Method for treating or preventing amyotrophic lateral sclerosis

Abstract

To provide methods for treating or preventing amyotrophic lateral sclerosis (ALS).SOLUTION: This disclosure provides methods for treating, ameliorating, or reversing at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject by increasing the amount of neuronal aspartate in spinal cord through administration of a therapeutically effective amount of a composition comprising a nucleic acid encoding ASPA or a functional fragment thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 724,780, filed August 30, 2018. The foregoing application is incorporated herein by reference.

[0002] The present invention relates generally to methods of treating or preventing amyotrophic lateral sclerosis (ALS), and more particularly to methods of treating, alleviating, ameliorating, or reversing ALS in an affected cell population by increasing the intracellular activity of aspartoacylase (ASPA), the rate-limiting component of the malate-aspartate shuttle that provides affected cells with the ability to utilize cytosolic NADH to facilitate mitochondrial oxidative phosphorylation in order to provide aspartate. [Background technology]

[0003] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig's disease, is a disease that causes the death of neurons that control voluntary muscles. It is characterized by adult-onset progressive dysfunction and loss of upper motor neurons in the motor cortex and lower motor neurons in the brainstem, spinal cord, and associated cords. Patients with or at risk for ALS experience progressively worsening weakness due to muscle stiffness, muscle spasms, and loss of muscle size. This results in difficulty speaking, swallowing, and ultimately breathing.

[0004] While there is no known cure for ALS, the FDA has approved two treatments specifically aimed at slowing the progression of the disease. Riluzole (Rilutek®) was the first FDA-approved drug for ALS and can extend lifespan by approximately 2–3 months in a limited clinical population with bulbar onset. Edaravone (Radicava®) is another FDA-approved treatment option for ALS, administered intravenously. Clinical data suggest that it improves ALS Functional Rating Scale (ALSFRS-R) scores compared with a placebo control. However, the long-term efficacy of edaravone in ALS patients has yet to be determined. Noninvasive ventilation may improve both quality of life and lifespan, but it is strictly palliative in nature. The disease can affect people of any age, but typically begins around age 60, and in hereditary cases, onset occurs around age 50. The average survival time from onset to death is 2–4 years. Approximately 10% of patients survive for more than 10 years, with most dying from respiratory failure.

[0005] Thus, there remains a pressing unmet need in the art to provide methods and reagents for preventing, treating, or reversing ALS. Summary of the Invention

[0006] The present disclosure addresses the above-mentioned needs in some aspects. In one aspect, the present disclosure provides a method for treating, ameliorating, or reversing at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a composition that increases the level or activity of aspartoacylase (ASPA) in the cells of the subject, for the purpose of providing a substrate for mitochondrial oxidative phosphorylation. In some embodiments, the administered composition increases the protein expression level of ASPA in the cells of the subject.

[0007] In some embodiments, the composition comprises a gene therapy composition. In some embodiments, the composition may comprise a nucleic acid encoding ASPA, or a functional fragment thereof, having an amino acid sequence at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO:1.

[0008] In some embodiments, the method includes introducing the nucleic acid into at least one cell of the subject by viral transduction. The composition can be provided with a virus or virus-like particle containing the nucleic acid. In some embodiments, the nucleic acid is carried on a recombinant adeno-associated virus (rAAV) vector, such as AAV9.

[0009] In some embodiments, the method comprises administering a composition to at least a portion of the spinal cord of the subject. In some embodiments, the composition is administered locally to the portion of the spinal cord of the subject.

[0010] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. The second therapeutic agent can be administered to the subject before, after, or simultaneously with the composition. In some embodiments, the second therapeutic agent is riluzole (6-(trifluoromethoxy)-2-benzothiazolamine), or a salt or solvate thereof. In some embodiments, the second therapeutic agent is edaravone (5-methyl-2-phenyl-4H-pyrazol-3-one), or a salt or solvate thereof. In some embodiments, the composition can be administered by a route selected from oral, parenteral, transdermal, pulmonary, intranasal, buccal, intrathecal, and intravenous.

[0011] In some embodiments, the subject is a mammal, such as a human. In some embodiments, the at least one cell is in the spinal cord of the subject. In some embodiments, the subject exhibits at least one symptom or mutation associated with ALS. In some embodiments, at least one symptom of ALS is mitochondrial dysfunction. Disruptions in mitochondrial structure, dynamics, and bioenergetics have been widely reported in ALS patients and model systems, and have been suggested to be directly involved in the pathogenesis of the disease.

[0012] In some embodiments, administration of the composition will enhance substrates for mitochondrial energy metabolism in the subject. In some embodiments, administration of the composition increases cell survival in the subject. In some embodiments, administration of the composition increases motor neuron survival in the subject. In some embodiments, administration of the composition extends life expectancy of the subject.

[0013] Also within the scope of this disclosure are kits for increasing the level or activity of ASPA in cells of a subject. The kits include an rAAV vector or virus-like particle, wherein the virus or virus-like particle contains a nucleic acid encoding ASPA or a functional fragment thereof, the nucleic acid having an amino acid sequence at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding ASPA or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the rAAV vector is AAV9. [Brief explanation of the drawings]

[0014] For the purpose of illustrating the invention, there are shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. For data shown in the figures, asterisks indicate the following levels of statistical significance: *p≦0.05, **p≦0.01, and ***p≦0.001.

[0015] [Figure 1] Neurons differentiated in vitro from iPSCs derived from an ALS patient after 40 days using established methodology are shown (Kiskinis et al., 2014, Cell Stem Cell). Cells are positive for the neuronal marker TuJ-1 and the motor neuron marker choline acetyltransferase (ChAT). [Figure 2] This figure shows the recovery of mitochondrial adenosine triphosphate (ATP) synthesis after treatment with AAV-ASPA. Motor neuron cultures generated from ALS iPSCs were transduced in culture at day 28 with adeno-associated viral vectors (AAV) to deliver either ASPA or GFP. On day 40, intact mitochondria were isolated from the cells and assayed for ATP synthesis. Naive wild-type, non-ALS cells (WT), naive ALS SOD1 mutant cells (ALS), AAV-GFP SOD1 mutant cells (ALS GFP), and AAV-ASPA SOD1 mutant cells (ALS ASPA) were assayed (n = 5 per group). A significant 1.5-fold increase in ATP synthesis rate (jiM ATP / min / jig of isolated mitochondria, using 15 jig per reaction) was observed in AAV-ASPA-treated ALS cells compared to AAV-GFP negative controls. **p<0.005, *p<0.05. [Figure 3] We show that NAA contains glycolytically derived acetyl coenzyme A (AcCoA) and aspartate, which is used to transfer cytosolic reducing equivalents (NADH) to the inner mitochondrial membrane. Both are substrates for mitochondrial ATP synthesis, and aspartate plays a key role in coupling glycolysis to oxidative phosphorylation via complexes I–V of the electron transport chain. Therefore, liberation of this substrate in neurons affected by neurodegenerative pathologies increases energy conservation. [Figure 4A](collectively, "Figure 4") shows the target neurons for metabolic gene therapy. An AAV reporter vector expressing green fluorescent protein (GFP) was delivered to the hippocampus of an adult mouse. Figure 4A shows a low-magnification confocal microscopy image of a GFP-expressing neuron labeled with an antibody against neuronal nuclear antigen (NeuN). Figure 4B shows a higher magnification of the area highlighted by the white arrow in panel A. Individual GFP-positive cell bodies colabeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a lighter shade in the black-and-white replica of the image) (Figure 4D), thereby confirming neuronal targeting in vivo. Therefore, using an appropriate AAV, therapeutic genes of interest can be delivered to neurons in the mammalian brain, provided they can be packaged appropriately. Data were generated using published methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4B] (collectively, "Figure 4") shows the target neurons for metabolic gene therapy. An AAV reporter vector expressing green fluorescent protein (GFP) was delivered to the hippocampus of an adult mouse. Figure 4A shows a low-magnification confocal microscopy image of a GFP-expressing neuron labeled with an antibody against neuronal nuclear antigen (NeuN). Figure 4B shows a higher magnification of the area highlighted by the white arrow in panel A. Individual GFP-positive cell bodies colabeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a lighter shade in the black-and-white replica of the image) (Figure 4D), thereby confirming neuronal targeting in vivo. Therefore, using an appropriate AAV, therapeutic genes of interest can be delivered to neurons in the mammalian brain, provided they can be packaged appropriately. Data were generated using published methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4C] (collectively, "Figure 4") shows the target neurons for metabolic gene therapy. An AAV reporter vector expressing green fluorescent protein (GFP) was delivered to the hippocampus of an adult mouse. Figure 4A shows a low-magnification confocal microscopy image of a GFP-expressing neuron labeled with an antibody against neuronal nuclear antigen (NeuN). Figure 4B shows a higher magnification of the area highlighted by the white arrow in panel A. Individual GFP-positive cell bodies colabeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a lighter shade in the black-and-white replica of the image) (Figure 4D), thereby confirming neuronal targeting in vivo. Therefore, using an appropriate AAV, therapeutic genes of interest can be delivered to neurons in the mammalian brain, provided they can be packaged appropriately. Data were generated using published methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4D] (collectively, "Figure 4") shows the target neurons for metabolic gene therapy. An AAV reporter vector expressing green fluorescent protein (GFP) was delivered to the hippocampus of an adult mouse. Figure 4A shows a low-magnification confocal microscopy image of a GFP-expressing neuron labeled with an antibody against neuronal nuclear antigen (NeuN). Figure 4B shows a higher magnification of the area highlighted by the white arrow in panel A. Individual GFP-positive cell bodies colabeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a lighter shade in the black-and-white replica of the image) (Figure 4D), thereby confirming neuronal targeting in vivo. Therefore, using an appropriate AAV, therapeutic genes of interest can be delivered to neurons in the mammalian brain, provided they can be packaged appropriately. Data were generated using published methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 5] This demonstrates the potential for targeting synaptic function using gene therapy. Endogenous NAA catabolized by recombinant ASPA delivered by AAV increases substrates for energy metabolism and increases the ATP available to support synaptic transmission in neurons. [Figure 6] This study demonstrates that the reducing power of NADH produced by cytosolic glycolysis is transported into the inner mitochondrial membrane space by aspartate aminotransferase, which transfers a hydrogen ion to aspartate to form malate. Aspartate moves from the mitochondria to the cytosol in exchange for glutamate. The cytosolic aspartate is then converted to malate, which carries the hydrogen ion of the cytosolic NADH and allows it to move freely into the mitochondria. Once inside, malate is converted back to aspartate by malate dehydrogenase, thereby liberating a hydrogen ion to form NADH, which can be used to drive mitochondrial oxidative phosphorylation. [Figure 7] We show that the free aspartate generated by the cleavage of NAA by ASPA becomes available for the malate-aspartate shuttle (MAS), which transports reduced NADH generated by glycolysis into mitochondria. This available NADH drives electron transport chain complexes I–V, culminating in newly synthesized ATP. [Figure 8]Schematic diagram of the methodology for demonstrating oxidative phosphorylation and ATP synthesis driven by free aspartate generated by ASPA in isolated spinal cord mitochondria. (1) 60 mm dishes of HeLa cells were transfected with plasmids expressing constitutively wild-type human ASPA (WT) or a nonfunctional mutant isoform (E285A). (2) Transfected cells were harvested 48 h later and mechanically lysed by sonication. (3) 50 μl of the lysate was added to a reaction mixture containing 5 mM purified NAA and incubated at 37°C for 2 h to allow the transfected ASPA enzyme to catabolize the NAA substrate. (4) The catabolic reaction was heat-inactivated, and 50 μl was added to isolated mitochondria in a reaction mix for a luminescence-based ATP synthesis assay. [Figure 9] The rate of ATP synthesis in mitochondria isolated from G93A SOD spinal cord and incubated with reaction mixtures derived from HeLa cells transfected with wild-type ASPA, HeLa cells transfected with E285A ASPA, or saline-treated HeLa cells is shown schematically in Figure 8. Addition of the wild-type ASPA-fueled reaction products significantly increased the rate of ATP synthesis (p = 0.039) proportional to the aspartate content of the aliquot (see Table 1). [Figure 10] Shown are the mean latencies at 12–16 weeks of age in SOD G93A mutant mice treated with either intrathecally administered saline or AAV9-ASPA. The average of three individual trials is shown along with the standard error of the mean (n = 15 / group). [Figure 11A](Collectively, "Figure 11") shows HPLC analysis of NAA and adenosine triphosphate (ATP): adenosine monophosphate (AMP) ratio (hereafter, ATP:AMP ratio) in the spinal cord of 16-week-old wild-type, saline-treated SOD, and AAV9-ASPA-treated SOD mice. A significant decrease in spinal cord NAA, along with an associated decline in the ATP:AMP ratio (B), was observed in saline-treated SOD mice compared to age-matched wild-type mice, suggesting a pathological decrease in NAA in response to reduced production of ATP (reflected in levels of AMP). NAA levels are further decreased in AAV9-ASPA-treated SOD spinal cords, but are associated with a corresponding increase in ATP:AMP, suggesting increased ATP synthesis. [Figure 11B] (Collectively, "Figure 11") shows HPLC analysis of NAA and adenosine triphosphate (ATP): adenosine monophosphate (AMP) ratio (hereafter, ATP:AMP ratio) in the spinal cord of 16-week-old wild-type, saline-treated SOD, and AAV9-ASPA-treated SOD mice. A significant decrease in spinal cord NAA, along with an associated decline in the ATP:AMP ratio (B), was observed in saline-treated SOD mice compared to age-matched wild-type mice, suggesting a pathological decrease in NAA in response to reduced production of ATP (reflected in levels of AMP). NAA levels are further decreased in AAV9-ASPA-treated SOD spinal cords, but are associated with a corresponding increase in ATP:AMP, suggesting increased ATP synthesis. [Figure 12]ATP synthesis rates in mitochondria isolated from wild-type, saline-treated, and AAV9-ASPA-treated spinal cords were shown. Mitochondria were assayed using a commercially available luminescence-based kit. ATP synthesis rates in mitochondria isolated from AAV9-ASPA-transduced SOD spinal cords were significantly superior to those in saline-treated SOD spinal cords, suggesting that providing NAA-derived aspartate via ASPA gene therapy is an effective means of enhancing energy metabolism and may offer associated benefits for motor function in ALS patients. Mean ATP synthesis rates, + / - standard error (n = 5 / group), are shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] In one aspect, the present invention relates to the unexpected discovery that increasing the expression of an aminoacylase, such as aspartoacylase (ASPA), in cells can be used to prevent, treat, or reverse one or more symptoms of ALS in a subject in need thereof. In certain embodiments, the compositions and methods of the present invention treat or prevent mitochondrial dysfunction in ALS patients. In other embodiments, the compositions and methods of the present invention enhance substrates for energy metabolism in ALS-affected cells. In yet other embodiments, the compositions and methods of the present invention promote cell survival in ALS patients. In yet other embodiments, the compositions and methods of the present invention promote motor neuron survival in ALS patients. In yet other embodiments, overexpression of ASPA in the spinal cord extends the life expectancy of ALS patients. In yet other embodiments, the treatment utilizes patient-derived motor neurons generated from induced pluripotent stem cells (iPSCs). In yet other embodiments, the treatment utilizes stem cells, progenitor cells, or patient-derived induced pluripotent stem cells (iPSCs) engineered to overexpress ASPA (or a functional fragment thereof) for transplantation into the affected area of ​​the patient's nervous system. In yet another embodiment, the construction of the ASPA expression and delivery construct is carried out ex vivo. In some embodiments, the expression of ASPA can show a reduced degeneration rate of ex vivo human muscle tissue explants compared to that of ALS subjects. In another embodiment, the treatment is a one-time gene therapy for ALS and related disorders.

[0017] Although the pathogenesis of major neurodegenerative diseases is multifactorial and incompletely defined, therapeutic strategies that enhance mitochondrial integrity have the potential to slow the progressive loss of higher-order functions. In the case of ALS in particular, mitochondrial dysfunction is thought to be directly or indirectly linked to all of the potential toxic mechanisms associated with ALS, such as excitotoxicity, loss of protein homeostasis, and axonal transport defects. Despite differences in the potential pathogenic mechanisms in the different in vitro and in vivo models studied, reduced mitochondrial electron transport chain (ETC) activity and ATP levels appear as common features in both familial and sporadic ALS.

[0018] The present invention provides a novel gene therapy intervention to provide ALS neurons with access to a specific energy substrate sequestered within the endogenous amino acid derivative, N-acetylaspartate (NAA) (Figure 3). NAA normally functions to conserve ATP in white matter-producing glial cells by uncoupling lipid synthesis from oxidative phosphorylation through the provision of AcCoA via catabolism by the glial hydrolase ASPA. Neurons do not naturally express ASPA and therefore cannot catabolize NAA. Catabolism of NAA by ASPA generates free acetate (used by glia to synthesize AcCoA) and aspartate. Unlike acetate, neuronal aspartate is a specific substrate for subunits of the malate-aspartate shuttle (Aralar1), and is therefore an essential component of the shuttle machinery for transferring glycolytic reducing equivalents to mitochondria (Figure 3). Free acetate is a poor substrate for neuronal energy metabolism because, compared to glial cells, there are relatively few available biochemical mechanisms capable of processing acetate for energy metabolism, and acetate cannot substitute for glucose in supporting neuronal function. Analysis of the flux of radiolabeled acetate provided to the nervous system has indeed demonstrated its predominant use by glial cells compared to neurons, and acetate is not involved in the transfer of cytosolic NADH to the mitochondrial ETC by Aralar1, a process that requires the conversion of aspartate to malate in the cytosol. In certain non-limiting embodiments, providing neurons with the ability to catabolize endogenous NAA promotes the conservation of spare ATP in the face of pathological metabolic abnormalities by providing aspartate for the shuttle of glycolytic reducing equivalents to mitochondria. In at least one embodiment, the present invention relates to a method for increasing the availability of aspartate for the shuttle of glycolytic reducing equivalents to mitochondria in a subject at risk of developing or suffering from ALS.

[0019] Adeno-associated virus (AAV) has emerged as a highly promising and attractive approach to gene delivery with an established clinical safety and efficacy profile and is highly efficient at targeting neurons. Advances in AAV vector design and associated administration technologies have enabled widespread gene delivery in the brain and spinal cord, making AAV highly suitable for the treatment of neurodegenerative diseases. In at least one embodiment, the present invention uses an AAV serotype to deliver a gene therapy composition containing a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO: 1.

[0020] The present disclosure demonstrates that expression of aminoacylase in spinal motor neurons of an ALS mouse model results in long-term improvements in motor function, as measured by accelerating rotarod performance in SOD(G93A) transgenic mice. The improved rotarod performance was associated with an increase in detectable spinal cord energy currency (ATP) and an associated increase in detectable free aspartate. This result suggests a mechanistic link between aspartate bioavailability and mitochondrial function and ETC function and energy currency generation in the face of ALS pathology. Specifically, the provision of excess bioavailable aspartate due to increased ASPA activity has been shown to promote the activity of the malate-aspartate shuttle, in which aspartate is rate-limiting, to generate energy currency in the form of ATP. In at least one embodiment, the methodology of the present invention increases aspartate bioavailability in the spinal cord of subjects at risk for or suffering from ALS.

[0021] In one aspect, the present disclosure provides a method for treating, improving, or reversing at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject in need thereof.The method comprises administering to the subject a therapeutically effective amount of a composition that increases the level or activity of aspartoacylase (ASPA) in the cells of the subject.In some embodiments, the administered composition increases the protein expression level of ASPA in the cells of the subject.In yet another embodiment, the method of the present invention relates to increasing NAA catabolism in the spinal cord of a patient suffering from ALS.

[0022] In another embodiment, the subject in need of the treatment of the present invention is a subject who has experienced a decline in motor function or is at risk of experiencing motor dysfunction associated with a condition other than cognitive impairment. In another embodiment, a subject at risk of motor dysfunction and exhibiting a hypermetabolic state, which may exhibit a metabolic ratio greater than 1 as measured according to the Harris and Benedict formula, is a candidate for the proposed intervention. In another embodiment, the subject in need may exhibit a defect related to spinal motor neurons, which have a high metabolic demand for maintaining action potentials, which may not be present in other diseases that do not primarily affect the motor system. In other embodiments, the subject in need may exhibit gradual onset, painless, progressive muscle weakness accompanied by stumbling, dropping objects, abnormal arm and / or leg fatigue, slurred speech, muscle spasms and spasms, and / or periods of uncontrollable laughing or crying. In some embodiments, subjects suffering from respiratory muscle weakness may require permanent ventilatory support to assist breathing.

[0023] In other embodiments, subjects in need of the treatment of the present invention may exhibit mutations that may be crucial for the proper diagnosis and development of ALS, including, for example, mutations in ALS2 (arsinin), TBK1 (TANK-binding kinase 1), TUBA4A (tubulin, alpha 4A), ANG (angiogenin), MATR3 (matrin-3), CHCHD10 (coiled-coil helix domain-containing 10), NEK1, PFN1 (profilin-1), C21ORF2, MOBP, SCFD1, SETX (senataxin), FUS, TDP43, VCP (valosin-containing protein), or enzymes associated with ALS (e.g., KIF5A, inesin family member 5A), and OPTN (optineurin). In another embodiment, the mutation may be in the C9ORF72 gene or may cause RNA accumulation that occurs when the gene is mutated. In yet another embodiment, subjects may initially be screened for the presence of such mutations. Moreover, in some embodiments, the screen is to identify at least any two or more of the above-identified mutations.

[0024] In another embodiment, the present invention relates to administering a therapeutically effective gene therapy agent to a subject exhibiting a mutation in ALS2, TBK1, TUBA4A, ANG, MATR3, CHCHD10, NEK1, PFN1, C21ORF2, MOBP, SCFD1, SETX, FUS, TDP43, VCP, or OPTN. In yet another aspect, a suitable screened subject can be administered a gene therapy composition comprising a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO: 1. In another embodiment, an identified subject can be administered a gene therapy composition comprising SEQ ID NO: 1 and AAV-9 and exhibits increased levels of NAA catabolism in the spinal cord. In at least one embodiment, the present invention relates to administering the disclosed gene therapy agents to patients exhibiting clinical symptoms of ALS, and to patients exhibiting ALS symptoms that may exhibit mutations in TBK1, TUBA4A, NEK1, C21ORF2, MOBP, SCFD1, FUS, and TDP43, or any combination of such mutations, by identifying at least one of the above-mentioned genetic mutations or genetic mutations occurring in proteins involved in cell axon dynamics and proteins involved in cellular clearance mechanisms.

[0025] In at least one embodiment, the present invention relates to administering a gene therapy agent of the present invention to a subject exhibiting a mutation in Cu / Zn superoxide dismutase (SOD1), particularly to increase the level of NAA catabolism in the spinal cord mitochondria of such patients exhibiting at least one clinical symptom of ALS. Only 5-10% of ALS cases have a genetic origin (familial ALS), and it has been suggested that only approximately 20% of familial ALS cases have mutations in Cu / Zn superoxide dismutase (SOD1). ALS-associated SOD1 mutations cause reduced protein stability. These mutations occur throughout the protein structure, including the active site, beta-sheets, and monomer interface. To this end, at least one embodiment of the present invention relates to a method of administering a gene therapy agent of the present invention to a patient exhibiting an SOD1 mutation. In yet another embodiment, the present invention relates to a method comprising identifying a subject with an SOD1 mutation and administering to the subject a composition comprising a nucleic acid encoding ASPA or a fragment thereof, the composition comprising the amino acid sequence of SEQ ID NO: 1 and AAV-9, and increasing the level of NAA catabolism in the spinal cord of such a subject.

[0026] In other embodiments, the subject in need thereof exhibits upper and lower motor neuron degeneration, with or without progressive brainstem degeneration, a decline in neurological status, or muscle weakness after electromyography (EMG) testing, compared with healthy subjects.However, such subjects do not exhibit a decline in dopamine receptor occupancy, do not respond to the administration of L-dopa, and do not exhibit symptoms related to cognitive loss, such as those of patients with Alzheimer's disease.

[0027] In some embodiments, the composition comprises a gene therapy composition. In some embodiments, the composition may comprise a nucleic acid encoding ASPA or a functional fragment thereof having an amino acid sequence at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding ASPA or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 1. [Table 1]

[0028] Also included within the scope of the present disclosure are variants, mutants, and homologs that share significant identity with ASPA, such as those that share at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the ASPA sequences described herein.

[0029] The terms "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs from another, normally related polypeptide in one or more amino acids. A variant may have "conservative" changes, in which the substituted amino acid has similar structural or chemical properties. Certain conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxyl side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. In rare cases, variants may have "non-conservative" changes (e.g., substitution of glycine with tryptophan). Similar minor changes may also include amino acid deletions or insertions (i.e., additions), or both. Guidance for determining which and how many amino acid residues can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNAStar software. Variants can be tested in functional assays. Preferred variants have changes (substitutions, deletions, etc.) of less than 10%, preferably less than 5%, and even more preferably less than 2%.

[0030] The term "homologue" or "homologous," when used in reference to polypeptides, refers to a high degree of sequence identity between two polypeptides, or a high degree of similarity between three-dimensional structures, or a high degree of similarity between active sites and mechanisms of action. In preferred embodiments, a homologue has greater than 60% sequence identity, more preferably greater than 75% sequence identity, and even more preferably greater than 90% sequence identity with a reference sequence. The term "substantial identity," as applied to polypeptides, means that two peptide sequences share at least 75% sequence identity when optimally aligned, such as by programs such as GAP or BESTFIT using default gap weights.

[0031] As used herein, expressing a gene means that the cell produces either the full-length polypeptide encoded by the gene or a functional fragment of the full-length polypeptide. The term "functional," when used in conjunction with "fragment," refers to a polypeptide that has a biological activity substantially similar to that of the entity or molecule of which it is a fragment. "Substantially similar" in this context means that at least 25%, at least 35%, or at least 50% of the relevant or desired biological activity of the corresponding wild-type peptide is retained. For example, a functional fragment of a polypeptide retains an enzymatic activity substantially similar to that of the full-length polypeptide encoded by the gene expressed in the cell.

[0032] "Overexpression" refers to the production of a gene product in a cell / organism that exceeds the level of production in a normal or non-transformed cell / organism. For example, it can refer to elevated (e.g., abnormal) levels of mRNA encoding a protein(s) (e.g., ASPA protein or a homolog thereof) and / or elevated levels of a protein(s) (e.g., ASPA) compared to a similar, corresponding, unmodified cell / organism that expresses basal levels of mRNA (e.g., encoding ASPA protein) or has basal levels of protein. In certain embodiments, ASPA or a homolog thereof can be overexpressed by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, or more in a cell / organism engineered to exhibit increased mRNA, protein, and / or ASPA activity.

[0033] Expression of ASPA can be induced by introducing one or more expression vectors carrying nucleic acids encoding the ASPA polypeptide or one or more of its fragments. The polypeptide or fragment can be inserted into an appropriate site in the vector (e.g., operably linked to a promoter). The expression vector is introduced into a selected host cell for amplification and / or polypeptide expression by well-known methods, such as transfection, transduction, infection, electroporation, microinjection, lipofection, or DEAE-dextran or other known techniques. These and other suitable methods are well known to those skilled in the art.

[0034] A wide variety of vectors can be used to express ASPA proteins. The ability of certain viruses to infect or enter cells through receptor-mediated endocytosis and to integrate into the host cell genome, resulting in stable and efficient expression of viral genes, makes them attractive candidates for transferring foreign nucleic acids into cells. Therefore, in certain embodiments, a viral vector is used to introduce a nucleotide sequence encoding an ASPA protein or a fragment thereof into a host cell for expression. The viral vector may contain a nucleotide sequence encoding an ASPA protein or a fragment thereof operably linked to one or more regulatory sequences, such as a promoter. Alternatively, the viral vector may not contain regulatory sequences and instead rely on regulatory sequences within the host cell to drive expression of the ASPA protein or a fragment thereof. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenoviral vectors, AAV vectors, and retroviral vectors.

[0035] For example, adeno-associated virus (AAV) can be used to introduce the nucleotide sequence encoding ASPA protein or its fragment into host cell for expression.AAV system has been previously described and is generally well known in the art (Kelleher and Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64, 1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992).The details of the generation and use of rAAV vector are described in, for example, United States Patent No. 5,139,941 and United States Patent No. 4,797,368, each of which is incorporated herein by reference in its entirety for all purposes.

[0036] In some embodiments, a retroviral expression vector can be used to introduce a nucleotide sequence encoding an ASPA protein or a fragment thereof into a host cell for expression. These systems have been previously described and are generally known in the art (Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). Examples of vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc / RSV, pcDNA3, Pbpv, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, β-actin, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, and retrovirus.

[0037] Combining a retrovirus with an appropriate packaging line can also be useful, allowing the capsid protein to function to infect target cells. Typically, cells and virus are cultured in culture medium for at least about 24 hours. Cells are then grown in culture medium for a short period of time, e.g., 24-73 hours, or at least two weeks, in some applications, and can be grown for five weeks or more before analysis. Commonly used retroviral vectors are "defective," meaning they are unable to produce the viral proteins necessary for productive infection. Vector replication requires propagation in a packaging cell line. The host cell specificity of a retrovirus is determined by the envelope protein env (pl20), which is provided by the packaging cell line. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types. Ecotropic packaging cell lines include BOSC23. Retroviruses with amphotropic envelope proteins, such as 4070A, can infect most mammalian cell types, including humans, dogs, and mice. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with xenotropic envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells. The vectors can contain genes that must subsequently be removed using a recombinase system, such as Cre / Lox, or cells expressing them can be destroyed by including genes that enable selective toxicity, such as herpesvirus TK and bcl-xs. An appropriate inducible promoter is activated in the desired target cell type, either the transfected cell or its progeny.

[0038] In some embodiments, genome editing technologies such as CRISPR / Cas9 system, designer zinc finger, transcription activator-like effector (TALE), or homing meganuclease can be used to induce the expression of the described ASPA protein in cells. Generally, "CRISPR / Cas9 system" refers collectively to the transcripts and other elements involved in the expression or induction of the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. One or more elements of the CRISPR system can be derived from type I, type II, or type III CRISPR systems. Alternatively, one or more elements of CRISPR system can be derived from a specific organism that contains endogenous CRISPR system, such as Streptococcus pyogenes.Generally, CRISPR system is characterized by an element (also called protospacer in endogenous CRISPR system) that promotes the formation of CRISPR complex at the site of target sequence.In some embodiments, genome editing technology such as CRISPR / Cas9 system, designer zinc finger, transcription activator-like effector (TALE) or homing meganuclease can be used to induce the expression of ASPA protein described in cells, and increase the substrate for mitochondrial oxidative phosphorylation.

[0039] In some embodiments, the method includes introducing a nucleic acid into at least one cell of a subject by viral transduction. The composition can be provided with a virus or virus-like particle containing the nucleic acid. In some embodiments, the nucleic acid is carried by a recombinant adeno-associated virus (rAAV) vector, such as AAV9.

[0040] In some embodiments, rAAV is artificially produced from its natural environment (e.g., from a host cell, tissue, or subject). For example, isolated AAV can be produced using recombinant methods. Such rAAV preferably has tissue-specific targeting ability, such that the AAV transgene is specifically delivered to one or more predetermined tissues. AAV capsid is an important factor in determining their tissue-specific targeting. Therefore, in at least one embodiment, recombinant AAV with a capsid suitable for spinal cord tissue can be selected. Methods for obtaining recombinant AAV with desired capsid proteins are described, for example, in Patent Application Publication No. 2003 / 0138772, the entire contents of which are incorporated herein by reference.

[0041] In another aspect, the present invention provides a method for treating, ameliorating, or reversing at least one symptom of ALS in a subject in need thereof by identifying a patient in need of increased intracellular aspartate levels, the method comprising administering to the subject a therapeutically effective amount of a composition that increases the level or activity of aspartoacylase (ASPA) in at least one cell of the subject, wherein the composition comprises a nucleic acid encoding ASPA or a functional fragment thereof, the composition having an amino acid sequence at least 75% identical to the sequence of SEQ ID NO:1.

[0042] In some embodiments, the method includes administering a composition to at least a portion of a subject's spinal cord. In some embodiments, the composition is administered locally to a portion of the subject's spinal cord. In some embodiments, the composition can be administered to the brainstem for use in treating ALS. In another embodiment, the composition of the present invention for use in treating ALS can be combined with a second pharmaceutical compound to treat or slow the progression of upper and lower motor neuron degeneration or improve upper and lower motor function. In some embodiments, the second pharmaceutical compound can be R(+)-N-propargyl-1-aminoindan in combination with 2-amino-6-trifluoromethoxybenzothiazole or a pharmaceutically acceptable salt thereof (including, but not limited to, mesylate, maleate, fumarate, tartrate, hydrochloride, hydrobromide, esylate, p-toluenesulfonate, benzoate, acetate, phosphate, and sulfate).

[0043] Gene therapy: Nucleic acids encoding protein(s) useful within the present invention can be used in gene therapy protocols for the treatment of diseases or disorders contemplated herein, such as diseases characterized by energy deficiency in cells of the central and peripheral nervous system that support motor function, including upper and lower motor neurons in the brain and spinal cord. In certain embodiments, the disease or disorder comprises amyotrophic lateral sclerosis (aggregation of superoxide dismutase). Improved constructs encoding the protein(s) can be inserted into appropriate gene therapy vectors and administered to patients to treat or prevent the disease or disorder.

[0044] Vectors, such as viral vectors, have been used in the prior art to introduce genes into a wide variety of different target cells. Typically, the vector is exposed to the target cells so that transformation can occur at a rate sufficient to provide a useful therapeutic or prophylactic effect from the expression of a desired polypeptide (e.g., a receptor). The transfected nucleic acid may be permanently integrated into the genome of each of the target cells, providing a long-term effect, or treatment may need to be repeated periodically.

[0045] A variety of vectors, both viral and plasmid, are known in the art (see, e.g., U.S. Pat. No. 5,252,479 and WO93 / 07282). In particular, numerous viruses have been used as gene transfer vectors, including parvoviruses such as SV40, vaccinia viruses, herpesviruses including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art use defective murine retroviruses. Several recently issued patents relate to methods and compositions for performing gene therapy (see, e.g., U.S. Pat. Nos. 6,168,916, 6,135,976, 5,965,541, and 6,129,705). Each of the aforementioned patents is incorporated herein by reference in its entirety.

[0046] AAV-mediated gene therapy: AAV, a parvovirus belonging to the Dependovirus genus, has several characteristics that make it particularly suitable for gene therapy applications. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells of various species. Importantly, AAV has not been associated with any human or animal disease and is not thought to alter the physiological properties of host cells upon integration.

[0047] Finally, AAV is stable under a wide range of physical and chemical conditions, making it suitable for manufacturing, storage, and transportation. The AAV genome, a linear, single-stranded DNA molecule containing approximately 4,700 nucleotides (the AAV-2 genome consists of 4,681 nucleotides, and the AAV-4 genome consists of 4,767), generally contains an internal non-repeated segment flanked at both ends by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides long (AAV-1 has a 143-nucleotide ITR) and have multiple functions, including functioning as origins of replication and as packaging signals for the viral genome. The internal non-repeated portion of the genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, enabling replication, assembly, and packaging of complete AAV virions. More specifically, at least four viral protein families, Rep78, Rep68, Rep52, and Rep40, are expressed from the AAV rep region, all named after their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3. AAV is a helper-dependent virus; that is, co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) is required to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome is inserted into the host cell chromosome or exists in an episomal form, but infectious virions are not produced.

[0048] Subsequent infection with a helper virus "rescues" the integrated genome, allowing it to replicate and package into viral capsids, thereby reconstituting infectious virions. While AAV can infect cells of different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells co-infected with canine adenovirus.

[0049] To generate infectious recombinant AAV (rAAV) containing heterologous nucleic acid sequences, an appropriate host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence but lacking the AAV helper function genes, rep and cap. The AAV helper function genes can then be provided in a separate vector. Alternatively, rather than providing a replication-competent helper virus (such as adenovirus, herpesvirus, or vaccinia), only the helper virus genes (i.e., accessory function genes) required for AAV production can be provided in the vector.

[0050] In summary, AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided in one or more vectors. The helper and accessory function gene products can then be expressed in host cells, where they act in trans in the rAAV vector containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence is then replicated and packaged as if it were a wild-type (wt) AAV genome, forming recombinant virions. When a patient's cells are infected with the resulting rAAV virions, the heterologous nucleic acid sequence enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes and accessory function genes, the rAAV cannot further replicate and package their genome. Furthermore, without a source of the rep and cap genes, wtAAV cannot form in the patient's cells.

[0051] In one embodiment of the present invention, suitable AAV serotypes or serotype variants include AAV1 through AAV12, such as AAV2, AAV2.5, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, as well as their capsid variants of AAV-based vectors, such as rationally engineered AAV9HR.

[0052] AAV-1 through AAV-11 have been described in the art (Mori, et al., 2004, Virology 330(2):375-83). AAV-2 is the most common serotype in the human population. One study estimated that at least 80% of the general population is infected with wt AAV-2 (Berns and Linden, 1995, Bioessays 17:237-245). AAV-3 and AAV-5 are also common in the human population, with infection rates of up to 60% (Georg-Fries, et al., 1984, Virology 134:64-71). Although AAV-1 and AAV-4 are simian isolates, both serotypes can transduce human cells (Chiorini, et al., 1997, J Virol 71:6823-6833; Chou, et al., 2000, Mol Ther 2:619-623). Of the six known serotypes, AAV-2 is the best characterized. For example, AAV-2 has been used in a wide range of in vivo transduction experiments and has been shown to transduce different tissue types, including mouse (U.S. Pat. No. 5,858,351; U.S. Pat. No. 6,093,392), dog muscle; mouse liver (Couto, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730; Couto, et al., 1997, J. Virol. 73:5438-5447; Nakai, et al., 1999, J. Virol. 73:5438-5447; and Snyder, et al., 1997, Nat. Genet. 16:270-276); mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); rabbit lung (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); and rodent photoreceptors (Flannery et al., 1997, Proc. Natl. Acad. Sci. USA 94:6916-6921).

[0053] The broad tissue tropism of AAV-2 can be exploited to deliver tissue-specific transgenes. For example, AAV-2 vectors have been used to deliver the following genes: the cystic fibrosis transmembrane conductance regulator gene to rabbit lungs (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); the factor NIII gene (Burton, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730); and the factor IX gene (Nakai, et al., 1999, J. Virol. 73:5438-5447; Snyder, et al., 1999, J. Virol. 73:5438-5447). al., 1997, Nat. Genet. 16:270-276; U.S. Patent No. 6,093,392) into mouse liver, dog, and mouse muscle (U.S. Patent No. 6,093,392); erythropoietin gene into mouse muscle (U.S. Patent No. 5,858,351); vascular endothelial growth factor (VEGF) gene into mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and aromatic 1-amino acid decarboxylase gene into monkey neurons. Expression of transgenes delivered by certain rAAVs has therapeutic effects in experimental animals: for example, expression of factor IX has been reported to restore phenotypic normality in a canine model of hemophilia B (U.S. Patent No. 6,093,392). Furthermore, expression of NEGF delivered by rAAV into mouse myocardium results in neovascularization (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806). al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and expression of AADC delivered by rAAV to the brains of monkeys with Parkinson's disease resulted in restoration of dopaminergic function.

[0054] The delivery of the protein to mammalian cells is achieved by first generating an AAV vector containing DNA encoding the protein, and then administering the vector to a mammal. Therefore, the present invention should be interpreted as including AAV vectors containing DNA encoding the protein. Upon understanding the present invention, the generation of AAV vectors containing DNA encoding these proteins will be clear to those skilled in the art.

[0055] In certain embodiments, the rAAV vectors of the present invention contain several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of the AAV ITR sequences, a promoter / enhancer element, a transcription termination signal, and any necessary 5' or 3' untranslated regions adjacent to the DNA encoding the protein or a biologically active fragment thereof. The rAAV vectors of the present invention may also contain a portion of the intron of the protein. Optionally, the rAAV vectors of the present invention also contain DNA encoding the mutant protein.

[0056] In certain embodiments, the vector comprises a promoter / regulatory sequence, including a promiscuous promoter, capable of driving high levels of heterologous gene expression in many different cell types. Such promoters include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter / enhancer sequence, the Rous sarcoma virus promoter / enhancer sequence, and the like. In certain embodiments, the promoter / regulatory sequence in the rAAV vectors of the present invention is the CMV immediate-early promoter / enhancer. However, the promoter sequence used to drive heterologous gene expression can also be an inducible promoter, such as, but not limited to, a steroid-inducible promoter, or a tissue-specific promoter, such as, but not limited to, the skeletal actin promoter and muscle creatine kinase promoter / enhancer, which are specific to muscle tissue.

[0057] In certain embodiments, the rAAV vectors of the invention comprise a transcription termination signal. While any transcription termination signal can be included in the vectors of the invention, in certain embodiments, the transcription termination signal is an SV40 transcription termination signal.

[0058] In certain embodiments, the rAAV vectors of the present invention comprise isolated DNA encoding the protein or a biologically active fragment of the protein. The present invention should be construed to include any mammalian sequence of the protein, whether known or unknown. Thus, the present invention should be construed to include genes from mammals other than humans, whose proteins function in a manner substantially similar to the human protein. Preferably, the nucleotide sequence comprising the gene encoding the protein is about 50% homologous to the gene encoding the protein, more preferably about 70% homologous, even more preferably about 80% or 85% homologous, and most preferably about 90%, 95%, or 99% homologous.

[0059] Furthermore, the present invention should be construed to include naturally occurring variants or recombinantly derived mutants of the wild-type protein sequence, which variants or mutants render the proteins encoded thereby as therapeutically effective as the full-length protein in the gene therapy methods of the present invention, or more therapeutically effective than the full-length protein.

[0060] The present invention should also be construed as including DNA encoding variants that retain the biological activity of proteins. Such variants include proteins or polypeptides that have been or can be modified using recombinant DNA technology, so that the proteins or polypeptides have additional properties that enhance their suitability for use in the methods described herein, for example, but not limited to, the variants confer enhanced stability to the protein in plasma and high specific activity to the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences, or by modifications that do not affect sequence, or both. For example, conservative amino acid changes can be made, which change the primary sequence of the protein or peptide but usually do not change its function.

[0061] The present invention is not limited to the particular rAAV vectors exemplified in the Examples, but rather should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV2.5, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, etc.

[0062] The invention also includes a method of treating a mammal having a disease or disorder, comprising administering to the mammal an rAAV vector comprising the protein in an amount effective to provide a therapeutic effect. Preferably, the mammal is a human.

[0063] Typically, the number of viral vector genomes / mammal administered in a single injection is approximately 1 x 10 8 ~Approx. 5×10 16 Preferably, the number of viral vector genomes / mammal administered in a single injection is in the range of about 1 x 10 10 ~Approx. 1×10 15 and more preferably, the number of viral vector genomes / mammal administered in a single injection is about 5×10 10 ~Approx. 5×10 15and most preferably, the number of viral vector genomes administered to a mammal in a single injection is about 5 x 10 11 ~Approx. 5×10 14 is.

[0064] When the methods of the invention involve multiple site simultaneous injections, or several multiple site injections, including injections at different sites over a period of several hours (e.g., from less than about 1 hour to about 2 or 3 hours), the total number of viral vector genomes administered can be the same as, or a fraction or multiple of, those recited in the single site injection method.

[0065] For administration of the rAAV vectors of the present invention via single-site injection, in certain embodiments, a composition containing the virus is injected directly into the subject's brain. For administration to mammals, the rAAV vector can be suspended in a pharmaceutically acceptable carrier, such as HEPES-buffered saline at a pH of about 7.8. Other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0066] The rAAV vectors of the invention may also be provided in the form of a kit, comprising, for example, a lyophilized preparation of the vector in a dry salt formulation, sterile water for suspension of the vector / salt composition, and instructions for suspension of the vector and its administration to a mammal.

[0067] Combination therapy Compositions identified using the methods described herein are useful in the methods of the invention in combination with one or more additional compounds or compositions useful in treating the diseases or alleviating the symptoms of the disorders contemplated herein. These additional compounds can include compounds identified herein or compounds known to treat, prevent, or alleviate the symptoms of the diseases or disorders contemplated herein, e.g., commercially available compounds.

[0068] Non-limiting examples of additional compounds include riluzole, edaravone, or their salts or solvates, or combinations thereof.Other compounds, including selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, alone or in combination with dextromethorphan and / or quinidine, can also be used in combination with the gene therapy regimen of the present invention.Therefore, synergistic effect can be calculated using suitable methods, such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the Loewe additivity equation (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each of the above equations can be applied to experimental data to generate corresponding graphs to help evaluate the effect of drug combinations. The corresponding graphs associated with the above equations are the concentration-effect curve, the isobologram curve, and the combination index curve, respectively.

[0069] Pharmaceutical Compositions and Formulations: The present invention also encompasses the use of the pharmaceutical compositions of the present invention for carrying out the methods of the present invention.Such pharmaceutical compositions can be provided in a form suitable for administration to a subject, and can include one or more pharmaceutically acceptable carriers, one or more additional components, or some combination thereof.At least one composition of the present invention can include a physiologically acceptable salt of the compound contemplated within the present invention, etc., in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0070] In one embodiment, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day, hi another embodiment, pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0071] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0072] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, nasal, buccal, ophthalmic, intrathecal, intracranial, intravenous, or other routes of administration. Other contemplated formulations include nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to those skilled in the art and will depend on any number of factors, including the type and severity of the disease being treated, the species and age of the animal or human patient being treated, and the like. Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then shaping or packaging the product into the desired single or multiple dosage unit, if necessary or desired.

[0073] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, for example, one-half or one-third of such a dosage. The unit dosage form can be for single daily administration or multiple daily administration (for example, about 1 to 4 times or more per day). When multiple daily administration is used, the unit dosage form can be the same or different for each dose.

[0074] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for human administration to make them suitable for administration to various animals are well understood, and those skilled in the art of veterinary pharmacology can design and implement such modifications with little, if any, experimentation. Subjects contemplated for administration of the pharmaceutical compositions of the present invention include, but are not limited to, commercially relevant mammals such as humans and other primates, cows, pigs, horses, sheep, cats, and dogs.

[0075] In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In another embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one composition of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, including, but not limited to, phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0076] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0077] The preparations can be used in a mixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, intrathecal, nasal, intravenous, subcutaneous, enteral, or any other suitable administration mode known in the art. Pharmaceutical preparations can be sterilized and, if necessary, can be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, coloring substances, flavoring substances, and / or aromatic substances. They can also be combined with other active agents, such as other analgesics, if necessary.

[0078] As used herein, "additional ingredients" includes, but is not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; biodegradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; bulking agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0079] The compositions of the present invention may contain a preservative in an amount of about 0.005% to 2.0% by weight of the total weight of the composition. Preservatives are used to prevent spoilage when exposed to environmental contaminants. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. An example of a preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0080] The compositions preferably contain antioxidants and chelating agents that inhibit the degradation of the compounds. Preferred antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid in a preferred range of about 0.01% to 0.3% by weight of the total composition, more preferably BHT in the range of 0.03% to 0.1% by weight. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total composition. Particularly preferred chelating agents include edetate (disodium edetate) and citric acid in a range of about 0.01% to 0.1% by weight of the total composition, more preferably 0.02% to 0.10% by weight. Chelating agents are useful for chelating metal ions in the compositions that may adversely affect the shelf life of the formulation. BHT and disodium edetate are particularly preferred antioxidants and chelating agents, respectively, for certain compounds, although other suitable and equivalent antioxidants and chelating agents may be substituted accordingly, as known to those skilled in the art.

[0081] Liquid suspensions can be prepared using conventional methods to suspend the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further contain one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions may further contain a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats and oils, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides with fatty acids, condensation products of long-chain aliphatic alcohols, condensation products of partial esters derived from fatty acids with hexitols, or condensation products of partial esters derived from fatty acids with hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0082] Liquid solutions of active ingredients in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, with the primary difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, "oily" liquids are those that contain carbon-containing liquid molecules and exhibit less polarity than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, and it should be understood that suspending agents do not necessarily aid in dissolving the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of the pharmaceutical preparations of the present invention can be prepared using known methods. Such formulations can be administered directly to a subject, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of a dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as bulking agents and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0083] The pharmaceutical compositions of the present invention can also be prepared, packaged, or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion. The oil phase can be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions can further contain one or more emulsifiers, such as naturally occurring gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soybean or lecithin phosphatides, esters or partial esters derived from the combination of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions can also contain additional ingredients, including, for example, sweeteners or flavoring agents.

[0084] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, depositing or bonding the chemical composition to a surface, incorporating the chemical composition into the structure of a material during its synthesis (i.e., with, for example, physiologically degradable materials), and absorbing aqueous or oily solutions or suspensions into absorbent materials, with or without subsequent drying.

[0085] Administration / Dosage: The dosage regimen can affect what constitutes an effective amount. The therapeutic formulation can be administered to a patient before or after the onset of symptoms associated with a disease or condition. Furthermore, several divided doses and staggered doses can be administered daily or sequentially, or the dose can be continuously infused or bolus injected. Furthermore, the dosage of the therapeutic formulation can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0086] The compositions of the present invention can be administered to a patient, preferably a mammal, more preferably a human, using known procedures at dosages and for periods of time effective to treat the patient's disease or condition. The effective amount of a therapeutic compound required to achieve a therapeutic effect can vary depending on factors well known in the medical field, such as the activity of the particular compound used; the timing of administration; the compound's excretion rate; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the state of the disease or disorder; and the age, sex, weight, condition, general health, and previous medical history of the patient being treated. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the present invention is about 0.01 to 50 mg / kg body weight / day. One of ordinary skill in the art would be able to examine the relevant factors and make the determination regarding the effective amount of a therapeutic compound without undue experimentation.

[0087] The compound can be administered to animals several times a day, or less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. It should be understood that the amount of compound administered per day can be administered, in non-limiting examples, every day, every other day, every two days, every three days, every four days, or every five days. For example, in every other day administration, a 5 mg / day dose is administered starting on Monday, the first subsequent 5 mg / day dose is administered on Wednesday, the second subsequent 5 mg / day dose is administered on Friday, etc. The frequency of administration will be readily apparent to those skilled in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0088] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being harmful to the patient.

[0089] A medical practitioner, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required; for example, the physician or veterinarian can start doses of the compounds of the invention used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0090] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the compound into dosage unit form.As used herein, "dosage unit form" refers to a physically discrete unit suitable as a unitary dose for the patient to be treated: each unit contains a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical vehicle.The dosage unit form of the present invention is determined by and directly depends on (a) the inherent characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such therapeutic compound for the treatment of cancer in patients.

[0091] In one embodiment, the compositions of the present invention are administered to a patient in dosages ranging from once to five or more times per day. In another embodiment, the compositions of the present invention are administered to a patient in dosage ranges including, but not limited to, once daily, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will vary from subject to subject depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, overall health, and other factors. Therefore, the present invention should not be construed as limited to any particular dosing regimen, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0092] The compound of the present invention to be administered may be administered in the following dosages: about 1 mg to about 7,500 mg, about 20 mg to about 7,000 mg, about 40 mg to about 6,500 mg, about 80 mg to about 6,000 mg, about 100 mg to about 5,500 mg, about 200 mg to about 5,000 mg, about 400 mg to about 4,000 mg, about 800 mg to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 ... The dose may range from about 100 mg to about 2,000 mg, from about 5 mg to about 1,000 mg, from about 10 mg to about 750 mg, from about 20 mg to about 600 mg, from about 30 mg to about 500 mg, from about 40 mg to about 400 mg, from about 50 mg to about 300 mg, from about 60 mg to about 250 mg, from about 70 mg to about 200 mg, from about 80 mg to about 150 mg, and all whole and partial increments therebetween.

[0093] In some embodiments, the dose of the compound of the invention is about 0.5 mg to about 5000 mg. In some embodiments, the dose of the compound of the invention used in the compositions described herein is less than about 5000 mg, or less than about 4000 mg, or less than about 3000 mg, or less than about 2000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of a second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and all whole and partial increments therebetween.

[0094] In one embodiment, the invention relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent, and instructions for using the compound to treat, prevent, or alleviate one or more symptoms of a disease or disorder in a patient.

[0095] The term "container" includes any receptacle for housing a pharmaceutical composition. For example, in one embodiment, the container is a package containing the pharmaceutical composition. In other embodiments, the container is not a package containing the pharmaceutical composition, i.e., the container is a receptacle such as a box or vial that contains a packaged or unpackaged pharmaceutical composition, as well as instructions for using the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. It should be understood that instructions for using the pharmaceutical composition may be included on the package containing the pharmaceutical composition, and thus the instructions enhance the functional relationship with the packaged product. However, it should be understood that the instructions may include information regarding the ability of the compound to perform its intended function, e.g., to treat, prevent, or alleviate a disease or disorder in a patient.

[0096] Route of administration: Routes of administration of any of the compositions of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., vaginal and perivaginal), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intracranial, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0097] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, and the like. It is understood that the formulations and compositions that may be useful in the present invention are not limited to the specific formulations and compositions described herein.

[0098] Oral administration For oral administration, tablets, dragees, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, or emulsions. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for tablet manufacture. Such excipients include inert diluents such as lactose, granulating and disintegrating agents such as cornstarch, binders such as starch, and lubricants such as magnesium stearate.

[0099] Tablets may be uncoated or may be coated using known methods to achieve delayed disintegration in the subject's gastrointestinal tract, thereby providing sustained release and absorption of the active ingredient. For example, tablets may be coated using materials such as glyceryl monostearate or glyceryl distearate. For further example, tablets may be coated using the methods described in U.S. Patent Nos. 4,256,108, 4,160,452, and 4,265,874 to form osmotically controlled release tablets. Tablets may further contain sweeteners, flavoring agents, coloring agents, preservatives, or some combination thereof to provide a pharmaceutically excellent and palatable preparation.

[0100] For oral administration, the compounds of the present invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders, fillers, lubricants, disintegrants, or wetting agents. If desired, tablets may be coated using a suitable method and coating material, such as the OPADRY™ film coating system (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400) available from Colorcon, West Point, Pa.

[0101] The liquid preparation for oral administration can be in the form of solution, syrup or suspension.Liquid preparation can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, methylcellulose or hydrogenated edible oils and fats), emulsifiers (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters or ethyl alcohol); and preservatives (for example, methyl or propyl parahydroxybenzoate or sorbic acid).The liquid preparation of the pharmaceutical composition of the present invention suitable for oral administration can be prepared, packaged and sold in liquid form or in the form of a dry product that is intended to be reconstituted with water or another suitable vehicle before use.

[0102] Parenteral administration As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically creating a hole in the tissue of a subject and administering the pharmaceutical composition through the hole in the tissue. Thus, parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injecting the composition, applying the composition through a surgical incision, applying the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is intended to include, but is not limited to, intracranial, subcutaneous, intravenous, intraperitoneal, intramuscular, intraspinal, intrasternal, intrathecal, brainstem injection, and kidney dialysis infusion techniques.

[0103] Pharmaceutical compositions suitable for parenteral administration include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powdered or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0104] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using, for example, non-toxic parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0105] Further dosage forms Additional dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the present invention also include those described in U.S. Patent Application Nos. 2003 / 0147952, 2003 / 0104062, 2003 / 0104053, 2003 / 0044466, 2003 / 0039688, and 2002 / 0051820. Additional dosage forms of the present invention also include those described in PCT Application Nos. WO03 / 35041, WO03 / 35040, WO03 / 35029, WO03 / 35177, WO03 / 35039, WO02 / 96404, WO02 / 32416, WO01 / 97783, WO01 / 56544, WO01 / 32217, WO98 / 55107, WO98 / 11879, WO97 / 47285, WO93 / 18755, and WO90 / 11757.

[0106] Sustained-Release Formulations and Drug Delivery Systems The sustained-release or sustained-release formulation of the pharmaceutical composition of the present invention can be prepared using conventional technology.In some cases, the dosage form used can be provided as a slow or sustained release of one or more active ingredients therein, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes or microspheres, or their combinations, to provide different ratios of desired release profiles.Suitable sustained-release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the pharmaceutical composition of the present invention.Therefore, the present invention includes single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps and caplets, which are adapted for sustained release.

[0107] Most sustained-release pharmaceutical products share a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed sustained-release formulation in pharmaceutical therapy will be characterized by the use of a minimum amount of active pharmaceutical ingredient to cure or control a condition in a minimum amount of time.

[0108] Advantages of sustained-release formulations include extended drug activity, reduced dosing frequency, and increased patient compliance. Additionally, sustained-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can therefore affect the occurrence of side effects.

[0109] Most sustained-release dosage forms are designed to initially release an amount of drug that quickly produces the desired therapeutic effect, and then gradually and continuously release other amounts of drug to maintain this level of therapeutic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

[0110] The sustained release of an active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "sustained-release ingredient" in the context of the present invention is defined herein as a compound(s), including but not limited to, a polymer, a polymer matrix, a gel, a permeable membrane, a liposome, or a microsphere, or a combination thereof, that promotes the sustained release of an active ingredient.

[0111] In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, fast-release, and sustained-release, including sustained-release, delayed-release, and pulsed-release formulations.

[0112] The term sustained release is used in its conventional sense to refer to a drug formulation that gradually releases drug over an extended period of time, which may, but does not necessarily, result in substantially constant blood levels of drug over an extended period of time, which may extend to a month or more and should result in a longer release than the same amount of drug administered in bolus form.

[0113] For sustained release, compound can be formulated with suitable polymer or hydrophobic material that provides compound with sustained release properties.Therefore, the compound for using the method of the present invention can be administered, for example, in the form of microparticles by injection or in the form of wafer or disk by implantation.In the preferred embodiment of the present invention, the compound of the present invention is administered to patients using sustained release formulation, alone or in combination with other pharmaceuticals.

[0114] The term delayed release is used herein in its conventional sense to refer to a drug formulation that initially releases drug after some delay following drug administration, which may, but does not necessarily, include a delay of from about 10 minutes up to about 12 hours.

[0115] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that releases drug in such a manner as to produce a pulsatile plasma profile of the drug following drug administration.

[0116] The term immediate release is used in its conventional sense to refer to a formulation that releases drug immediately after drug administration.

[0117] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any whole or partial increments thereof.

[0118] As used herein, rapid elimination refers to any period up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any whole or partial increments thereof.

[0119] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the claims appended hereto. For example, it is understood that variations of reaction conditions, including but not limited to reaction times, reaction sizes / volumes, and experimental reagents such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, using art-recognized substitutes and no more than routine experimentation, are within the scope of this application.

[0120] definition To aid in understanding the detailed description of the compositions and methods according to the present disclosure, several explicit definitions are provided to facilitate clear disclosure of the various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0121] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0122] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full-length coding sequence or any portion of a coding sequence, so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. The term "portion" when used with respect to a gene refers to a fragment of that gene. The size of the fragment can vary from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0123] The term "gene" encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends, approximately 1 kb apart, so that the length of the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and may contain regulatory elements such as enhancers. Introns are removed, or "spliced ​​out," from the nuclear or primary transcript; therefore, introns are absent in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0124] "Gene transfer" and "gene delivery" refer to a method or system for reliably inserting specific nucleic acid sequences into target cells.

[0125] The term "recombinant" when made with respect to a nucleic acid molecule refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term "recombinant" when made with respect to a protein or polypeptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0126] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that allows the latter to be expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are adjacent and, where necessary to link two protein coding regions, are in the same reading frame.

[0127] As used herein, the term "in vitro" refers to events that occur not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0128] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0129] As used herein, "treatment" or "treating," or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any treatment-related improvement in or effect on one or more diseases, conditions, or symptoms being treated. In the case of prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0130] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition, but is at risk of or susceptible to developing the disorder or condition.

[0131] The term "disease" as used herein is intended to be generally synonymous with, and is used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that all reflect an abnormal condition that prevents one of the human or animal body parts from functioning normally, is typically manifested by distinguishing signs and symptoms, and reduces the length or quality of life of a human or animal.

[0132] The terms "reduce," "decreased," "reduction," "reduce," or "inhibit" are all generally used herein to mean a statistically significant reduction. However, for the avoidance of doubt, "decreased," "reduction," or "reduce," or "inhibit" means a reduction of at least 10% compared to the reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% reduction, or a reduction of less than 100% (the absence of a level compared to a reference sample), or any reduction of 10-100% compared to the reference level.

[0133] As used herein, the term "modulate" is meant to refer to any change in a biological state, ie, an increase, a decrease, etc.

[0134] The terms "increased," "increase," "enhancement," or "activation" are all used herein to generally mean a statically significant increase. For the avoidance of doubt, the terms "increased," "increase," "enhancement," or "activation" mean an increase of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of less than or equal to 100%, or any increase of 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase of 2-fold to 10-fold or more compared to a reference level.

[0135] The terms "effective amount," "effective dose," or "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount of the drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing a disease or at risk of disease recurrence, inhibits the onset or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the onset or recurrence of a disease can be evaluated using a variety of methods known to skilled practitioners, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0136] Doses are often expressed in relation to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight," even if the term "body weight" is not explicitly mentioned.

[0137] The term "agent" is used herein to refer to a chemical compound, a mixture of compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological material such as a bacterial, plant, fungal, or animal (e.g., mammalian) cell or tissue. The activity of such agents may qualify them as "therapeutic agents," which are biologically, physiologically, or pharmacologically active substances or substances that act locally or systemically in a subject.

[0138] The terms "therapeutic agent," "therapeutic potential agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and counteracting the disease, symptom, disorder, or pathological condition altogether.

[0139] As used herein, "combination" therapy, unless otherwise clear from the context, means to include the administration of two or more therapeutic agents in a coordinated manner, including but not limited to simultaneous administration. Specifically, combination therapy includes both simultaneous administration (e.g., administration of a combination drug or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is somehow conditioned on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after another therapeutic agent has been administered and allowed to act for a predetermined period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0140] The terms "sample," "test sample," and "patient sample" may be used interchangeably herein. A sample may be a serum, urine plasma, amniotic fluid, cerebrospinal fluid, cell (e.g., antibody-producing cells), or tissue sample. Such samples may be used directly as obtained from a patient or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to alter the characteristics of the sample in some manner as discussed herein or otherwise known in the art. As used herein, the terms "sample" and "biological sample" generally refer to biological material that is tested for and / or suspected of containing an analyte of interest, such as an antibody. A sample may be any tissue sample from a subject. A sample may contain proteins from a subject.

[0141] As used herein, "homology" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both molecules is occupied by the same monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a 10-subunit-long polymer) are homologous, the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous. As an example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

[0142] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes that gene product to be produced in a cell substantially only when an inducer corresponding to that promoter is present in the cell.

[0143] As used herein, the terms "inhibit" and "antagonize" refer to reducing by a measurable amount or completely preventing the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein. Inhibitors are compounds, e.g., antagonists, that bind to proteins, genes, and mRNAs and partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize, or downregulate stability, expression, function, and activity.

[0144] "Instructional material," as that term is used herein, includes publications, records, drawings, or any other medium of expression that can be used to communicate the utility of any of the compositions and / or compounds of the present invention in a kit. The kit's instructional material can, for example, be affixed to a container containing any of the compositions of the present invention or shipped together with a container containing any of the compositions. Alternatively, the instructional material can be shipped separately from the container with the intention that the recipient use the instructional material and any of the compositions cooperatively. Delivery of the instructional material can be, for example, by physical delivery of a publication or other medium of expression that communicates the utility of the kit, or alternatively can be achieved by electronic transmission by computer, such as e-mail, or downloading from a website.

[0145] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0146] An "isolated nucleic acid" refers to a nucleic acid segment or fragment separated from sequences that flank it in its naturally occurring state, i.e., a DNA fragment removed from sequences that normally flank the fragment, i.e., the sequences that flank the fragment in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., the RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule independent of other sequences (i.e., as a cDNA or genomic or cDNA fragment generated by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0147] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may include introns to the extent that nucleotide sequences that encode proteins may, in some versions, contain introns.

[0148] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intrathecal, or infusion techniques.

[0149] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful within the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients, that facilitate administration of the compound to an organism.

[0150] Multiple techniques exist in the art for administering compounds, including but not limited to intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0151] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the composition and that is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0152] The phrase "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting a compound(s) of the present invention within or to a subject so that it may perform its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and glycols such as propylene glycol. Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifiers; coloring agents; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delayers that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the compositions.

[0153] As used herein, the phrase "pharmaceutically acceptable salts" refers to salts of the administered compound prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

[0154] "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides.

[0155] Conventional notation is used herein to depict polypeptide sequences, with the left end of the polypeptide sequence being the amino terminus and the right end of the polypeptide sequence being the carboxyl terminus. As used herein, a "peptidomimetic" is a compound that contains non-peptide structural elements that can mimic the biological action of a parent peptide. Peptidomimetics may or may not contain peptide bonds.

[0156] The term "promoter" as used herein is defined as a DNA sequence that is recognized by the synthetic machinery of a cell, or introduced synthetic machinery, and is required to initiate the specific transcription of a polynucleotide sequence.

[0157] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0158] As used herein, the term "recombinant polypeptide" is defined as a polypeptide produced by using recombinant DNA methodology. As used herein, the term "recombinant DNA" is defined as DNA produced by joining pieces of DNA from different sources.

[0159] As used herein, the term "RNA" is defined as ribonucleic acid. The terms "specifically bind" or "specifically binds," as used herein, mean that a first molecule (e.g., an antibody) binds preferentially to a second molecule (e.g., a particular antigenic epitope), but not necessarily exclusively to that second molecule.

[0160] As used herein, "subject" refers to a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. In certain embodiments, the subject is a human. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0161] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0162] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct position and orientation relative to the polynucleotide, so as to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.As used herein, "variant" refers to a nucleic acid sequence or peptide sequence that differs in sequence from the reference nucleic acid sequence or peptide sequence, respectively, but retains the essential properties of the reference molecule.The change in the sequence of the nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitution, addition, deletion, fusion, and truncation.The change in the sequence of the peptide variant is typically limited or conservative, so that the sequences of the reference peptide and the variant are very similar overall, and are identical in many regions.

[0163] A variant and a reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A nucleic acid or peptide variant may be a naturally occurring variant, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring nucleic acid and peptide variants can be produced by mutagenesis techniques or direct synthesis.

[0164] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0165] As used herein, the term "virus" is defined as a particle consisting of nucleic acid (RNA or DNA) enclosed in a protein coat, with or without an outer lipid envelope, that is capable of transfecting cells with that nucleic acid.

[0166] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0167] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0168] The terms "including," "comprising," "containing," or "having," and variations thereof, unless expressly stated otherwise, are meant to encompass the items listed thereafter and equivalents thereof, as well as additional subject matter.

[0169] The phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. are used repeatedly. Such phrases do not necessarily refer to the same embodiment, although they may if the context indicates otherwise.

[0170] The term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0171] The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definition of the present invention.

[0172] As used herein, the term "approximately" or "about," when applied to one or more values, refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about," unless otherwise stated or apparent from the context, refers to a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value (except where such number exceeds 100% of possible values). Unless otherwise indicated herein, the term "about" is intended to include values ​​near the stated range, e.g., weight percent, that are equivalent in terms of the function of the individual component, composition, or embodiment.

[0173] Whenever values ​​and ranges are provided herein, it is understood that all values ​​and ranges subsumed within those values ​​and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application.

[0174] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set. Exceptions may occur where explicit disclosure or context clearly dictates otherwise.

[0175] Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to further clarify the invention and do not impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0176] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the method steps can be performed simultaneously or sequentially. When method steps are performed sequentially, the steps can be performed in any order unless otherwise indicated.

[0177] Where a method includes a combination of steps, any and all combinations or subcombinations of the steps are included within the scope of the disclosure unless otherwise stated herein.

[0178] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it does not contradict this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing contained herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0179] It is to be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. [Example]

[0180] Example 1 Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without purification. As described in this paper, patient-derived induced pluripotent stem cells (iPSCs) were obtained from an NIH-funded biorepository (www.nimhgenetics.org / available_data / ipsc / ). We used both normal healthy cells and cells from individuals with a mutation in SOD1 (N139K) diagnosed with familial ALS. A differentiation protocol has been established that results in the culture of motor neurons after 40 days of treatment with a defined regimen of growth factors and specialized media (Figure 1). Treatment (ASPA) and control (GFP) genes were packaged into AAV vectors, and initial cohorts of cells were treated to assess their effects on mitochondrial function. Mitochondria isolated from SOD1 mutant motor neurons were shown to have a lower ATP synthesis rate compared to wild-type cells, but transduction of these mutant cells with AAV-ASPA significantly restored mitochondrial ATP synthesis rate compared to AAV-GFP controls, as assessed by a luminescence-based in situ assay (Figure 2).

[0181] Example 2 Analysis of the stimulation of ATP synthesis by ASPA-derived free aspartate in mitochondria isolated from the spinal cord of 16-week-old SOD G93A mice. Mitochondria were isolated from 16-week-old SOD G93A whole spinal cords using mechanical homogenization and differential centrifugation. Mitochondria were kept on ice until use in the assay. The rate of ATP synthesis was analyzed in 30 μg of isolated mitochondria using a commercially available luminescence-based kit. A reaction mixture containing 1.0 mM malate, 1.0 mM glutamate, 10 mM NADH, and 0.2 mM ADP was prepared in a solution containing luciferase and luciferin, and 30 μg of mitochondria was added. The addition of 2 U / ml aspartate aminotransferase and 3 U / ml malate dehydrogenase drives MAS and initiates ATP synthesis. In this example, free aspartate was used in place of the reaction product generated by incubating lysates from cells overexpressing either wild-type ASPA or a nonfunctional mutant ASPA with 5 mM NAA, according to the scheme in Figure 8. Addition of wild-type ASPA reaction products to SOD spinal cord mitochondria significantly increased the rate of ATP synthesis as measured by luminescence over a 3-minute period, whereas addition of the nonfunctional E285A ASPA mutant reaction product did not (Figure 9). The aspartate content of these reaction products was assessed by HPLC (Table 1), indicating a >800-fold increase in aspartate in the wild-type ASPA reaction product.

[0182] Table 1 shows the aspartate content of the reaction product after 2 hours of incubation of 50 μl lysate from HeLa cells transfected with wild-type ASPA plasmid (WT) or nonfunctional E285 ASPA (E285A) with 5 mM NAA. Reactions were performed in a 200 μl mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.5 mM DTT, 0.05% IPEGAL CA630, and 5 mM NAA. The reaction was terminated by heating to 95°C for 3 minutes. 20 μl of the reaction mixture was assayed for aspartate concentration by HPLC analysis of OPA-derivatized samples. Five individual samples were assayed for each transfection group. [Table 2]

[0183] Example 3 AAV-ASPA treatment of SOD G93A mice Eight-week-old male SOD G93A mice were given 1x10 11 Mice were transduced with the AAV9-CBh-ASPA vector genome (vg). Mice were anesthetized by inhalation anesthesia (isoflurane, 4% induction, maintenance dose titrated to effect), and the vector was delivered in a volume of 5 μl. Control animals received 5 μl of saline (0.9%) via the same route of administration (ROA). From 9 to 16 weeks of age, animals were tested on an accelerating rotarod (4-40 rpm) once a week. The mean fall latency over three consecutive 3-minute trials was recorded (with a 30-second rest period between each trial). Weeks 9 to 11 were designated the training session, and fall latencies at weeks 12, 13, 14, 15, and 16 were compared between the saline-treated and AAV9-ASPA-treated groups (n = 15 per group). Rotarod analysis was performed by an individual blinded to the treatment groups. Mutant mice treated with AAV9-ASPA showed long-term improvements in rotarod performance (latency to fall), which were statistically significant at 15 and 16 weeks of age (p=0.0218 and 0.0271, respectively).

[0184] Increased NAA catabolism in AAV9-ASPA-treated SOD G93A animals is associated with an increased ATP:AMP ratio.

[0185] NAA is characteristically decreased in association with increasing pathological energy deficits in a wide range of neurodegenerative diseases, including ALS. The central hypothesis of the current study is that this metabolic response is due to an attempt to uncouple NAA synthesis from mitochondrial oxidative phosphorylation and ATP synthesis, due to the shared requirement for free aspartate by both synthetic processes. Therefore, as shown in Figure 9, the liberation of aspartate from endogenous NAA by exogenously supplied ASPA would be expected to support mitochondrial ATP synthesis via a shuttle mechanism that facilitates the import of cytosolic aspartate into mitochondria for use in the mitochondrial electron transport chain (ETC). The current intervention is expected to support this hypothesis, with data consistent with NAA-supplied aspartate acting as a fuel for the ETC, measured as an increase in available energy currency. The key metric in this context is the function of the ASPA transgene. The function of AAV-delivered ASPA was confirmed in spinal cords isolated from 16-week-old SOD G93A animals immediately following rotarod analysis at 16 weeks of age. Age-matched C57BL / 6J wild-type (non-SOD) male mice were used as a calibration reference control. Spinal cords of AAV9-ASPA and saline-controlled SOD G93A mice were analyzed for NAA, AMP, and ATP content by HPLC. Snap-frozen whole spinal cords were obtained from 16-week-old animals immediately after rotarod analysis at 16 weeks of age. Whole spinal cords were homogenized in precipitation solution using a mechanical dispersion element and extracted with chloroform. Samples prepared in this manner were aliquoted and stored at -80°C for subsequent analysis. Absolute molar concentrations of target metabolites were calculated using standard curves generated from purified reference standards. In line with previously reported reductions in this metabolite in both clinical and animal model populations, 16-week-old saline-treated SOD G93A mice exhibited reduced spinal NAA compared to wild-type controls (p=0.0015, n=5 / group, Figure 11). Spinal cords of saline-treated mutant mice also showed a reduced ATP:AMP ratio (p=0.0134), suggesting that ATP hydrolysis (i.e., use) exceeded its synthesis.Spinal cords treated with SOD G93A AAV9-ASPA showed an additional 1.7-fold reduction in NAA compared to saline-treated controls (p=0.011, n=5 / group), associated with a significant increase in the ATP:AMP ratio (p=0.0045), suggesting the function of the AAV-delivered ASPA transgene, increasing ATP production and improving energy status. This implies that increased NAA catabolic products resulting from AAV9-ASPA transduction support bioavailable energy currency, with associated benefits for motor function (Figure 10).

[0186] Spinal cord mitochondria treated with AAV9-ASPA exhibit increased levels of ATP synthesis relative to available NAA-derived aspartate.

[0187] Intact mitochondria were isolated from the spinal cords of saline- and AAV9-ASPA-treated SOD G93A mutant mice, as well as from 16-week-old age-matched wild-type controls, and used to assess ATP synthesis rate using a luminescence-based assay. Mitochondria from each cohort were provided with ADP, and the rate of conversion to ATP was assessed over a 3-minute period and expressed as the average ATP synthesis rate (nM / min / mg of mitochondrial protein) (Figure 12). The ATP synthesis rate of saline-treated spinal cord mitochondria from 16-week-old SOD G93A mice was reduced 1.7-fold compared to age-matched wild-type controls (p=0.00058), indicating a pathological energy crisis. The ATP synthesis rate of AAV9-ASPA-treated SOD G93A mitochondria was significantly increased compared to saline controls (p=0.0022) (Figure 10B), consistent with an improved ATP:AMP ratio, demonstrating that the promotion of mitochondrial oxidative metabolism by ASPA-catabolized aspartate can reverse the decline in motor function.

[0188] While the present invention has been described with emphasis on illustrative embodiments, it will be apparent to those skilled in the art that variations in composition and method may be used, and that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

1. 1. A therapeutically effective amount of a composition for use in treating a subject at risk of developing or suffering from ALS by increasing the amount of neuronal aspartate in the spinal cord and brain by increasing NAA catabolism in the spinal cord and brain for the benefit of mitochondrial function, the composition comprising a nucleic acid encoding aspartoacylase (ASPA) or a functional fragment thereof, the nucleic acid having an amino acid sequence at least 90% identical to the sequence of SEQ ID NO: 1, carried in a recombinant adeno-associated virus (rAAV) vector; The composition for use, wherein the subject is identified by screening the subject for ALS.

2. The composition for use according to claim 1 , wherein the ASPA or functional fragment thereof does not include one containing the full-length amino acid sequence of SEQ ID NO:

1.

3. 3. The composition for use according to claim 1 or 2, wherein administration of the composition increases NAA catabolism in brain and spinal cord tissue and enhances motor neuron survival in the subject.

4. 4. The composition for use according to any one of claims 1 to 3, wherein the subject is administered a second therapeutic agent.

5. 5. The composition for use of claim 4, wherein the second therapeutic agent is administered to the subject before, after, or simultaneously with the composition.

6. 6. The composition for use according to claim 5, wherein the second therapeutic agent is riluzole, edaravone, or a salt or solvate thereof, or a combination thereof.

7. The composition for use according to claim 1 , wherein the composition is provided as a gene therapy composition.

8. 8. The composition for use according to any one of claims 1 to 7, wherein the nucleic acid is introduced into at least one cell of the subject by viral transduction, preferably the composition is provided with a virus or virus-like particle comprising the nucleic acid.

9. 9. The composition for use according to any one of claims 1 to 8, wherein the composition is administered to at least a portion of the brain and spinal cord of the subject.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the composition is administered by a route selected from oral, parenteral, transdermal, pulmonary, intranasal, buccal, intrathecal, and intravenous, preferably the intrathecal route.

11. The composition for use according to any one of claims 1 to 10, wherein the subject is a mammal, or preferably a human.

12. 12. The composition for use of any one of claims 1 to 11, wherein treating a subject comprises treating at least one symptom of ALS or mitochondrial dysfunction.

13. 13. The composition for use according to any one of claims 1 to 12, wherein the rAAV vector is an rAAV1 vector.

14. 1. A kit for use in treating a subject at risk of developing or suffering from ALS by increasing the amount of neuronal aspartate in the spinal cord and brain by increasing NAA catabolism in the spinal cord and brain for the benefit of mitochondrial function, comprising: The kit comprises an rAAV1 vector or a virus-like particle comprising an rAAV1 vector; A kit, wherein the virus or virus-like particle comprises a nucleic acid encoding aspartoacylase (ASPA) or a functional fragment thereof, the nucleic acid having an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:

1.

15. A composition for use according to any one of claims 1 to 13, or a kit according to claim 14, wherein the functional fragment has the enzymatic activity of ASPA.

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