern, which spontaneously increases their firing rate.

[0006] For reasons that are not yet fully understood, dopamine-producing neurons in the substantia nigra begin to die in PD patients, causing dopamine deficiency in polysynaptic neurons and a loss of signaling through dopamine receptors. Additionally, the loss of DA neurons causes a reduction in gamma-aminobutyric acid (GABA) inhibitory input to the subthalamic nucleus (STN), leading to increased activity in the STN. The STN then signals other cells in the basal ganglia for increased activity. When GABA levels fall below a certain threshold, dopamine depletion occurs in the brain. The loss of dopamine alters the activity of neurons in the basal ganglia, causing uncontrolled firing of neurons. When dopamine levels fall below a certain point (approximately 80% decrease), PD symptoms, such as uncontrolled muscle movements and tremors, begin to occur.

[0007] PD is characterized by progressive deterioration of muscle movements, poor balance and coordination, and uncontrollable tremors. Standard treatment for PD involves oral administration of the dopamine precursor L-3,4-dihydroxyphenylalanine (levodopa or L-dopa), which eliminates PD-related symptoms but does not ultimately prevent the degeneration of dopaminergic cells. Therefore, currently used PD treatments only reduce PD symptoms without slowing or halting disease progression.

[0008] Administration of L-dopa allows PD patients to experience "on-time," a period during which PD symptoms are adequately controlled. When the effects of L-dopa wear off, PD symptoms recur. This period is called off-time. Measurement of on-time and off-time is usually calculated by asking patients to complete a medication diary. In this diary, patients record the time it takes for L-dopa to work and wear off. In the early stages of PD, patients' on-time is approximately 16 hours with L-dopa administration. However, as the disease progresses, the amount of on-time gradually decreases, even with larger doses of the drug. In addition, side effects associated with long-term administration of L-dopa can be quite severe and include mental changes such as depression, hallucinations, mania, delusions, agitation, and excessive sleepiness. Administration of L-dopa can also have adverse effects in patients with cardiovascular or pulmonary disease, renal disease, liver disease, or endocrine disease. Some of the side effects associated with chronic L-dopa administration can be mitigated by coadministration of N-amino-α-methyl-3-hydroxy-L-tyrosine monohydrate, an inhibitor of aromatic amino acid decarboxylase (AADC), the enzyme that decarboxylates L-dopa to dopamine. However, this drug combination can still cause nausea, dyskinesia, psychosis, and hypotension.

[0009] One of the major obstacles to treating PD with small-molecule drugs is the inability of most systemically administered drugs to cross the blood-brain barrier. One approach focuses on increasing the lipid content of polypeptides to facilitate their transport across the blood-brain barrier. Another approach focuses on enhancing the permeability of capillaries in the brain. However, neither of these approaches has resolved the blood-brain barrier crossing problem. Other approaches to treating PD, such as transplantation of engineered cells that produce dopamine into the brain and deep brain stimulation, are characterized by severe side effects, including high mortality rates, increased likelihood of serious infections, and potential brain damage. More importantly, none of the available drugs address the underlying cause of PD or provide PD patients with increased on-time without severe side effects.

[0010] Given the current limitations in the treatment of PD, there remains an unmet need for (1) nontransient treatments for PD, particularly in PD patients who do not respond to current standard treatments, (2) nontransient treatments that effectively extend on-time in PD patients without causing significant side effects, and (3) reliable methods for identifying patient populations that may be most receptive to nontransient treatments for PD. Summary of the Invention

[0011] In one aspect, the disclosure provides a method of treating PD in a subject in need thereof, the method comprising: (a) identifying a subject having an on-time of less than about 10 hours per day, preferably less than about 8 hours per day; and (b) administering to the subject a composition comprising a therapeutically effective amount of one or more vectors to the patient's subthalamic nucleus, wherein each vector comprises a nucleic acid sequence encoding glutamic acid decarboxylase (GAD), and the subject's on-time is increased. In one embodiment, the one or more vectors are introduced bilaterally into the patient's subthalamic nucleus.

[0012] In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein the subject has an on-time of less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, or less than 4 hours per day prior to treatment.

[0013] In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein one or more vectors comprise a nucleic acid sequence encoding GAD-65 and / or a nucleic acid sequence encoding GAD-67. In an embodiment, two vectors are administered, one vector comprising a nucleic acid encoding GAD-65 and the other vector comprising a nucleic acid encoding GAD-67. In a further embodiment, the vector comprising a nucleic acid encoding GAD-65 and the vector comprising a nucleic acid encoding GAD-67 are administered in a ratio of about 1:2 to about 2:1, preferably about 1:1.

[0014] In one embodiment, the present disclosure provides a method of treating PD in a subject in need thereof, wherein one or more vectors used in the method comprise a nucleic acid sequence encoding GAD-65 and a nucleic acid sequence encoding GAD-67. In one embodiment, the amino acid sequence of human GAD-65 is provided as SEQ ID NO: 1 (Genbank Accession Nos. NM000818; M81882). In another embodiment, the amino acid sequence of human GAD-65 is provided as SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encodes a protein 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 the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In embodiments, the nucleic acid sequence encoding GAD-65 comprises SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the nucleic acid sequence encoding GAD-65 comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In one embodiment, the amino acid sequence of human GAD-67 is provided as SEQ ID NO:5 (Accession No. M81883). In another embodiment, the amino acid sequence of human GAD-67 is provided as SEQ ID NO:7. In some embodiments, the nucleic acid sequence encodes a protein 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 the sequence of SEQ ID NO:5 or SEQ ID NO:7. In embodiments, the nucleic acid sequence encoding GAD-67 comprises SEQ ID NO:6. In embodiments, the nucleic acid sequence encoding GAD-67 comprises SEQ ID NO:8. In embodiments, the nucleic acid sequence encoding GAD-67 comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0015] In one embodiment, the present disclosure provides a method of treating PD in a subject in need thereof, wherein one or more vectors used in the method are viral vectors. In one embodiment, the present disclosure provides a method of treating PD in a subject in need thereof, wherein the disclosed viral vector is an adeno-associated viral (AAV) vector.

[0016] In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein the composition comprises at least 1x10 11 In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein the composition comprises at least 3 x 10 vector genomes / ml. 11 In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein the composition comprises at least 1 x 10 vector genomes / ml. 12 Contains vector genomes / ml.

[0017] In one embodiment, the disclosure provides a method of treating PD in a subject in need thereof, wherein the subject exhibits an average increase in on-time of at least 20%, at least 30%, or at least 40% at 12 months after treatment compared to before treatment. In embodiments, the subject has an increase in on-time of about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours at 12 months after treatment. [Brief explanation of the drawings]

[0018] [Figure 1]Increase in "on" time after AAV-GAD gene therapy. Subjects who received either AAV-GAD (GAD) or sham surgery (sham) to the STN recorded hourly on and off times in a daily diary for 2 weeks at each listed time point (1, 3, 6, and 12 months after surgery). Average daily on and off times determined from the diary over the 2-week period were compared with baseline diaries obtained within 30 days before surgery to determine changes in on-time hours after surgery. Analysis of variance revealed an overall difference between groups over the entire 12-month study period (ANOVA; p = 0.44). Two-tailed t-tests demonstrated significant increases in on time at 3 and 12 months after surgery (p < 0.05), with a strong trend toward significance at 1 month. The average increase in on time ranged from 1 to 2.5 hours across the study in the AAV-GAD group, with minimal or no change in the sham group at any time point. [Figure 2] Correlation between baseline on-time and change in on-time after AAV-GAD gene therapy. At 12 months after surgery, the change in average daily on-time from baseline for each subject in the AAV-GAD treatment group was correlated with the change in clinical score for the same subject over the same period. The clinical score used was Part 3 of the Unified Parkinson's Disease Rating Scale (UPDRS), the gold standard assessment of motor function for Parkinson's disease patients. This shows a strong correlation between baseline on-time and on-time increase at 12 months after subthalamic AAV-GAD gene therapy (r = -0.59), indicating that fewer hours of on-time at baseline (more severe patients) predicted a greater increase in on-time hours after treatment (greater response). Subjects with low baseline on-time showed an increase in on-time after treatment. Patients with on-time less than 8 hours had the greatest response to AAV-GAD therapy, with a mean increase of 3.5 hours per day in on-time. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure provides methods for treating PD in a subject in need thereof, as well as methods for identifying PD patients who would be most receptive to such methods. In one aspect, a method is provided for identifying a subject having an on-time of less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, or less than 4 hours per day, and treating PD by administering a therapeutically effective amount of GAD (e.g., by expressing GAD-65 and GAD-67) to the patient's subthalamic nucleus to increase on-time.

[0020] Patient selection In one aspect, the present disclosure provides methods for treating patients who can most benefit from the treatments provided herein. In some embodiments, the patient has about 10 hours or less of on-time per day. In some embodiments, the patient has about 9 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, or about 5 hours or less of on-time per day. At the onset of the disease, typically within the first 3-5 years of diagnosis, a single dose of L-dopa allows the patient to have about 16 hours of on-time. As the disease progresses, the duration of on-time gradually decreases, even with increasing L-dopa drug dosage. In the intermediate stage of PD, typically about 5-10 years after diagnosis, 50-70% of patients suffer from L-dopa-induced motor complications. At this stage, the benefits of L-dopa wear off at about 4 hours or less, and the patient begins to fluctuate between on-time and off-time motor responses. As the disease progresses, the duration of response after a single dose of L-dopa gradually shortens, eventually reflecting the plasma half-life of L-dopa (approximately 60-90 minutes) in advanced PD patients (typically more than 10 years after diagnosis). In some cases, patients may fail to respond to a given dose of L-dopa. The most challenging clinical situation is described as a true "on-off" or yo-yo phenomenon, in which patients rapidly fluctuate between on-time and off-time states that appear unrelated to L-dopa dose. Dyskinesias and off-time periods are severe at this stage, and they can be difficult or impossible to satisfactorily control with available therapies. In addition, intermediate- and advanced-stage patients suffer from adverse side effects due to increased L-dopa drug use.

[0021] The standard treatment for patients with stage 3 or higher PD is a combination of L-dopa and an AADC inhibitor. This therapy temporarily relieves PD symptoms, allowing patients to regain control of their functions. As used herein, the period during which a PD patient experiences relief from PD symptoms is referred to as "on-time." PD symptoms relieved during the on-time period include, but are not limited to, loss of motor control, pain, slurred speech, tremor, and balance problems. As the effects of the medication wear off, tremor and other PD symptoms return. As used herein, the period during which a patient experiences a recurrence of PD symptoms due to wear-off of the medication is referred to as "off-time."

[0022] Measurement of on-time and off-time can be determined by a medication diary kept by the patient. The patient records the time when they feel the medication is working, their motor function is under control, when they feel the medication is wearing off, and when a relapse of PD symptoms occurs. Clinicians use the timing recorded in the diary to determine the PD patient's on-time and off-time at a specific point in time. On-time and off-time can also be measured using wearable devices, including, but not limited to, motion sensors, accelerometers, and posture detection devices, for example, by measuring and tracking hypokinetic and hyperkinetic characteristics associated with PD.

[0023] In embodiments, the patient has had PD for at least three years before receiving a therapy described herein. In some embodiments, the patient has had PD for at least four years, or at least five years. In embodiments, the patient receiving a therapy described herein is in stage 2, stage 3, stage 4, or stage 5 of the disease. PD progression can be divided into five stages. Stage 1 of PD is characterized by impairment of facial expression, speech, and / or locomotor activity. Symptoms initially affect only one side of the body (unilateral involvement) and usually result in minimal or no functional impairment. During stage 2 of PD, both hemispheres of the brain are affected by the disease. As a result, tremors gradually become bilateral and may affect the patient's midline. Additional symptoms of stage 2 PD can include loss of facial expression on both sides, decreased eye blinking, speech abnormalities, soft, monotonous voice, slurred speech, stiffness or rigidity of the trunk muscles that may result in neck or back pain, hunched posture, and a general slowing of activities of daily living. Stage 3 is characterized by loss of balance and slowed movements. Balance is impaired by an inability to make the rapid, automatic, and involuntary adjustments needed to prevent falls, and falls are common at this stage. Patients in stage 4 have severe, limiting symptoms. Patients may be able to stand unassisted but may require a walker for movement. Stage 5 is the most advanced and debilitating stage of PD. Leg stiffness may make standing or walking impossible. Patients require a wheelchair or are bedridden. At this stage of PD, patients require 24-hour care for all activities.

[0024] In embodiments, the patient or patients selected for the treatments disclosed herein have a UPDRS Part III score of about 20 or greater, about 30 or greater, or about 40 or greater while on medication, and / or have motor complications caused by the administration of L-dopa. PD stage can be measured using the Unified Parkinson's Disease Rating Scale (UPDRS) (see, e.g., Metman et al., Mov. Disord. 19:1079-1084 (2004)). The UPDRS scale includes a series of assessments of typical PD symptoms. The scale consists of four parts: Part I assesses behavioral problems such as intellectual decline, hallucinations, and depression; Part II assesses the patient's perception of their ability to perform activities of daily living, including dressing, walking, and eating; Part III encompasses the assessment of motor impairments, including assessment of tremor, slowness (bradykinesia), stiffness (rigidity), and balance; and Part IV encompasses several treatment complications, including assessment of involuntary movements (dyskinesia), painful spasms (dystonia), and irregular drug responses (motor fluctuations). Part III, or the motor examination, is scored by a clinician through a structured neurological examination. It consists of 14 items that can be scored from 0 (normal) to 4 (severe). The scores are added to give an overall score of involuntary movements. Clinicians use the scores obtained from the assessment of the items listed in Part III to determine the severity of PD and appropriate treatment.

[0025] How to treat PD In embodiments, the present disclosure provides methods for treating, preventing, and / or reducing the severity or extent of PD by administering to a subject in need thereof a therapeutically effective amount of a composition or compositions comprising one or more vectors comprising a nucleic acid encoding GAD-65 and / or a vector comprising a nucleic acid encoding GAD-67. In embodiments, a method for treating a PD patient comprises administering to a patient in need thereof one or more compositions, wherein the one or more compositions comprise a vector or vectors for expression of GAD-65 and / or a vector for expression of GAD-67.

[0026] As used herein, "treating," "treat," or "treatment" refers to delaying, alleviating, ameliorating, or reducing at least one symptom of a disease or disorder, or reversing one or more symptoms after the onset of a disease or disorder. The purpose of treatment is to prevent, reduce, or arrest an undesirable physical condition, disorder, or disease, or to obtain a beneficial clinical result.

[0027] The terms "prevent," "prevention," and the like refer to acting before the onset of an obvious disease or disorder to prevent the onset of the disease or disorder, or to minimize the extent of the disease or disorder, or to delay the course of its development.

[0028] "Cure" and like terms mean to cure, get well, or restore health, or allow a period of time without recurrence of disease, such that the risk of recurrence is small.

[0029] The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause a clinically significant improvement in a subject's condition, or to delay or minimize or alleviate one or more symptoms associated with a disease or disorder, or to effect a desired beneficial change in physiology in a subject.

[0030] In embodiments, subjects undergoing a therapy described herein (e.g., receiving a composition comprising a therapeutically effective amount of one or more vectors comprising a nucleic acid encoding GAD-65 and / or a vector comprising a nucleic acid encoding GAD-67) have an average increase in on-time of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, or about 4 hours at 12 months after receiving the therapy. In embodiments, subjects have an increase in on-time of about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% at 12 months after receiving the therapy.

[0031] Standard treatment for PD involves administering L-dopa, a dopamine precursor, to replenish dopamine. However, increasing GABA levels also allows higher concentrations of GABA to restore or increase dopamine levels, thereby stopping these uncontrolled movements. In embodiments, the present disclosure provides methods for increasing GABA levels in the subthalamic nucleus by administering one or more vectors containing glutamic acid decarboxylase (GAD) isoforms. GABA is produced in the brain by GAD, which catalyzes the decarboxylation of glutamate to GABA and CO2. The mammalian brain expresses two distinct isoforms of GAD, GAD-65 and GAD-67, named for their respective molecular weights of 65 and 67 kDa. These isoforms combine to provide a dual system for regulating neuronal GABA levels. In humans, the genes for GAD-67 and GAD-65 are located on different chromosomes (GAD1 and GAD2 genes are located on chromosomes 4 and 10, respectively). GAD-65 and GAD-67 show significant differences in their expression levels in different brain regions. While GAD-67 is uniformly expressed throughout the brain, GAD-65 expression is primarily concentrated in axon terminals. These two enzymes together maintain the majority of the physiological supply of GABA in mammals. Human GAD-65 cDNA encodes a polypeptide of 585 amino acid residues (GenBank accession numbers NM000818; M81882). In one embodiment, the amino acid sequence of human GAD-65 is provided as SEQ ID NO: 1. In another embodiment, the amino acid sequence of human GAD-65 is provided as SEQ ID NO: 3. Human GAD-67 encodes a polypeptide of 594 amino acid residues (GenBank accession number M81883). In one embodiment, the amino acid sequence of human GAD-67 is provided as SEQ ID NO: 5 (GenBank accession number M81883). In another embodiment, the amino acid sequence of human GAD-67 is provided as SEQ ID NO: 7.

[0032] In embodiments, the vector comprises a nucleic acid sequence encoding a GAD isoform. In one embodiment, the nucleic acid sequence encodes GAD-65. In one embodiment, the nucleic acid sequence encodes GAD-67. In embodiments, the vector comprises a nucleic acid sequence encoding GAD-65 and a nucleic acid sequence encoding GAD-67. In embodiments, the nucleic acid sequence encoding GAD-65 comprises a sequence encoding the protein of SEQ ID NO: 1. In other embodiments, the nucleic acid sequence encoding GAD-65 comprises a sequence encoding the protein of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encodes a protein 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 the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In one embodiment, the nucleic acid sequence encoding GAD-65 comprises SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the nucleic acid sequence encoding GAD-65 comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0033] In embodiments, the nucleic acid sequence encoding GAD-67 comprises a sequence encoding the protein of SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, the nucleic acid sequence encodes a protein 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 the sequence of SEQ ID NO:5 or SEQ ID NO:7. In embodiments, the nucleic acid sequence encoding GAD-67 comprises SEQ ID NO:6. In embodiments, the nucleic acid sequence encoding GAD-67 comprises SEQ ID NO:8. In embodiments, the nucleic acid sequence encoding GAD-67 comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0034] As used herein, the term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. For example, if a position in the compared nucleotide sequence is occupied by the same base, the molecules are identical at that position. The degree of 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 and polynucleotides encoding such polypeptides that are at least 85%, 90%, 95%, 98%, or 99% identical to the specific polypeptides described herein and preferably exhibit substantially the same function 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 ALIGN programs (version 2.0). The well-known Smith-Waterman algorithm can also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources. In comparing sequences, these methods take into account various substitutions, deletions, and other modifications.

[0035] In some embodiments, the GAD-65 encoding sequence and / or the GAD-67 encoding sequence are codon optimized.

[0036] The present disclosure provides methods for treating, preventing, and / or reducing the severity or degree of PD symptoms by administering to a subject in need thereof a therapeutically effective amount of a composition comprising a first vector comprising a nucleic acid encoding GAD-65 and a second vector comprising a nucleic acid encoding GAD-67. In embodiments, the ratio of the two vectors in the composition (one encoding GAD-65 and the other encoding GAD-67) is about 2:1 to about 1:2. For example, when the vector is an AAV vector, the ratio of viral particles comprising a nucleic acid encoding GAD-65 to viral particles comprising a nucleic acid encoding GAD-67 is about 2:1 to about 1:2, particularly about 1:1.

[0037] In some embodiments, the method comprises administering a first composition comprising a vector comprising a therapeutically effective amount of a nucleic acid encoding GAD-65 and a second composition comprising a vector comprising a therapeutically effective amount of a nucleic acid encoding GAD-67. In some embodiments, the first AAV vector and the second AAV vector are administered simultaneously. In some embodiments, the first AAV vector is administered before the second AAV vector. In some embodiments, the second AAV vector is administered before the third AAV vector.

[0038] In embodiments, provided herein are methods for treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof, wherein the neurodegenerative disease or disorder is mediated by GABA deficiency. In one aspect, the present disclosure provides a method for treating a disease or disorder of the central nervous system associated with dopaminergic hypoactivity, disease, injury, or chemical damage. In one embodiment, the neurodegenerative disease or disorder is a cognitive disorder. In one embodiment, the neurodegenerative disease or disorder is PD.

[0039] vector In one aspect, a method for treating PD in a subject in need thereof is provided, the method comprising administering to the subject one or more vectors comprising a nucleic acid sequence encoding GAD. As used herein, a vector is a vehicle for delivering genetic material into cells. In an embodiment, the vector is a nucleic acid, including but not limited to a plasmid, an episome, an RNA molecule, or a DNA molecule. In an embodiment, the nucleic acid is circular. In an embodiment, the nucleic acid is linear. In an embodiment, the vector is a viral vector.

[0040] Vectors useful in the methods and compositions disclosed herein include vectors capable of autonomous replication (episomal vectors) and / or vectors designed for gene expression in cells (expression vectors). In certain embodiments, the vectors described herein are expression vectors. Expression vectors allow for the expression of nucleic acids in target cells. Expression vectors can contain both prokaryotic sequences that allow the vector to propagate in bacteria and eukaryotic sequences that facilitate the expression of encoded polypeptides in eukaryotic cells. The various methods utilized in the preparation of plasmids and transformation of host organisms are known in the art.

[0041] In some embodiments, the GAD-expressing vector can be delivered via ex vivo gene therapy, replacing lost cells with transplanted cells that express GAD. An advantage of using such cells is the reduced likelihood of immune resistance, since the patient's own cells can be used in the autologous transplant procedure.

[0042] In some embodiments, the vectors can be delivered using non-viral delivery systems, such as colloidal dispersion systems, e.g., macromolecule complexes, nanocapsules, microparticles, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0043] In certain embodiments, the vector described herein is a viral vector.Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, AAVs), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses, such as picornaviruses, alphaviruses, adenoviruses, double-stranded DNA viruses, including herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox).Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses may include avian leukosis-sarcoma, mammalian type C, type B, type D viruses, HTLV-BLV complex, lentiviruses, and spumaviruses.

[0044] In embodiments, the vector containing the nucleic acid encoding GAD is a viral vector used in gene therapy. The GAD transgene can be incorporated into any type of viral vector used in gene therapy, such as recombinant retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus-1.

[0045] In one embodiment, recombinant AAV (rAAV) is the vector(s) used for GAD transgene delivery. AAV particles contain a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. AAV cannot replicate without a helper virus, which can be adenovirus, vaccinia, or herpesvirus. In the absence of a helper virus, AAV assumes a latent state and inserts its genome into host cell chromosomes. Subsequent infection with a helper virus rescues the latent integrated copy, which then replicates to produce infectious viral progeny.

[0046] Recombinant AAV (rAAV) vectors contain recombinant viral genomes and capsid proteins. rAAV genomes containing GAD transgene(s) can be assembled from polynucleotides encoding the transgene(s), appropriate regulatory elements, and viral elements necessary for packaging the rAAV genome. General methods for constructing rAAV genomes are readily known in the art. AAV-based expression vectors can be constructed with AAV inverted terminal repeats (ITRs) flanked by restriction sites that can be used for subcloning transgenes, either directly using available restriction sites or by excising the transgene with a restriction enzyme, followed by polishing the ends and ligating, optionally with a linker, into the AAV expression vector. The GAD transgene can be incorporated into an AAV-based expression vector along with one or more expression control elements, including, for example, an enhancer, promoter, and / or posttranscriptional regulatory sequences (PREs) flanking the AAV ITRs.

[0047] Methods for generating rAAV vectors with specific capsid proteins are known in the art. Viral particles can be generated by providing the components necessary for packaging the rAAV genome into capsids in trans, or the necessary components can be provided by engineered host cells. Both methods use standard molecular biology techniques known to those skilled in the art. Some or all of the necessary elements can be under the control of either an inducible or constitutive promoter. The recombinant AAV genome, rep sequence, cap sequence, and helper functions for producing rAAV can be delivered to packaging host cells using any appropriate genetic elements (vectors). Typically, recombinant AAV is produced by transfecting host cells with the recombinant AAV genome (including the transgene) packaged into AAV particles, AAV helper function vectors, and accessory function vectors. The AAV helper function vectors encode AAV helper function sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Accessory function vectors typically encode nucleotide sequences for non-AAV-derived viral and / or cellular functions required for AAV replication, including, but not limited to, elements involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and AAV capsid assembly.

[0048] As used herein, terms such as "AAV1," "AAV2," "AAV3," and "AAV4" refer to AAV vectors that contain inverted terminal repeats (ITRs) from AAV1, AAV2, AAV3, or AAV4, respectively, and capsid proteins from AAV1, AAV2, AAV3, or AAV4, respectively. Terms such as "AAV2 / 1," "AAV2 / 8," and "AAV2 / 9" refer to pseudotyped AAV vectors that contain ITRs from AAV2 and capsid proteins from AAV1, AAV8, or AAV9, respectively.

[0049] The AAV vectors described herein generally comprise a rAAV genome encoding one or more GAD transgenes operably linked to one or more regulatory elements in a manner that allows transcription, translation, and / or expression of the transgene in a target cell or tissue, and that is flanked by 5' and 3' ITRs. The ITR sequences are typically about 145 bp in length. AAV ITR sequences can be modified, for example, by inserting, deleting, or substituting one or more nucleotides using standard molecular biology techniques, provided that the modification of the ITR sequence does not interfere with AAV vector function (e.g., efficient encapsidation of the rAAV genome). AAV ITRs can be derived from any of several AAV serotypes. The AAV ITR sequences at the 3' and 5' ends can be the same or can be derived from different AAV serotypes.

[0050] The expression control element or regulatory element operably linked to the transgene can include a promoter or enhancer, such as the chicken beta-actin promoter or cytomegalovirus enhancer, among others described herein. The recombinant AAV genome is generally encapsidated by capsid proteins (e.g., from the same AAV serotype from which the ITRs are derived or from an AAV serotype different from that from which the ITRs are derived). In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence, encoding GAD-65 or GAD-67. Exemplary AAV vector components that can be used in conjunction with the compositions and methods of the present disclosure are described herein.

[0051] Any suitable AAV serotype or combination of AAV serotypes can be used in the methods and compositions of the present disclosure. Because the methods and compositions of the present disclosure are for the treatment and cure of neurodegenerative diseases or disorders, AAV serotypes that target at least the central nervous system can be used in some embodiments, including, but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

[0052] In addition, the rAAV genome contains expression control elements operably linked to the GAD transgene(s) in a manner that allows for its transcription, translation, and / or expression in cells infected with the virus. As used herein, "operably linked" refers to the relationship between two or more nucleic acid sequences in which a particular nucleic acid sequence (e.g., a control element) affects a characteristic of another nucleotide sequence (e.g., affects expression of a transgene). Operatively linked sequences include both expression control elements contained in or adjacent to the GAD transgene and expression control elements that act in trans or at a distance to control transgene expression. As used herein, expression control elements include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and, if desired, sequences that improve secretion of the encoded product. For example, as used herein, a nucleic acid sequence (e.g., a GAD coding sequence) and a regulatory sequence are considered to be operably linked if they are covalently linked in such a manner that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. In one embodiment, human GAD-65 and / or GAD-67 are under the control of a cytomegalovirus enhancer-chicken β-actin promoter and a woodchuck post-transcriptional regulatory element.

[0053] The promoter operably linked to the GAD transgene can be either inducible or constitutive. Inducible promoters allow for the control of gene expression and can be exogenously controlled by conditions or compounds. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone-insect, and the tetracycline-repressible system. A constitutive promoter is an unregulated promoter that allows continuous transcription of its associated gene. Examples of constitutive promoters include, but are not limited to, the chicken beta-actin promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, and the β-actin promoter.

[0054] In embodiments, the native promoter for the GAD transgene, or a fragment thereof, may be used when transgene expression that mimics native expression is preferred. In another embodiment, a tissue-specific promoter is used to enable expression in a specific tissue for targeted gene therapy. Tissue-specific promoters, such as neuron-specific and glial-specific promoters, allow proteins to be expressed in a specific tissue of interest. In one embodiment, the promoter is tissue-specific and essentially active only in the central nervous system or has higher activity in the central nervous system. In one embodiment, the promoter may be specific to a particular cell type or neuron. In another embodiment, the promoter is specific to cells located in a specific region of the brain, such as the cortex, subthalamic nucleus, stranium, substantia nigra, and / or hippocampus.

[0055] Some suitable neuron-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), S100 promoter (GenBank Accession No. M65210), and glutamine synthase promoter (GenBank Accession No. X59834).

[0056] In some embodiments, the viral vector is an rAAV vector, e.g., rAAV2-retro, AAV10, AAV2 / 10, AAV9, or AAV2 / 9. In some embodiments, a composition comprising a nucleic acid encoding GAD-65 and / or GAD-67 is administered to a patient with progressing PD. In some embodiments, the administered composition comprises a nucleic acid encoding GAD-65 and GAD-67. In some embodiments, the method comprises simultaneously or sequentially administering one or more compositions comprising a vector comprising a nucleic acid encoding GAD-65 and a nucleic acid encoding GAD-67. In some embodiments, the method comprises simultaneously or sequentially administering one or more compositions comprising a nucleic acid encoding GAD-65, a nucleic acid encoding GAD-67, and another drug used to treat PD.

[0057] An effective amount of rAAV is an amount sufficient to infect a sufficient number of cells in the target tissue in the subject to which the rAAV is administered. An effective amount of rAAV can be defined as an amount sufficient to have a therapeutic benefit to the subject, for example, to improve one or more symptoms of a disease in the subject. The effective amount may vary depending on the species, age, weight, and health condition of the subject and CNS tissue being targeted. The effective amount may also vary depending on the mode of administration. In some cases, multiple doses of rAAV are administered to achieve an effective amount of the intended therapeutic benefit. The effective amount may vary depending on the serotype of rAAV. In certain cases, an effective amount of rAAV is 10 or more per subject. 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the concentration of the AAV vector / vectors containing the GAD transgene(s) administered is about 1x10 genome copies. 11 , about 2x10 11 , about 3x10 11 , about 4x10 11 , about 5x10 11 , about 6x10 11 , approximately 7x10 11 , about 8x10 11 , approximately 9x10 11 , approximately 1x10 12 , or about 1x10 13 vector genomes / ml. Pharmaceutical Compositions In one embodiment, the present disclosure provides pharmaceutical compositions comprising one or more vectors containing a nucleic acid encoding GAD-65 and one or more vectors containing a nucleic acid encoding GAD-67. These compositions can be administered alone or in combination with other drugs to treat subjects suffering from PD. A pharmaceutical composition may refer to a composition containing a vector or vectors and a pharmaceutically acceptable carrier, and optionally other materials, such as one or more inactive ingredients (e.g., detectable agents or labels) or one or more active ingredients. A pharmaceutically acceptable carrier may include one or more physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The composition may include ingredients such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, or mixtures thereof. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate-buffered saline, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers). Carbohydrates such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and myo-inositol may also be used as excipients.

[0058] The carrier may also include a pH adjuster, such as a buffer or a salt prepared from an organic acid or base. Examples of buffers include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, and phosphate buffers. Additional carriers may include polymeric excipients or additives, such as polyvinylpyrrolidone, Ficoll (polymeric sugar), dextrates (e.g., cyclodextrins, e.g., 2-hydroxypropyl-quadrature-cyclodextrin), polyethylene glycol, flavorings, antibacterial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, e.g., TWEEN® 20 and TWEEN® 80), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0059] Administration method The method for delivering one or more GAD transgenes to a subject can include administering rAAV by a single or multiple administration routes. For example, rAAV can be administered to a subject by intravenous injection of an effective amount of rAAV that crosses the blood-brain barrier. For example, intrathecal administration or intracerebral administration by intracerebroventricular injection can also be used to deliver an effective amount of rAAV to the CNS. In a non-limiting example, an effective amount of rAAV can be co-administered by two different administration routes, for example, by intrathecal administration and intracerebral administration. Co-administration can be performed at approximately the same time or at different times.

[0060] The term "intrathecal administration" refers to administering a drug into the spinal canal or subarachnoid space to reach the cerebrospinal fluid (CSF). The term "intracranial administration" refers to administering a drug in and / or around the brain. Intracerebral administration includes, but is not limited to, administering a drug to the brain, pons, cerebellum, intracranial cavity, and meninges surrounding the brain. Intracerebral administration may include administration to the dura mater, arachnoid substance, and pia mater of the brain. Intracerebral administration, in some cases, may involve administering a drug to the cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain. Intracerebral administration, in some cases, may involve administering a drug to the ventricles of the brain, such as the right ventricle, left ventricle, third ventricle, or fourth ventricle.

[0061] Intracerebral injection can involve direct injection into and / or around the brain. In some cases, intracerebral administration involves injection using a stereotactic procedure for precise delivery. Stereotactic microinjection techniques are well known in the art and have been used in the art to precisely deliver vectors to specific parts of the brain. This procedure involves the use of a computer and a three-dimensional scanning device. In this method, the subject to be treated is positioned within a stereotactic frame and then imaged using high-resolution MRI to determine the three-dimensional positioning of the specific area to be treated. The MRI images can then be used to determine the exact target site for microinjection of the composition. The stereotactic computer software provides three-dimensional coordinates precisely registered to the stereotactic frame. For intracranial delivery, a burr hole is drilled above the entry site, and a stereotactic device is used to position the needle and ensure implantation at the desired depth. For bilateral delivery, a document manager manually confirms the target site with the surgeon to avoid any errors, and a cooling period can be implemented. Stereotactic microinjection can be used to deliver vectors to any specific part of the brain, including, but not limited to, the hippocampus, cortex, subthalamic nucleus, and / or substantia nigra. In some cases, a microinjection pump can be used to deliver the rAAV compositions described herein. The injection rate can vary between 1 μl / min and 100 μl / min, depending on various factors, such as the subject's age, subject's weight / size, AAV serotype, and the selected brain region. In another embodiment, the vector is delivered using other delivery methods suitable for localized delivery, such as local penetration of the blood-brain barrier.

[0062] The dosage regimen required for therapy can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the subject's responsiveness to the therapy.

[0063] The vectors described herein can be used in combination with one or more therapeutic agents other than GAD. For example, this therapy can be used in combination with various other compounds, including, but not limited to, dopamine replacement therapy (e.g., levodopa or carbidopa), dopamine agonists (e.g., pramipexole, ropinerole, bromocriptine), MAO inhibitors (e.g., selegiline and rasagiline), catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone and tolcapone), and any agent known in the art for treating one or more symptoms associated with PD described herein. The vector or vectors can be administered to a subject suffering from PD simultaneously with or separately from other agents used to treat the disease.

[0064] The kits described herein can include any combination of the agents, compositions, components, reagents, administration devices or mechanisms, or other entities provided herein. For example, the kits described herein can include one or more AAV-GAD vectors and one or more of a carrier composition, an administration device, and a combination therapeutic agent. The kits can further include a device for facilitating delivery, such as a syringe for injection, or a tool for facilitating delivery of the therapeutic composition to the brain, e.g., the substantia nigra. Any of the kits provided herein can be included in a container, pack, or dispenser along with instructions for administration. [Example]

[0065] Example 1: Study Design Identifying the PD patient population most receptive to the disclosed treatment methods.

[0066] Patient selection Sixty-six patients with progressive PD were screened for eligibility to participate in a randomized, double-blind, sham-controlled, multicenter, phase 2 trial of STN AAV2-GAD gene therapy; 45 were randomized. All patients had progressive, levodopa-responsive PD, as defined by the UK Parkinson's Disease Society criteria. In addition to levodopa, use of other medications for this disorder was permitted as long as there was no change in dose or medication type for at least 4 weeks prior to enrollment. An overnight drug break and a Unified Parkinson's Disease Rating Scale (UPDRS) Part 3 total subscore (motor score) of 25 or greater were required. Additional inclusion criteria were age 30–75 years, PD symptom duration of at least 5 years, and levodopa responsiveness of at least 12 months. Patients were required to have been unable to undergo neurosurgery, had previously used dopamine receptor blockers, had focal neurological deficits, or had abnormal cranial MRI. 18 F-fluorodeoxyglucose PET scans had to be compatible with PD according to criteria for metabolic brain patterns specific to PD, which excluded patients with atypical parkinsonism or an indeterminate pattern. Patients were also excluded because of cognitive impairment, as defined by a Mattis Dementia Rating Scale score of less than 130.

[0067] Randomization A statistician and programmer at PharmaNet Inc. (each with no further role in the study) generated the randomization code. Prior to randomization, all subjects underwent resting-state metabolic brain imaging using FDG PET. After screening to exclude atypical parkinsonism, 45 subjects with PD were randomized 1:1 to receive either STN AAV2-GAD gene therapy (n = 22) or sham surgery (n = 23). When patients arrived in the operating room, the neurosurgeon opened an envelope containing the computer-generated randomized treatment assignment (1:1 ratio) for either AAV2-GAD or sham treatment. Patients, caregivers, and researchers were masked to the treatment assignment. For sham-assigned patients, the operating room team performed a previously rehearsed plan to simulate the same bilateral stereotaxic procedure performed for the AAV2-GAD group. Patients treated with sham surgery underwent partial-thickness burr holes after the stereotaxic frame was placed. The simulation included the sounds of microelectrode electrophysiological recordings, and the infusion pump and external catheter used to infuse standard saline into the burr hole site were used exactly as in patients receiving AAV2-GAD infusions. Masking was carefully planned for all information regarding treatment assignment, and no deviations occurred at any site. All assessors were masked to treatment assignment and had no access to isolated postoperative images and surgical records. Patients were consulted about their treatment assignment at all visits from the third postoperative day onward. Of the 45 initial subjects, 8 were excluded from data analysis before unblinding due to drug delivery failure (pump failure, inaccurate targeting of the STN) based on predefined criteria.

[0068] Post-treatment selection of patient population for analysis Subjects and investigators were blinded to treatment status for at least 6 months after the procedure; six subjects in the treatment group and two subjects in the sham group were excluded from analysis due to missing surgical targets or catheter / pump malfunction. At baseline, there were no group differences in age, sex, UPDRS motor assessment, or cognitive testing (P > 0.07). Subjects were rescanned blinded 6 months after surgery (except for one subject in each group) and at the end of the 12-month clinical trial. After the final participant completed the 6-month blinded follow-up, subjects were simultaneously unblinded. Because surgical procedures were interleaved over a 1-year period, the majority of participants (16 of 22 in the sham group (73%) and 11 of 20 in the GAD group (55%)) underwent imaging at 12 months after unblinding; the remaining six sham and nine GAD subjects remained blinded at this 12-month time point.

[0069] Example 2: Viral Vector Construction AAV-GAD plasmids were generated containing DNA encoding the human GAD-65 or GAD-67 open reading frame under the control of the cytomegalovirus enhancer-chicken β-actin promoter and woodchuck posttranscriptional regulatory elements. Recombinant AAV genomes were packaged in human embryonic kidney (HEK) 293 cells and purified by heparin affinity chromatography according to standard procedures, as previously described. The final formulation buffer was 1x phosphate-buffered saline. Genomic vector titers were measured by absolute quantification on an ABI7000 sequence detection system (Applied Biosystems, Foster City, CA, USA).

[0070] Example 3: Vector Delivery Viruses encoding GAD-65 or GAD-67 were mixed at a 1:1 ratio and incubated at 1 × 10 in 2× phosphate-buffered saline. 11 Viral genome (vg) / mL (low dose), 3 × 10 11 vg / mL (medium dose), and 1 × 10 12The drug was diluted to 0.05 mg / mL (high dose). Bulk collection and final formulated products were rigorously tested in lot release testing in accordance with FDA guidelines. Biosafety tests for mycoplasma, endotoxin, sterility, and adventitious viruses, as well as general safety testing, were performed (AppTec Laboratory Services, Philadelphia, USA). Sham-based treatment was performed using normal saline.

[0071] The subthalamic nucleus (STN) was localized using a Leksell stereotactic frame and MRI image guidance. Standard intraoperative microelectrode recordings were performed with the subject awake to verify the exact location of the STN. The tip of the microelectrode was then withdrawn to the location believed to be the center of the STN. After microelectrode removal, a guide tube was inserted 10 mm above the center of the nucleus. A catheter with a 10 mm tip and a diameter of 200 μM I was flushed with AAV2-GAD infusion solution and inserted into the estimated center of the nucleus. The catheter was locked in place with a cap containing a ripcord that tethered the catheter for post-procedure release at the bedside. To avoid catheter blockage, the scalp was closed after the initial catheter placement. The dose of AAV2-GAD (35 μl of 1 x 10 s) per hemisphere was 100 mg / h. 12 AAV2-GAD (1x10 genomes / ml) was administered bilaterally to the subthalamic nucleus of PD subjects. Procedures were performed on both sides of the scalp. A timeout occurred before the start of surgery on each side of the brain, and a study coordinator or other surgical team member confirmed that the coordination was recorded by the surgeon and documented in writing before entering the brain. Another group of PD subjects received a dose of AAV2-GAD (1x10 genomes / ml in 35 μl) per hemisphere. 11 A separate group of PD subjects received a dose of AAV2-GAD (35 μl of 3 x 10 genomes / ml) unilaterally into the subthalamic nucleus. 11 A separate group of PD subjects received a dose of AAV2-GAD (35 μl of 1x10 genomes / ml) unilaterally into the subthalamic nucleus. 12Subjects received 1000mg of ribosomal RNA (1000mg / ml) unilaterally into the subthalamic nucleus. After completion of the infusion, a sectioned head CT scan was performed to determine the location of the catheter tip. Post-catheter CT and MRI scans were performed on all subjects at 24- and 48-hour intervals after treatment.

[0072] Example 3: Determining "on-time" after treatment Subjects who underwent either AAV-GAD (GAD) or sham surgery (sham) into the subthalamic nucleus recorded hourly on and off times in a daily diary for two weeks at each listed time period (1, 3, 6, and 12 months after surgery). The average daily on and off times determined from the diary over the two-week period were compared with baseline diaries taken within 30 days before surgery using a two-tailed t-test to determine the change in the number of on-time hours after surgery. At 12 months after surgery, the change in average daily on time from baseline for each subject in the AAV-GAD treatment group correlated with the change in clinical scores for the same subjects over the same period. The clinical score used was Part 3 of the UPDRS. array SEQ ID NO: 1 MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYN WELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKGAAAL GIGTDSVILIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYD TGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEIERLGQDL SEQ ID NO: 2 SEQ ID NO: 3 MSPIHHHHHHLVPRGSEASNSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDF HYPNELLQEYNWELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSL KKGAAALGIGTDSVILKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDL SYDTGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL SEQ ID NO:4 SEQ ID NO:5 MASSTPSSSATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRTNSLEEKSRLVSAFRERQSSKNLLSCENSDRDARFRRTETDFSNLFARDLLPAKNGEEQTVQFLLEVVDILLNYVRKTFDRSTKVLDFHHPHQLL EGMEGFNLELSDHPESLEQILVDCRDTLKYGVRTGHPRFFNQLSTGLDIIGLAGEWLTSTANTNMFTYEIAPVFVLMEQITLKKMREIVGWSSKDGDGIFSPGGAISNMYSIMAARYKYFPEVKTKGMAAVPKLVLFTSEQSHYSIKKA GAALGFGTDNVILIKCNERGKIIPADFEAKILEAKQKGYVPFYVNATAGTTVYGAFDPIQEIADICEKYNLWLHVDAAWGGGLLMSRKHRHKLNGIERANSVTWNPHKMMGVLLQCSAILVKEKGILQGCNQMCAGYLFQPDKQYDVS YDTGDKAIQCGRHVDIFKFWLMWKAKGTVGFENQINKCLELAEYLYAKIKNREEFEMVFNGEPEHTNVCFWYIPQSLRGVPDSPQRREKLHKVAPKIKALMMESGTTMVGYQPQGDKANFFRMVISNPAATQSDIDFLIEEIERLGQDL SEQ ID NO:6 SEQ ID NO:7 MASSTPSSSATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRTNSLEEKSRLVSAFKERQSSKNLLSCENSDRDARFRRTETDFSNLFARDLLPAKNGEEQTVQFLLEVVDILLNYVRKTFDRSTKVLDFHHPHQLL EGMEGFNLELSDHPESLEQILVDCRDTLKYGVRTGHPRFFNQLSTGLDIIGLAGEWLTSTANTNMFTYEIAPVFVLMEQITLKKMREIVGWSSKDGDGIFSPGGAISNMYSIMAARYKYFPEVKTKGMAAVPKLVLFTSEQSHYSIKKA GAALGFGTDNVILIKCNERGKIIPADFEAKILEAKQKGYVPFYVNATAGTTVYGAFDPIQEIADICEKYNLWLHVDAAWGGGLLMSRKHRHKLNGIERANSVTWNPHKMMGVLLQCSAILVKEKGILQGCNQMCAGYLFQPDKQYDVS YDTGDKAIQCGRHVDIFKFWLMWKAKGTVGFENQINKCLELAEYLYAKIKNREEFEMVFNGEPEHTNVCFWYIPQSLRGVPDSPQRREKLHKVAPKIKALMMESGTTMVGYQPQGDKANFFRMVISNPAATQSDIDFLIEEIERLGQDL SEQ ID NO:8 SEQ ID NO:9 MASSTPSSSATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRTNSLEEKSRLVSAFKERQSSKNLLSCENSDRDARFRRTETDFSNLFARDLLPAKNGEEQ TVQFLLEVVDILLNYVRKTFDRSTKVLDFHHPHQLLEGMEGFNLELSDHPESLEQILVDCRDTLKYGVRTGHPRFFNQLSTGLDIIGLAGEWLTSTANTNMPSDMRECWLLR

Claims

1. 1. A method of treating Parkinson's disease (PD) in a subject in need thereof, comprising: (a) identifying subjects with less than 10 hours of on-time per day; (b) administering to the subject a composition comprising a therapeutically effective amount of one or more vectors to the patient's subthalamic nucleus, wherein each vector comprises a nucleic acid sequence encoding glutamic acid decarboxylase (GAD), and wherein the administering increases on-time in the subject; The method comprising:

2. 10. The method of claim 1, wherein the subject has less than 8 hours of on-time per day prior to treatment.

3. 3. The method of any one of claims 1 to 2, wherein the subject has a score of 30 or greater on UPDRS Part III in an off-drug state.

4. The method of any one of claims 1 to 3, wherein the one or more vectors are introduced bilaterally into the subthalamic nucleus of the patient.

5. The method of any one of claims 1 to 4, wherein the one or more vectors comprise a nucleic acid sequence encoding GAD-65 and a nucleic acid sequence encoding GAD-67.

6. 6. The method of claim 5, wherein the composition comprises a vector encoding GAD-65 and a vector encoding GAD-67 in a ratio of about 1:

1.

7. The method of any one of claims 1 to 6, wherein the one or more vectors are viral vectors.

8. 8. The method of claim 7, wherein the viral vector is an adeno-associated viral (AAV) vector.

9. The composition is at least 1×10 11 9. The method of claim 7 or 8, comprising vector genomes / ml.

10. The composition is at least 3×10 11 9. The method of claim 7 or 8, comprising vector genomes / ml.

11. The composition is at least 1×10 12 9. The method of claim 7 or 8, comprising vector genomes / ml.

12. 12. The method of any one of claims 1-11, wherein the subject exhibits at least a 40% increase in on-time 12 months after treatment.

13. 16. The method of any one of claims 1-15, wherein the subject exhibits at least a 30% increase in on-time 12 months after treatment.

14. 16. The method of any one of claims 1-15, wherein the subject exhibits at least a 20% increase in on-time 12 months after treatment.

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