Treatment or prevention methods for amyotrophic lateral sclerosis (ALS)

Abstract

We provide medicines to treat amyotrophic lateral sclerosis (ALS). [Solution] A pharmaceutical is provided for treating a subject at risk of developing ALS or who is suffering from ALS, comprising a therapeutically effective amount of a composition comprising a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75% identical to a specific sequence supported on a recombinant adeno-associated virus (rAAV) vector, wherein the subject is identified by screening for ALS, and the pharmaceutical is administered to the subject's brain, spinal cord, or brainstem.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 724,780, filed on 30 August 2018 under Section 119(e) of the U.S. Patent Act. The aforementioned application is incorporated herein by reference.

[0002] The present invention relates, in general terms, to methods for treating or preventing amyotrophic lateral sclerosis (ALS), and more specifically, to methods for treating, alleviating, improving, or reversing ALS by increasing the intracellular activity of aspartoacylase (ASPA), the rate-limiting component of the malate-aspartate shuttle, which provides affected cells with the ability to utilize cytosolic NADH to promote mitochondrial oxidative phosphorylation in affected cell populations, for the purpose of providing aspartic acid. [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 functional impairment and the loss of upper motor neurons in the motor cortex, as well as lower motor neurons in the brainstem, spinal cord, and associated tracts. Patients with ALS, or at risk of developing it, experience progressive weakness that worsens with muscle stiffness, spasms, and decreased muscle size. As a result, they have difficulty speaking, swallowing, and eventually breathing.

[0004] While there is no known cure for ALS, the FDA has approved two therapies specifically aimed at slowing the progression of the disease. Riluzole (Rilutek®) is the first FDA-approved drug for ALS and can extend life by approximately 2-3 months in a finite clinical population with medullary onset. Edaravone (Radicava®) is another FDA-approved treatment option for ALS administered intravenously. Clinical data suggest improvement in ALS Functional Rating Scale (ALSFRS-R) scores compared to a control placebo group. However, the long-term efficacy of edaravone in ALS patients has not yet been determined. Non-invasive ventilation may improve both quality of life and lifespan, but it is essentially strictly palliative. The disease can occur in people of any age, but it usually begins around age 60, and around age 50 in hereditary cases. The average survival time from onset to death is 2-4 years. About 10% survive for more than 10 years, most of whom die from respiratory failure.

[0005] Therefore, there remains an urgent and unmet need in the art to provide methods and reagents for preventing, treating, or reversing ALS. [Overview of the project]

[0006] This disclosure addresses the above-mentioned needs in several embodiments. In one embodiment, this disclosure provides a method for treating, improving, or reversing at least one symptom of amyotrophic lateral sclerosis (ALS) in a subject requiring it. The method involves administering to a subject a therapeutically effective amount of a composition that increases the level or activity of aspartoacylase (ASPA) in the cells of the subject, with the aim 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 that is at least 75%, 85%, 95%, or 99% identical to the sequence of SEQ ID NO: 1.

[0008] In some embodiments, the method involves introducing nucleic acids into at least one target cell by viral transduction. The composition can provide a virus or virus-like particle containing nucleic acids. In some embodiments, the nucleic acids are supported on a recombinant adeno-associated virus (rAAV) vector such as AAV9.

[0009] In some embodiments, this method involves administering the composition to at least a portion of the target spinal cord. In some embodiments, the composition is administered topically to a portion of the target spinal cord.

[0010] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. The second therapeutic agent may be administered to the subject before, after, or concurrently 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-pyrazole-3-one), or a salt or solvate thereof. In some embodiments, the composition may be administered by a route selected from oral, parenteral, transdermal, transpulmonary, intranasal, buccal, intrathecal, and intravenous.

[0011] In some embodiments, the subject is a mammal such as a human. In some embodiments, at least one cell is located within the subject's spinal cord. 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 to mitochondrial structure, dynamics, and bioenergy 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 the substrates for mitochondrial energy metabolism in the subject. In some embodiments, administration of the composition will increase cell survival in the subject. In some embodiments, administration of the composition will increase motor neuron survival in the subject. In some embodiments, administration of the composition will extend the life expectancy of the subject.

[0013] Also within the scope of this disclosure are kits for increasing the level or activity of ASPA in target cells within the scope of this disclosure. The kit comprises an rAAV vector or virus-like particles, the virus or virus-like particles 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 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 drawing]

[0014] For the purpose of illustrating the present invention, specific embodiments of the present invention are shown in the drawings. However, the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. For the data shown in the drawings, asterisks indicate the following levels of statistical significance: *p≦0.05, **p≦0.01, and ***p≦0.001.

[0015] [Figure 1] Using established methodologies, we show neurons differentiated in vitro from iPSCs derived from ALS patients after 40 days (Kiskinis et al., 2014, Cell Stem Cell). The cells are positive for the neuronal marker TuJ-1 and the motor neuron marker choline acetyltransferase (ChAT). [Figure 2] This study demonstrates the recovery of mitochondrial adenosine triphosphate (ATP) synthesis after treatment with AAV-ASPA. Motor neuron cultures generated from ALS iPSCs were transduced in culture on day 28 with adeno-associated virus vector (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 / group). A significant 1.5-fold increase in ATP synthesis rate (jiM ATP / min / jig of isolated mitochondria, 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] NAA has been shown to contain acetyl coenzyme A (AcCoA), derived from glycolysis, and aspartate, which is used to transport the cytosolic reduction equivalent (NADH) to the inner mitochondrial membrane. Both are substrates for mitochondrial ATP synthesis, and aspartate plays a crucial role in linking glycolysis to oxidative phosphorylation by electron transport chain complexes I-V. Therefore, releasing this substrate in neurons affected by neurodegenerative diseases increases energy conservation. [Figure 4A]Figure 4 (collectively referred to as "Figure 4") shows 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 microscope image of GFP-expressing neurons 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 co-labeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a faint shade in the black and white copy of the image) (Figure 4D), thus supporting in vivo neuronal targeting. Therefore, using an appropriate AAV, the therapeutic gene can be delivered to neurons in the mammalian brain to the extent that it can be appropriately packaged. The data were generated using publicly available methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4B] Figure 4 (collectively referred to as "Figure 4") shows 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 microscope image of GFP-expressing neurons 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 co-labeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a faint shade in the black and white copy of the image) (Figure 4D), thus supporting in vivo neuronal targeting. Therefore, using an appropriate AAV, the therapeutic gene can be delivered to neurons in the mammalian brain to the extent that it can be appropriately packaged. The data were generated using publicly available methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4C] Figure 4 (collectively referred to as "Figure 4") shows 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 microscope image of GFP-expressing neurons 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 co-labeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a faint shade in the black and white copy of the image) (Figure 4D), thus supporting in vivo neuronal targeting. Therefore, using an appropriate AAV, the therapeutic gene can be delivered to neurons in the mammalian brain to the extent that it can be appropriately packaged. The data were generated using publicly available methodologies (Francis, et al., 2006, Journal of Neuroscience Research 84(1):151-169; Francis, et al., 2011, Glia 59(10):1435-1446). [Figure 4D] Figure 4 (collectively referred to as "Figure 4") shows 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 microscope image of GFP-expressing neurons 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 co-labeled with NeuN (Figure 4C) are shown emitting a merged yellow signal (appearing as a faint shade in the black and white copy of the image) (Figure 4D), thus supporting in vivo neuronal targeting. Therefore, using an appropriate AAV, the therapeutic gene can be delivered to neurons in the mammalian brain to the extent that it can be appropriately packaged. The data were generated using publicly available 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 targeting of synaptic function using gene therapy. Endogenous NAA, catabolized by recombinant ASPA delivered by AAV, increases the 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 glycolysis in the cytosol is transported to the inner mitochondrial membrane space by aspartate aminotransferase, which transfers hydrogen ions to aspartate to produce malate. Aspartate moves from the mitochondria to the cytosol in exchange for glutamate. Subsequently, the aspartate in the cytosol is converted to malate, which can carry hydrogen ions from cytosolic NADH and move freely into the mitochondria. Once inside, malate is converted back to aspartate by malate dehydrogenase, thereby releasing hydrogen ions to form NADH, which can then be used to promote mitochondrial oxidative phosphorylation. [Figure 7] This study demonstrates that free aspartate, generated by the cleavage of NAA by ASPA, becomes available to the malate-aspartate shuttle (MAS), which transports reduced NADH produced by glycolysis to the mitochondria. This available NADH then drives electron transport chain complexes I-V, culminating in the newly synthesized ATP. [Figure 8]This is a schematic diagram of the methodology for demonstrating oxidative phosphorylation and ATP synthesis driven by free aspartate produced by ASPA in isolated spinal cord mitochondria. (1) HeLa cells in a 60 mm dish were transfected with a plasmid of constitutively expressed wild-type human ASPA (WT) or a non-functional mutant isoform (E285A). (2) Transfected cells were harvested after 48 hours and mechanically lysed by sonication. (3) 50 μl of lysate was added to a reaction mixture containing 5 mM purified NAA and incubated at 37°C for 2 hours to catabolize the NAA substrate with the transfected ASPA enzyme. (4) The catabolic reaction was inactivated by heat and 50 μl was added to isolated mitochondria in a reaction mix for a luminescence-based ATP synthesis assay. [Figure 9] Figure 8 schematically shows the ATP synthesis rates in mitochondria incubated with reaction mixtures derived from HeLa cells isolated from G93A SOD spinal cord and transfected with wild-type ASPA, HeLa cells transfected with E285A ASPA, or HeLa cells treated with physiological saline. Addition of the reaction product fueled by wild-type ASPA significantly increased the ATP synthesis rate in proportion to the aspartic acid content of the aliquot (p=0.039) (see Table 1). [Figure 10] This shows the mean latency at 12–16 weeks of age in SOD G93A mutant mice treated with either intrathecal saline or AAV9-ASPA. The mean of three individual trials is shown along with the standard error of the mean (n=15 / group). [Figure 11A](Collectively referred to as "Figure 11") shows the HPLC analysis of N-acetylaspartate (NAA) and adenosine triphosphate (ATP):adenosine monophosphate (AMP) ratio (hereinafter referred to as ATP:AMP ratio) in the spinal cords of 16-week-old mice of wild type, SOD treated with physiological saline, and SOD treated with AAV9-ASPA. A). A significant decrease in spinal cord NAA was observed in saline-SOD mice compared to age-matched wild type, along with a related decrease in the ATP:AMP ratio (B), suggesting a pathological decrease in NAA in response to a decrease in ATP production for use (reflected in the level of AMP). The level of NAA further decreased in the spinal cords of SOD treated with AAV9-ASPA, but was associated with a corresponding increase in ATP:AMP, suggesting an increase in ATP synthesis. [Figure 11B] (Collectively referred to as "Figure 11") shows the HPLC analysis of N-acetylaspartate (NAA) and adenosine triphosphate (ATP):adenosine monophosphate (AMP) ratio (hereinafter referred to as ATP:AMP ratio) in the spinal cords of 16-week-old mice of wild type, SOD treated with physiological saline, and SOD treated with AAV9-ASPA. A). A significant decrease in spinal cord NAA was observed in saline-SOD mice compared to age-matched wild type, along with a related decrease in the ATP:AMP ratio (B), suggesting a pathological decrease in NAA in response to a decrease in ATP production for use (reflected in the level of AMP). The level of NAA further decreased in the spinal cords of SOD treated with AAV9-ASPA, but was associated with a corresponding increase in ATP:AMP, suggesting an increase in ATP synthesis. [Figure 12]Shows the ATP synthesis rate in mitochondria isolated from wild-type, saline-treated SOD, and SOD spinal cord treated with AAV9-ASPA. Mitochondria were assayed using a commercially available luminescence-based kit. The ATP synthesis rate in mitochondria isolated from AAV9-ASPA-transduced SOD spinal cord was significantly superior to that of SOD spinal cord mitochondria treated with saline, suggesting that the supply of NAA-derived aspartic acid via ASPA gene therapy is an effective means of enhancing energy metabolism and presents relevant advantages for the motor function in ALS. Mean ATP synthesis rate, standard error + / - presented (n = 5 / group).

Mode for Carrying Out the Invention

[0016] The present invention relates to the unexpected discovery that, in one embodiment, increasing the expression of aminoacylases such as aspartoacylase (ASPA) in cells can be used to prevent, treat, or reverse one or more symptoms of ALS in subjects requiring it. 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 another embodiment, the compositions and methods of the present invention promote cell survival in ALS patients. In yet another embodiment, the compositions and methods of the present invention promote motor neuron survival in ALS patients. In yet another embodiment, overexpression of ASPA in the spinal cord extends the life expectancy of ALS patients. In yet another embodiment, the treatment utilizes patient-derived motor neurons generated from induced pluripotent stem cells (iPSCs). In yet another embodiment, 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 further embodiments, ASPA expression and construction of the delivery construct are performed ex vivo. In some embodiments, ASPA expression may show a reduced rate of degeneration of human muscle tissue explants ex vivo compared to those of ALS subjects. In another embodiment, the treatment is a one-dose gene therapy for ALS and related disorders.

[0017] While the etiology of major neurodegenerative diseases is multifactorial and not fully defined, therapeutic strategies that enhance mitochondrial integrity may slow the progressive loss of higher-order functions. In particular, in the case of ALS, mitochondrial dysfunction is thought to be directly or indirectly related to all hypothetical toxic mechanisms associated with ALS, including excitotoxicity, loss of protein homeostasis, and defects in axonal transport. Despite differences in hypothetical pathogenic mechanisms in different in vitro and in vivo models studied, decreased mitochondrial electron transport chain (ETC) activity and ATP levels appear as common features in both familial and sporadic ALS.

[0018] This invention provides a novel gene therapy intervention to supply neurons in ALS with a means to access specific energy substrates 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 cleaving lipid synthesis from oxidative phosphorylation via the supply of AcCoA through catabolism by the glial hydrolase ASPA. Neurons do not spontaneously express ASPA and therefore cannot catabolize NAA. Catabolism of NAA by ASPA yields free acetate (used by glia for AcCoA synthesis) and aspartate. Unlike acetate, aspartate in neurons is a specific substrate of a subunit of the malate-aspartate shuttle (Aralar1) and is therefore an essential component of the shuttle mechanism for transporting glycolytic reducing equivalents to mitochondria (Figure 3). Free acetate is insufficient as a substrate for neuronal energy metabolism because, compared to glial cells, there are relatively few available biochemical mechanisms that can process acetate for energy metabolism, and acetate cannot replace glucose in supporting neuronal function. Analysis of the flux of radiolabeled acetate supplied to the nervous system has demonstrated, in fact, that it is primarily used by glial cells compared to neurons, and that acetate is not involved in the transport of cytosolic NADH to mitochondrial ETC by Aralar1, a process that requires the conversion of aspartate to malate in the cytosol. In certain non-limiting embodiments, reserve ATP storage in the face of pathological metabolic abnormalities is promoted by providing aspartate for the shuttle of glycolytic reduction equivalents to mitochondria by providing neurons with the ability to catabolize endogenous NAA. In at least one embodiment, the present invention relates to a method for increasing the availability of aspartate for the shuttle of glycolytic reduction equivalents to mitochondria in subjects at risk of developing or suffering from ALS.

[0019] Adeno-associated viruses (AAVs) have emerged as a highly promising and attractive approach to gene delivery with an established clinical safety and efficacy profile, and are highly efficient at targeting neurons. Advances in AAV vector design and associated administration techniques have enabled broad gene delivery in the brain and spinal cord, making AAVs highly suitable for the treatment of neurodegenerative diseases. In at least one embodiment, the present invention utilizes an AAV serotype for delivering 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 Sequence ID No. 1.

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

[0021] In one embodiment, the 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 a therapeutically effective amount of a composition to the subject 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, subjects requiring treatment of the present invention are those who have experienced a decline in motor function or are at risk of experiencing motor dysfunction accompanied by a medical condition different from cognitive impairment. In another embodiment, subjects at risk of motor dysfunction and exhibiting a hypermetabolic state that may show a metabolic ratio greater than 1 when measured based on the Harris-Benedict equation are candidates for the proposed intervention. In another embodiment, subjects in need may exhibit defects related to motor neurons in the spinal cord with a high metabolic demand for maintaining action potentials that may not be presented by other diseases that do not primarily affect the motor system. In another embodiment, subjects in need may exhibit progressive, painless, progressive muscle weakness accompanied by stumbling, dropping objects, abnormal fatigue of the arms and / or legs, slurred speech, muscle spasms and contractions, and / or periods of uncontrollable laughter or crying. In some embodiments, subjects suffering from respiratory muscle weakness may require permanent ventilatory support to assist breathing.

[0023] In other embodiments, subjects requiring treatment of the present invention may exhibit mutations that may be decisive in the proper diagnosis and onset of ALS, including mutations in, for example, ALS2 (arsin), 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 (balosin-containing protein), or enzymes associated with ALS (e.g., KIF5A, inesin family member 5A), and OPTN (optinulin). In yet another embodiment, the mutation may be in the C9ORF72 gene or may cause an accumulation of RNA that occurs when the gene is mutated. In yet another embodiment, subjects may be screened for the presence of such mutations. Furthermore, in some embodiments, the screening is for identifying at least two or more of the mutations identified above.

[0024] In another embodiment, the present invention relates to administering a therapeutically effective gene therapy to subjects exhibiting mutations in ALS2, TBK1, TUBA4A, ANG, MATR3, CHCHD10, NEK1, PFN1, C21ORF2, MOBP, SCFD1, SETX, FUS, TDP43, VCP, or OPTN. In yet another embodiment, a suitable screened subject may 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 yet another embodiment, an identified subject may be administered a gene therapy composition comprising SEQ ID NO: 1 and AAV-9, exhibiting increased levels of NAA catabolism in the spinal cord. In at least one embodiment, the present invention relates to administering a disclosed gene therapy to patients exhibiting clinical symptoms of ALS, and to patients exhibiting ALS symptoms that may show mutations in TBK1, TUBA4A, NEK1, C21ORF2, MOBP, SCFD1, FUS, and TDP43, or any combination of such mutations, by identifying at least one of the gene mutations occurring in proteins involved in cell axon dynamics and proteins involved in cell clearance mechanisms.

[0025] In at least one embodiment, the present invention relates to administering the gene therapy of the present invention to subjects exhibiting a mutation in Cu / Zn superoxide dismutase (SOD1), and in particular to increasing the level of NAA catabolism in spinal mitochondria of such patients presenting with at least one clinical manifestation of ALS. Only 5–10% have a genetic origin (familial ALS), and it has been suggested that only about 20% of familial ALS cases have a mutation in Cu / Zn superoxide dismutase (SOD1). Mutations in SOD1 associated with ALS cause a decrease in protein stability. These mutations occur throughout the protein structure, including the active site, β-sheet, and monomer interface. For this purpose, at least one embodiment of the present invention relates to a method for administering the gene therapy of the present invention to patients exhibiting a SOD1 mutation. In yet another embodiment, the present invention relates to a method comprising identifying a subject having a SOD1 mutation and administering to the subject a composition comprising a nucleic acid encoding ASPA or a fragment thereof, comprising the amino acid sequences of SEQ ID NO: 1 and AAV-9, and increasing the level of NAA catabolism in the spinal cord of such subject.

[0026] In other embodiments, the subjects in question exhibit upper and lower motor neuron degeneration, with or without progressive brainstem degeneration, impaired neuronal status, or muscle weakness after electromyography (EMG) testing, compared to healthy patients. However, such patients do not exhibit decreased dopamine receptor occupancy, are unresponsive to L-dopa administration, and do not exhibit symptoms associated with cognitive loss, such as those seen in 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] Furthermore, the scope of this disclosure includes variants, mutants, and homologs having significant identity with ASPA. For example, such variants and homologs have at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequence of ASPA described herein.

[0029] When used in relation to polypeptides, the terms “variant” and “mutant” refer to an amino acid sequence in which one or more amino acids differ from another normally associated polypeptide. A variant may have a “conservative” change in which the substituted amino acids have similar structural or chemical properties. Certain conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains are serine and threonine; the group of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Very rarely, 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 in determining which and how many amino acid residues can be substituted, inserted, or deleted without loss of 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 less than 10%, preferably less than 5%, and more preferably less than 2% change (substitution, deletion, etc.).

[0030] The terms “homologous” or “homologous,” when used in reference to polypeptides, refer 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, homologous sequences have more than 60% sequence identity with a reference sequence, more preferably more than 75%, and even more preferably more than 90%. The term “substantial identity” as applied to polypeptides means that two peptide sequences share at least 75% sequence identity when optimally aligned by a program such as GAP or BESTFIT using default gap weights.

[0031] As used herein, gene expression means that a 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 having biological activity substantially similar to that of the entity or molecule that is the 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 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 at levels exceeding those of normal or untransformed cells / organisms. For example, it could refer to high levels (e.g., abnormal levels) of mRNA encoding a protein(s) (e.g., ASPA protein or its homologs) and / or high levels of protein(s) (e.g., ASPA) compared to a similar corresponding unmodified cell / organism expressing basal levels of mRNA (e.g., one encoding the ASPA protein) or a similar corresponding unmodified cell / organism having basal levels of the protein. In certain embodiments, ASPA or its homologs may be overexpressed 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 cells / organisms designed to exhibit increased mRNA, protein, and / or ASPA activity.

[0033] ASPA expression can be induced by introducing one or more expression vectors having nucleic acids encoding one or more ASPA polypeptides or fragments thereof. The polypeptides or fragments thereof can be inserted into appropriate sites in the vector (e.g., operably ligated to a promoter). The expression vectors are introduced into host cells selected for amplification and / or polypeptide expression by well-known methods such as transfection, transduction, infection, electroporation, microinjection, lipofection, or the DEAE-dextran method or other known techniques. These methods and other suitable methods are well known to those skilled in the art.

[0034] A wide variety of vectors can be used for ASPA protein expression. Due to their ability to infect or enter cells via receptor-mediated endocytosis and their ability to integrate into the host cell genome and stably and efficiently express viral genes, they are attractive candidates for transporting foreign nucleic acids into cells. Therefore, in certain embodiments, viral vectors are used to introduce nucleotide sequences encoding the ASPA protein or fragments into host cells for expression. Viral vectors may contain one or more regulatory sequences, such as nucleotide sequences encoding the ASPA protein or fragments operably linked to a promoter. Alternatively, viral vectors may not contain regulatory sequences and instead rely on regulatory sequences within the host cell to drive the expression of the ASPA protein or fragments. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenovirus vectors, AAV vectors, and retroviral vectors.

[0035] For example, adeno-associated viruses (AAVs) can be used to introduce nucleotide sequences encoding the ASPA protein or fragments thereof into host cells for expression. AAV systems have been previously described and are 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). Details relating to the generation and use of rAAV vectors are described, for example, in U.S. Patents 5,139,941 and 4,797,368, which are incorporated herein by reference in their entirety for all purposes.

[0036] In some embodiments, retroviral expression vectors can be used to introduce nucleotide sequences encoding the ASPA protein or fragments thereof into host cells for expression. These systems have been previously described and are generally well 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, herpesvirus, retrovirus, etc.

[0037] The combination of retroviruses with appropriate packaging strains can also be useful, as the capsid protein functions to infect target cells. Typically, cells and viruses are cultured in culture medium for at least about 24 hours. Cells are then grown in culture medium for a shorter period, e.g., 24–73 hours, or at least 2 weeks, in some applications, and can be grown for 5 weeks or more before analysis. Commonly used retroviral vectors are "defective," meaning they cannot produce the viral proteins necessary for replication and infection. Vector replication requires growth in a packaging cell line. The host cell specificity of a retrovirus is determined by the envelope protein env(pl20). The envelope protein is provided by the packaging cell line. There are at least three types of envelope proteins: allotropic, bitropic, and heterotropic. Retroviruses packaged with allotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types. An allotropic packaging cell line is BOSC23. Retroviruses with bispecific envelope proteins, such as 4070A, can infect most mammalian cell types, including human, canine, and mouse cells. Bispecific packaging cell lines include PA12 and PA317. Retroviruses packaged with heterospecific envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells. Vectors may contain genes that must be subsequently removed using a recombinase system such as Cre / Lox, or cells expressing them may be disrupted by containing genes that enable selective toxicity, such as herpesvirus TK, bcl-xs. Appropriate inducible promoters are activated in the desired target cell type, which is either the transfected cell or its offspring.

[0038] In some embodiments, genome editing technologies such as the CRISPR / Cas9 system, designer zinc fingers, activator-like effectors (TALEs), or homing meganucleases can be used to induce the expression of described ASPA proteins in cells. Generally, the “CRISPR / Cas9 system” refers collectively to transcripts and other elements involved in the expression or induction of the activity of CRISPR-related (”Cas”) genes, including the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (including “direct repeats” and partial direct repeats processed by tracrRNA with respect to the endogenous CRISPR system), the guide sequence (also referred to as “spacers” with respect to the endogenous CRISPR system), or other sequences and transcripts from the CRISPR locus. One or more elements of the CRISPR system may originate from the type I, type II, or type III CRISPR system. Alternatively, one or more elements of the CRISPR system may originate from specific organisms, including endogenous CRISPR systems such as Streptococcus pyogenes. Generally, the CRISPR system is characterized by elements (also called protospacers in relation to the endogenous CRISPR system) that promote the formation of the CRISPR complex at the site of a target sequence. In some embodiments, genome editing technologies such as the CRISPR / Cas9 system, designer zinc fingers, activator-like effectors (TALEs), or homing meganucleases can be used to induce the expression of described ASPA proteins in cells, increasing the substrates for mitochondrial oxidative phosphorylation.

[0039] In some embodiments, the method involves introducing nucleic acids into at least one target cell by viral transduction. The composition may provide a virus or virus-like particle containing nucleic acids. In some embodiments, the nucleic acids are supported on a recombinant adeno-associated virus (rAAV) vector such as AAV9.

[0040] In some embodiments, rAAVs are artificially generated from their natural environment (e.g., from host cells, tissues, or subjects). For example, isolated AAVs can be generated using recombinant methods. Such rAAVs preferably have tissue-specific targeting capabilities such that the AAV transgene is specifically delivered to one or more predetermined tissues. The AAV capsid is a key factor in determining their tissue-specific targeting. For this purpose, in at least one embodiment, recombinant AAVs having a capsid suitable for spinal cord tissue can be selected. A method for obtaining recombinant AAVs having a desired capsid protein is described, for example, in Japanese Patent Application Publication No. 2003 / 0138772, which is incorporated herein by reference in its entirety.

[0041] In another embodiment, the present invention provides a method for treating, improving, or reversing at least one symptom of ALS in a subject requiring an increase in intracellular aspartate levels, by identifying such patient, 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, having an amino acid sequence at least 75% identical to the sequence of SEQ ID NO: 1.

[0042] In some embodiments, the method involves administering the composition to at least a portion of the target spinal cord. In some embodiments, the composition is administered topically to a portion of the target spinal cord. In some embodiments, the composition may be administered to the brainstem for use in the treatment of ALS. In another embodiment, the composition of the present invention for use in the treatment of ALS may be combined with a second pharmaceutical compound to treat upper and lower motor neuron degeneration, slow its progression, or improve upper and lower motor function. In some embodiments, the second pharmaceutical compound may be R(+)-N-propargyl-1-aminoindan in combination with 2-amino-6-trifluoromethoxybenzothiazole or any 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 useful proteins(s) within the present invention may 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 systems that support motor function, including upper and lower motor neurons of the brain and spinal cord. In certain embodiments, the disease or disorder includes amyotrophic lateral sclerosis (superoxide dismutase aggregation). Improved constructs encoding protein(s) may 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 conventionally used to introduce genes into a wide variety of target cells. Typically, the vector is exposed to target cells in such a way that transformation can occur at a rate sufficient to provide a useful therapeutic or preventive effect from the expression of the desired polypeptide (e.g., a receptor). The transfected nucleic acid may be permanently integrated into the genome of each target cell, providing long-term effects, or the treatment may need to be repeated periodically.

[0045] Various vectors, both viral and plasmid vectors, are known in the art (see, for example, U.S. Patent No. 5,252,479 and WO93 / 07282). In particular, numerous viruses, including pavova viruses such as SV40, vaccinia viruses, herpesviruses including HSV and EBV, and retroviruses, have been used as gene transfer vectors. Many gene therapy protocols in the prior art use defective mouse retroviruses. Several recently issued patents relate to methods and compositions for carrying out gene therapy (see, for example, U.S. Patents No. 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 genus Dependovirus, possesses 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 is not associated with any human or animal disease and is not thought to alter the physiological properties of host cells when incorporated.

[0047] Finally, AAV is stable under a wide range of physical and chemical conditions, making it suitable for manufacturing, storage, and transport requirements. 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-repeat segment flanked by terminal inversion sequences (ITRs) at both ends. The ITR is approximately 145 nucleotides long (AAV-1 has an ITR of 143 nucleotides) and has multiple functions, including functioning as an origin of replication and as a packaging signal for the viral genome. The internal non-repeat portion of the genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode the replication and capsid gene products, enabling the replication, assembly, and packaging of the complete AAV virion. More specifically, at least four families of viral proteins, Rep78, Rep68, Rep52, and Rep40, are expressed from the AAV rep region, all named after their apparent molecular weight. 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 vacciniavirus) is required to form a functionally complete AAV virion. In the absence of co-infection with a helper virus, AAV either inserts its viral genome into the host cell's chromosome or establishes a latent state where the viral genome exists in episomal form, but no infectious virion is produced.

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

[0049] To produce infectious recombinant AAV (rAAV) containing heterologous nucleic acid sequences, an appropriate host cell line can be transfected with an AAV vector containing heterologous nucleic acid sequences but lacking AAV helper functional genes, rep, and cap. The AAV helper functional genes can then be provided to another vector. Furthermore, instead of providing a reproducible helper virus (such as an adenovirus, herpesvirus, or vaccinia), only the helper virus genes necessary for AAV production (i.e., accessory functional genes) can be provided to the vector.

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

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

[0052] AAV-1 to AAV-11 are described in the relevant technical field (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). AAV-1 and AAV-4 are monkey isolates, but both serotypes can be transduced into 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 (US Patent No. 5,858,351, US Patent No. 6,093,392), canine 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-specificity of AAV-2 can be utilized 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); factor NIII gene (Burton, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730); and factor IX gene (Nakai, et al., 1999, J. Virol. 73:5438-5447; Snyder, et al. The gene has been introduced into mouse liver, dog, and mouse muscle (US Patent No. 6,093,392) (Su, et al., 1997, Nat. Genet. 16:270-276; U.S. Patent No. 6,093,392); the erythropoietin gene into mouse muscle (US Patent No. 5,858,351); the vascular endothelial growth factor (VEGF) gene into mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and the aromatic 1-amino acid decarboxylase gene into monkey neurons. Expression of transgenes delivered by specific 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 (US Patent No. 6,093,392). Furthermore, expression of NEGF delivered to mouse cardiomyocytes by rAAVs results in neovascularization (Su, et al. (al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and the expression of AADC delivered by rAAV to the brains of monkeys with Parkinson's disease resulted in the restoration of dopaminergic function.

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

[0055] In certain embodiments, the rAAV vector of the present invention comprises several essential DNA elements. In certain embodiments, these DNA elements comprise at least two copies of the AAV ITR sequence, a promoter / enhancer element, a transcription termination signal, and any necessary 5' or 3' untranslated region adjacent to the DNA encoding the protein or a bioactive fragment thereof. The rAAV vector of the present invention may also comprise a portion of the introns of the protein. Optionally, the rAAV vector of the present invention may also comprise the DNA encoding the mutant protein.

[0056] In certain embodiments, the vector includes a promoter / regulatory sequence containing 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, cytomegalovirus (CMV) pre-early promoter / enhancer sequences and Roussarcoma virus promoter / enhancer sequences. In certain embodiments, the promoter / regulatory sequence in the rAAV vector of the present invention is a CMV pre-early promoter / enhancer. However, the promoter sequence used to drive heterologous gene expression may also be an inducible promoter, e.g., a steroid-inducible promoter, or a tissue-specific promoter, e.g., a skeletal actin promoter and a muscle creatine kinase promoter / enhancer, which are muscle tissue-specific, etc.

[0057] In certain embodiments, the rAAV vector of the present invention includes a transcription termination signal. Any transcription termination signal may be included in the vector of the present invention, but in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.

[0058] In certain embodiments, the rAAV vector of the present invention comprises isolated DNA encoding the protein or a biologically active fragment of the protein. The present invention should be interpreted as comprising any mammalian sequence of the protein, whether known or unknown. Accordingly, the present invention should be interpreted as comprising a gene of a non-human mammal in which the protein functions substantially similarly to a human protein. Preferably, the nucleotide sequence comprising the gene encoding the protein is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% or 85% homologous, and most preferably about 90%, 95%, or 99% homologous to the gene encoding the protein.

[0059] Furthermore, the present invention should be interpreted as including naturally occurring variants or recombinant variants of wild-type protein sequences, wherein the variant or variant makes the protein it encodes as therapeutically effective as, or even more therapeutically effective than, the full-length protein in the gene therapy of the present invention.

[0060] The present invention should also be interpreted as including DNA encoding variants that preserve the biological activity of a protein. Such variants include proteins or polypeptides modified or modifiable using recombinant DNA technology, so that the protein or polypeptide has additional properties that enhance its suitability for use in the methods herein, for example, but not limited to, variants conferring enhanced stability to the protein in plasma and high specific activity of the protein. Analogues may differ from naturally occurring proteins or peptides by conserved amino acid sequence differences, by modifications that do not affect the sequence, or both. For example, conserved amino acid changes may occur that alter the primary sequence of the protein or peptide but usually do not alter its function.

[0061] The present invention is not limited to the specific rAAV vectors illustrated in the experimental examples. Rather, the present invention should be interpreted as 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., as well as any suitable AAV vector.

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

[0063] Typically, the number of viral vector genomes / mammals administered in a single injection is approximately 1 × 10⁶. 8 ~Approx. 5×10 16 This is within the range. Preferably, the number of viral vector genomes / mammals administered by a single injection is approximately 1 × 10⁶. 10 ~Approx. 1×10 15 More preferably, the number of viral vector genomes / mammals administered in a single injection is approximately 5 × 10⁶. 10 ~Approx. 5×10 15Therefore, most preferably, the number of viral vector genomes administered to a mammal by a single injection is approximately 5 × 10⁶. 11 ~Approx. 5×10 14 That is the case.

[0064] If the method of the present invention involves simultaneous injection at multiple sites, or several multi-site injections including injections at different sites over a period of several hours (e.g., less than about 1 hour to about 2 or 3 hours), the total number of viral vector genomes administered may be the same as, a part of, or a multiple of those listed for single-site injection.

[0065] For the administration of the rAAV vector of the present invention by single-site injection, in certain embodiments, the virus-containing composition is injected directly into the brain of the target. For administration to mammals, the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES-buffered saline at pH 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 organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0066] The rAAV vector of the present invention may also be provided in the form of a kit, which includes, for example, a lyophilized preparation of the vector in a dry salt formulation, sterile water for suspending the vector / salt composition, and instructions for suspending the vector and administering it to a mammal.

[0067] Combination therapy The compositions identified using the methods described herein are useful in the methods of the present invention in combination with one or more additional compounds or compositions useful for treating the diseases or alleviating the symptoms of the disorders intended herein. These additional compounds may include the compounds identified herein or compounds known to treat, prevent, or reduce the symptoms of the diseases or disorders intended herein, such as commercially available compounds.

[0068] Non-limiting examples of additional compounds include riluzole, edaravone, or their salts or solvates, or combinations thereof. Other compounds containing selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, either alone or in combination with dextromethorphan and / or quinidine, can also be used in combination with the gene therapy regimen of the present invention. Thus, synergistic effects can be calculated using appropriate 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). The equations described above can be applied to experimental data to generate corresponding graphs, which can be used to evaluate the effects of drug combinations. The corresponding graphs associated with the equations described above are the concentration-effect curve, the isobologram curve, and the combination exponential 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 may be provided in a form suitable for administration to a subject and may comprise one or more pharmaceutically acceptable carriers, one or more additional components, or several combinations thereof. At least one composition of the present invention may comprise a physiologically acceptable salt of a compound intended in the present invention in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0070] In one embodiment, a pharmaceutical composition useful for carrying out the method of the present invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In another embodiment, a pharmaceutical composition useful for carrying out the present 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 the pharmaceutical composition of the present invention vary depending on the identity, size, and condition of the target being treated, as well as the route through which the composition is administered. For 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 can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, transpulmonary, intranasal, buccal, ophthalmic, intrathecal, intracranial, intravenous, or other routes of administration. Other intended formulations include hypothetical nanoparticles, liposome preparations, resealed red blood cells containing active ingredients, and immunological formulations. The route of administration(s) 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 type and age of the animal or human patient being treated, etc. Formulations of the pharmaceutical compositions described herein can be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods include associating the active ingredient with a carrier or one or more other accessory components, and then, if necessary or desirable, shaping or packaging the product into desired single or multi-dose units.

[0073] As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a given amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to the subject, or a convenient fraction of such a dose, for example, half or one-third of such a dose. The unit dosage form may be for once-daily administration or multiple-daily administration (e.g., about one to four times or more per day). If multiple-daily administration is used, the unit dosage form may be the same or different for each dose.

[0074] The descriptions of pharmaceutical compositions provided herein primarily concern pharmaceutical compositions suitable for ethical administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. It is well understood that modifying pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals is possible, and those skilled in the art of veterinary pharmacology can design and carry out such modifications with little experiment, if any. The subjects to whom the pharmaceutical compositions of the present invention are intended to be administered include, but are not limited to, humans and other primates, cattle, pigs, horses, sheep, cats, and dogs, and other commercially relevant mammals.

[0075] In one embodiment, the composition of the present invention is formulated using one or more pharmaceutically acceptable excipients or carriers. In other embodiments, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of at least one composition of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and organic acid salts. 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, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol, in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0077] The formulations can be used in mixtures 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 mode of administration known in the art. The pharmaceutical formulations may be sterilized and, if necessary, mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic buffering, colorants, flavorings, and / or aromatics. They may also be combined with other activators, such as other analgesics, if necessary.

[0078] As used herein, “additional components” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; biodegradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, lubricants; buffers; salts; thickeners; bulking agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional components” that may 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 are 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. The preservative is used to prevent spoilage when exposed to contaminants in the environment. Examples of useful preservatives according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, 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 composition preferably contains antioxidants and chelating agents that inhibit the degradation of the compound. 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 weight of the composition, and more preferably BHT in a 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 weight of the composition. Particularly preferred chelating agents include edetates (disodium edetate) and citric acid in a range of about 0.01% to 0.1% by weight of the total weight of the composition, more preferably 0.02% to 0.10% by weight. The chelating agent is useful for chelating metal ions in the composition that may adversely affect the shelf life of the formulation. BHT and disodium edetate are particularly preferred antioxidants and chelating agents for certain compounds, respectively, but as is known to those skilled in the art, other suitable and equivalent antioxidants and chelating agents may therefore be substituted.

[0081] Liquid suspensions can be prepared using conventional methods for achieving suspension of active ingredients in aqueous or oily vehicles. 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 include one or more additional components, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, lubricants, preservatives, buffers, salts, flavorings, colorants, and sweeteners. Oily suspensions may further include thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible oils and fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides and fatty acids, condensation products of long-chain aliphatic alcohols, condensation products of fatty acid-derived partial esters and hexitol, or condensation products of fatty acid-derived partial esters and anhydrous hexitol (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners 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, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, “oily” liquids contain carbon-containing liquid molecules and exhibit properties of lower polarity than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain any of the components described with respect to liquid suspensions, and it should be understood that the suspending agent does not necessarily help dissolve the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, vegetable oils such as almond oil, oily esters, ethyl alcohol, 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 may 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 to them. Each of these formulations may further contain one or more of the following: dispersants or humectants, suspending agents, and preservatives. Additional excipients such as bulking agents and sweeteners, flavoring agents, or coloring agents may also be included in these formulations.

[0083] The pharmaceutical compositions of the present invention may also be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase may be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions may further contain one or more emulsifiers, such as naturally occurring rubbers such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soy or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and anhydrous hexitol such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional components, such as sweeteners or flavorings.

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

[0085] Administration / Medication: The administration plan may affect what constitutes the effective dose. Therapeutic formulations may be administered to patients before or after the onset of symptoms associated with the disease or condition. Furthermore, several divided doses and time-staggered doses may be administered daily or sequentially, or the dose may be administered by continuous infusion or bolus injection. In addition, the dose of the therapeutic formulation may be increased or decreased proportionally, as indicated by the urgency of the therapeutic or prophylactic situation.

[0086] The administration of the compositions of the present invention to patients, preferably mammals, more preferably humans, may be carried out using known procedures in doses and for durations effective in treating the patient's disease or condition. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors well known in the medical field, such as the activity of the particular compound used; the timing of administration; the excretion rate of the compound; 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 status, and prior medical history of the patient being treated. The administration plan may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. A non-limiting example of the effective dose range of the therapeutic compound of the present invention is about 0.01 to 50 mg / kg body weight / day. Those skilled in the art will be able to examine the relevant factors and make a determination regarding the effective amount of the therapeutic compound without excessive experimentation.

[0087] The compound may be administered to animals several times a day, or less frequently, for example, once a day, once a week, once every two weeks, once a month, or once every few months, or even less than once a year. It should be understood that the amount of the compound administered per day may, in non-limiting examples, be daily, every other day, every two days, every three days, every four days, or every five days. For example, in an every-other-day administration, a 5 mg / day dose may be initiated on Monday, followed by a first subsequent 5 mg / day dose on Wednesday, and a second subsequent 5 mg / day dose on Friday. The frequency of administration will depend on any number of factors, such as the type and severity of the disease being treated, the type and age of the animal, etc., and will be readily apparent to those skilled in the art.

[0088] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be modified to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and mode of administration, without being harmful to the patient.

[0089] A physician with ordinary skills in the art, such as an internist or veterinarian, can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, an internist or veterinarian can start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0090] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate compounds into dosage units. As used herein, “dosage unit” refers to a physically distinct unit suitable as a unit dose for a patient being treated: each unit contains a predetermined amount of the therapeutic compound calculated to produce the desired therapeutic effect in conjunction with the necessary pharmaceutical vehicle. The dosage units of the present invention are determined and directly depend 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 to formulate / manufacture such therapeutic compounds for the treatment of cancer in a patient.

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

[0092] The administered amounts of the compound of the present invention are approximately 1 mg to 7,500 mg, approximately 20 mg to 7,000 mg, approximately 40 mg to 6,500 mg, approximately 80 mg to 6,000 mg, approximately 100 mg to 5,500 mg, approximately 200 mg to 5,000 mg, approximately 400 mg to 4,000 mg, approximately 800 mg to 3,000 mg, approximately 1 mg to 2,500 mg, and approximately 2 mg. The dosage ranges from approximately g to 2,000 mg, 5 mg to 1,000 mg, 10 mg to 750 mg, 20 mg to 600 mg, 30 mg to 500 mg, 40 mg to 400 mg, 50 mg to 300 mg, 60 mg to 250 mg, 70 mg to 200 mg, and 80 mg to 150 mg, and can be any full or partial increment in between.

[0093] In some embodiments, the dose of the compound of the present invention is about 0.5 mg to about 5000 mg. In some embodiments, the dose of the compound of the present 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 1000 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 the 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 any full or partial increment in between.

[0094] In one embodiment, the present invention relates to a packaged pharmaceutical composition comprising a container for containing a therapeutically effective amount of the compound of the present invention, either alone or in combination with a second pharmaceutical product, 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 container 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; that is, the container is a box or vial or similar container containing a packaged or unpackaged pharmaceutical composition, as well as instructions for use of the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. Instructions for use of the pharmaceutical composition may be included on the package containing the pharmaceutical composition, and it should be understood that the instructions enhance the functional relationship with the packaged product. However, it should be understood that the instructions may include information about the compound's intended function, for example, its ability to treat, prevent, or alleviate a patient's disease or disorder.

[0096] Route of administration: Routes of administration of any composition of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, tongue, (trans)chuccal, (trans)urethral, ​​vagina (e.g., transvaginal and perivaginal), nasal (intra) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intracranial, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0097] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersants, suspensions, liquids, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma preparations, lozenges, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosol preparations for inhalation, compositions and preparations for intravesical administration, etc. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the specific formulations and compositions described herein.

[0098] Oral administration Tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable for oral administration. Other formulations suitable for oral administration include, but are not limited to, powders or granules, 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 and non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include inert diluents such as lactose, granulators and disintegrants such as corn starch, 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 target 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. Further example, tablets may be coated using the methods described in U.S. Patents 4,256,108, 4,160,452, and 4,265,874 to form osmotically controlled release tablets. Tablets may further contain sweeteners, flavorings, colorings, preservatives, or combinations thereof to provide a pharmaceutically superior 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 using pharmaceutically acceptable excipients such as binders, fillers, lubricants, disintegrants, or wetting agents. If necessary, the tablets may be coated using appropriate methods and coating materials, such as the OPADRY™ film coating system available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400).

[0101] Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible oils and fats), emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl parahydroxybenzoic acid or sorbic acid). Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, and sold in liquid form or in the form of dry products intended for reconstitution 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 creating a physical hole in the target tissue and administering the pharmaceutical composition through that hole in the tissue. Therefore, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of the composition through a surgical incision, application of the composition through a non-surgical wound penetrating the tissue, etc. In particular, parenteral administration is intended to include, but is not limited to, intracranial, subcutaneous, intravenous, intraperitoneal, intramuscular, intraspinal, intrasternal, intrathecal, brainstem injection, and renal dialysis infusion techniques.

[0103] Formulations of pharmaceutical compositions suitable for parenteral administration include an 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 forms suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, for example in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and portable sustained-release or biodegradable formulations. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before parenteral administration of the reconstituted composition.

[0104] Pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile, injectable formulations may be prepared, for example, using water or a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solutions, and fixative oils such as synthetic monoglycerides or diglycerides. Other useful parenterally administered formulations include those containing the active ingredient as a component in microcrystalline form, liposome preparations, or biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0105] Further forms of administration 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 Applications 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 applications 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 Sustained-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage form used may be provided as a slow or sustained release of one or more active ingredients, for example, using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres, or combinations thereof, to provide a desired release profile in different ratios. Suitable sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the present invention. Accordingly, single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps, and caplets adapted for sustained release, are included in the present invention.

[0107] Most sustained-release drugs share the common goal of improving pharmacotherapy beyond what is achieved by their corresponding uncontrolled drugs. Ideally, the use of optimally designed sustained-release formulations in pharmaceutical treatment is characterized by the use of minimal active pharmaceutical ingredients to cure or control a condition in minimal time.

[0108] The advantages of sustained-release formulations include extended drug activity, reduced administration frequency, and improved patient compliance. Furthermore, sustained-release formulations can be used to influence other properties such as the time of action onset or drug levels in the blood, and therefore can influence the occurrence of side effects.

[0109] Most sustained-release formulations are designed to initially release a rapid amount of the drug to produce the desired therapeutic effect, and then gradually and continuously release other amounts of the drug to maintain this level of therapeutic effect over a longer period. To maintain this constant level of drug in the body, the drug must be released from the formulation at a rate that replaces the amount of drug being metabolized and excreted from the body.

[0110] The sustained release of the active ingredient can be stimulated by various inducing factors, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. In the context of the present invention, the term “sustained-release ingredient” is defined herein as a compound (one or more) including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres, or combinations thereof, that promotes the sustained release of the active ingredient.

[0111] In certain embodiments, the formulations of the present invention may be short-acting, rapidly disappearing, and sustained-release formulations, such as sustained-release, delayed-release, and pulsed-release formulations.

[0112] The term "sustained-release" is used in its original sense to refer to drug formulations that release the drug gradually over a long period, resulting in a substantially constant level of drug in the blood over a long period, though not necessarily. The duration can be more than a month, and should result in a longer release than the same amount of drug administered in bolus form.

[0113] For sustained release, the compound may be formulated using a suitable polymer or hydrophobic material that provides the compound with sustained-release properties. Therefore, the compound for use in the method of the present invention may be administered, for example, by injection in the form of microparticles, or by implantation in the form of wafers or disks. In a preferred embodiment of the present invention, the compound of the present invention is administered to a patient, either alone or in combination with another pharmaceutical product, using a sustained-release formulation.

[0114] The term "delayed release" is used herein in its conventional sense to refer to a drug formulation that first releases the drug after some delay following drug administration, which may, but not necessarily, range from approximately 10 minutes to up to approximately 12 hours.

[0115] The term "pulsed release" is used herein in its conventional sense to refer to a drug formulation that releases the drug in a manner that generates a pulsed plasma profile of the drug after administration.

[0116] The term "immediate release" is used in its original sense to refer to formulations that release the drug immediately after administration.

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

[0118] As used herein, rapid elimination means any period of time up to approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes after drug administration, and any period including these, as well as any and all or partial increments thereof.

[0119] Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific procedures, embodiments, claims, and examples described herein by means of routine experiments. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims appended herein. For example, modifications using known substitutes in the art, and performed by routine experiments alone, of reaction conditions including but not limited to reaction time, reaction size / volume, experimental reagents such as solvents and catalysts, pressure, atmospheric conditions such as a nitrogen atmosphere, and reducing / oxidizing agents, should be understood to be within the scope of this application.

[0120] definition To aid in understanding the detailed descriptions of compositions and methods in this disclosure, several express definitions are provided to facilitate the clear disclosure of the various aspects of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains.

[0121] As used herein, “expression” refers to the process by which polynucleotides are transcribed from a DNA template (to mRNA or other RNA transcripts, etc.) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may collectively be referred to as “gene products.” If the polynucleotides originate from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0122] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing the coding sequence necessary for the production of RNA, polypeptides, or their precursors (e.g., proinsulin). Functional polypeptides may be encoded by the full-length coding sequence or any portion of the coding sequence, as long as the desired activity or functional properties of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) are preserved. When used in relation to a gene, the term "part" 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. Therefore, "nucleotides containing at least a portion of a gene" may include a gene fragment or the entire gene.

[0123] The term “gene” includes the coding region of a structural gene and includes sequences located adjacent to both the 5' and 3' coding regions at a distance of approximately 1 kb from each end, so that the gene corresponds to the length of full-length mRNA. The sequence located at 5' of the coding region and present on the mRNA is called the 5' untranslated sequence. The sequence located at 3' or downstream of the coding region and present on the mRNA is called the 3' untranslated sequence. The term “gene” includes both the cDNA and genomic morphology of a gene. The genomic morphology or clone of a gene includes the coding region 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 introns may contain regulatory elements such as enhancers. Introns are removed or “spliced” from the nuclear transcript or primary transcript, and therefore introns are not present in the messenger RNA (mRNA) transcript. mRNA functions during translation and specifies the sequence or order of amino acids in the nascent polypeptide.

[0124] "Genetic transfer" and "gene delivery" refer to methods or systems for reliably inserting specific nucleic acid sequences into target cells.

[0125] When the term "recombinant" is used in reference to nucleic acid molecules, it refers to nucleic acid molecules composed of nucleic acid segments that have been joined together by molecular biological techniques. When the term "recombinant" is used in reference to proteins or polypeptides, it refers to protein molecules expressed using recombinant nucleic acid molecules.

[0126] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are close together and, if two protein-coding regions need to be joined, they are within the same reading frame.

[0127] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, or during cell culture, rather than within a multicellular organism.

[0128] As used herein, the term "in vivo" refers to events occurring within multicellular organisms such as non-human animals.

[0129] As used herein, “treatment” or “to treat,” or “to alleviate” or “to improve,” are interchangeable. These terms refer to an approach to obtain beneficial or desirable outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefit means improvement or effect on any treatment of one or more diseases, conditions, or symptoms. In the case of preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0130] Terms such as "prevent," "prevention," "preventive treatment," and "preventive therapy" refer to reducing the likelihood of developing a disability or condition in individuals who are not yet affected but are at risk of developing or are prone to developing such a condition.

[0131] As used herein, the term “disease” is intended to be generally synonymous with and interchangeable with the terms “disorder” and “condition” (as in “medical condition”), all of which reflect an abnormal condition in which one or any part of the body of a human or animal is impaired from functioning normally, typically manifested by distinguishing between signs and symptoms, and which reduces the lifespan or quality of life of a human or animal.

[0132] The terms “decrease,” “lower,” “decrease,” “reduce,” or “inhibit” are all used herein to generally mean a decrease of a statistically significant amount. However, to avoid misunderstanding, “decrease,” “decrease,” “reduce,” or “inhibit” means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of 100% or less (absence of a level compared to a reference sample), or any decrease of 10 to 100% compared to a reference level.

[0133] As used herein, the term “regulate” means any change in a biological state, i.e., an increase, a decrease, etc.

[0134] The terms “increased,” “enhancement,” “boosting,” or “activation” are all used herein to generally mean an increase of a statically significant amount. To avoid misunderstanding, the terms “increased,” “enhancement,” or “boosting,” 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 100% or less, or any increase between 10% and 100% compared to a reference level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level.

[0135] The terms “effective dose,” “effective dosage,” or “effective administration” are defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The “therapeutic effective dose” or “therapeutic effective administration” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional impairment or disability due to the distress of the disease. The “preventive effective dose” or “preventive effective administration” of a drug is the amount of the drug, when administered alone or in combination with another therapeutic agent to a subject at risk of developing or relapsing the disease, that suppresses the onset or relapse of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or suppress the onset or relapse of the disease can be evaluated using a variety of methods known to experienced practitioners, for example, in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the drug in in vitro assays.

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

[0137] The term “drug” is used herein to refer to compounds, mixtures of compounds, biological macromolecules (nucleic acids, antibodies, proteins or parts thereof, e.g., peptides), or extracts made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals). The activity of such drugs may appropriately define a drug as a “therapeutic agent,” which is a biological, physiological, or pharmacologically active substance (or substance) that acts locally or systemically on a target.

[0138] The terms “therapeutic drug,” “therapeutic drug,” or “therapeutic agent” are used interchangeably and refer to a molecule or compound that, when administered to a subject, confers some beneficial effect. Beneficial effects include: the feasibility of a diagnostic determination; improvement of a disease, symptom, disorder, or pathological condition; reduction or prevention of the onset of a disease, symptom, disorder, or condition; and overall offsetting of a disease, symptom, disorder, or pathological condition.

[0139] As used herein, “combination” therapy means the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous administration, unless otherwise evident from the context. 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 in some way conditioned upon the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and acted upon for a predetermined period. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0140] "Sample," "test sample," and "patient sample" are interchangeable terms used herein. Samples may be serum, urinary plasma, amniotic fluid, cerebrospinal fluid, cell (e.g., antibody-producing cells), or tissue samples. Such samples may be used directly as obtained from a patient, or they may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the properties of the sample in any manner discussed herein or in any other manner 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, “homologous” refers to the subunit sequence identity between two polymer molecules, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two molecules are homologous at a given position if the subunit positions in both are occupied by the same monomeric subunit, for example, if the positions in each of two DNA molecules are occupied by adenine. Homologousity between two sequences is a direct function of the number of matching or homologous positions. For example, two sequences are 50% homologous if half of the positions in them (e.g., five positions in a polymer with 10 subunit lengths) are homologous. Two sequences are 90% homologous if 90% of the positions (e.g., 9 out of 10) are matching or 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 identifying a gene product, causes that gene product to be produced in a cell only if an inducer substantially corresponding to its promoter is present in the cell.

[0143] As used herein, the terms “inhibit” and “antagonize” mean reducing or completely preventing the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein by a measurable amount. Inhibitors are compounds, for example, antagonists, that bind to proteins, genes, and mRNA to partially or completely block a stimulus, reduce, prevent, delay, inactivate, desensitize, or downregulate the stability, expression, function, and activity of a stimulus, gene, and mRNA.

[0144] Where the term “instructional materials” is used herein, includes publications, records, figures, or any other expressive medium that may be used to convey the usefulness of any composition and / or compound of the present invention contained in the kit. The kit’s instructional materials may, for example, be affixed to the container containing any composition of the present invention, or shipped together with the container containing any composition. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the recipient use the instructional materials in conjunction with any composition. Delivery of the instructional materials may, for example, be by physical delivery of publications or other expressive medium that conveys the usefulness of the kit, or it may be achieved alternatively by, for example, electronic transmission by computer, such as email, or download from a website.

[0145] "Isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from its naturally occurring coexisting substances is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0146] "Isolated nucleic acid" refers to a nucleic acid segment or fragment separated from its adjacent sequences in its naturally occurring state; that is, a DNA fragment typically removed from sequences adjacent to the fragment, i.e., sequences adjacent to the fragment in the genome where it naturally occurs. This term also applies to nucleic acids substantially purified from other components naturally associated with the nucleic acid, i.e., RNA or DNA or proteins naturally associated with it in cells. Therefore, this term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or the genomic DNA of prokaryotes or eukaryotes, or existing as a distinct molecule independently of other sequences (i.e., as cDNA or genome or cDNA fragments produced by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0147] Unless otherwise specified, “nucleotide sequences encoding amino acid sequences” are degenerate types and include all nucleotide sequences encoding the same amino acid sequence. The phrase “nucleotide sequences encoding proteins or RNA” may include introns to the extent that protein-coding nucleotide sequences may contain introns in some types.

[0148] Parenteral administration of the composition 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 in the present invention and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates the administration of the compound to a living organism.

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

[0151] As used herein, the term “pharmaceutically acceptable” means a material such as a carrier or diluent that does not negate the biological activity or properties of a composition and is relatively non-toxic; that is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition containing it.

[0152] The term “pharmaceutically acceptable carrier” includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid extenders, diluents, excipients, solvents, or encapsulating materials, that are involved in transporting or delivering the compound(s) of the present invention within or to a subject so that they can perform their intended function. Typically, such compounds are transported or delivered from one organ or part of body to another. Each salt or carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Some examples of materials that can function 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, ethylcellulose, 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; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; 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 coatings, antimicrobial and antifungal agents, as well as absorption retarders, etc., that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the composition.

[0153] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of a administered compound prepared from a pharmaceutically acceptable nontoxic acid, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

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

[0155] In this specification, conventional notation is used to describe polypeptide sequences, where the left end of a polypeptide sequence is the amino terminus and the right end is the carboxyl terminus. As used herein, "peptide mime" is a compound containing non-peptide structural elements that can mimic the biological action of a parent peptide. Peptide mimes may or may not contain peptide bonds.

[0156] As used herein, the term “promoter” is defined as a DNA sequence that is recognized by a cellular synthetic mechanism, or an introduced synthetic mechanism, and is required to initiate the specific transcription of a polynucleotide sequence.

[0157] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product operably ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for the expression of the gene product. A promoter / regulatory sequence may, for example, express a 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 methods. As used herein, the term “recombinant DNA” is defined as DNA produced by combining DNA fragments from different sources.

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

[0160] As used herein, “Subject” refers to human or non-human mammals. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and mouse mammals. In certain embodiments, the subject is human. A “tissue-specific” promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or identified by a gene, causes its gene product to be produced within a cell only if the cell is substantially the tissue type corresponding to its promoter.

[0161] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which exogenous nucleic acids are transferred to or introduced into host cells. “Transfected,” “transformed,” or “transduced” cells are those that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include the primary target cell and its progeny.

[0162] As used herein, the phrases “transcriptionally controlled” or “operatably linked” mean that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide. “Variant,” as the term is used herein, is a nucleic acid or peptide sequence that differs in sequence from the reference nucleic acid or peptide sequence, respectively, but retains the essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and shortenings. Since changes in the sequence of a peptide variant are typically limited or conserved, the sequences of the reference peptide and the variant are very similar overall and identical in many regions.

[0163] Variants and reference peptides may have different amino acid sequences due to one or more substitutions, additions, or deletions in any combination. Nucleic acid or peptide variants may be naturally occurring variants, such as allele variants, or variants not known to exist naturally. Nucleic acid and peptide variants not found in nature can be produced by mutagenesis or direct synthesis.

[0164] A “vector” is a composition of substances containing isolated nucleic acids that can be used to deliver isolated nucleic acids into the interior of 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. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The 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, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0165] As used herein, the term “virus” is defined as a particle consisting of nucleic acid (RNA or DNA) encapsulated in a protein coat, with or without an external lipid envelope, that can transfect cells with its nucleic acid.

[0166] Throughout this disclosure, various aspects of the invention can be represented in a range form. It should be understood that the use of ranges is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose not only all possible subranges but also the individual numbers within those ranges. For example, a range description such as 1-6 should be considered to specifically disclose not only subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, but also the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0167] Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless explicitly indicated otherwise in the context.

[0168] The terms “including,” “comprising,” “containing,” or “having,” and their variations, unless otherwise stated, mean to encompass the items listed thereafter and their equivalents, as well as any additional subject matter.

[0169] Phrases such as "in one embodiment," "in various embodiments," and "in several embodiments" are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may refer to it unless otherwise indicated in the context.

[0170] The terms "and / or" or " / " mean any one of the items, any combination of items, or all items to which the term relates.

[0171] The term "substantially" does not exclude "completely"; for example, a composition that "substantially does not contain" Y does not have to contain Y completely. The term "substantially" may be omitted from the definitions of this invention as needed.

[0172] As used herein, the terms “approximately” or “about” refer to values ​​similar to the reference values ​​described, when applied to one or more such values. In some embodiments, unless otherwise stated or evident from the context, the terms “approximately” or “about” refer 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 lower than the reference values ​​described (except where such numbers exceed 100% of the possible values). Unless otherwise indicated herein, the term “about” is intended to include values ​​close to the described range, e.g., by weight percentage, that are equivalent with respect to the function of the individual components, compositions, or embodiments.

[0173] Wherever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed within those values ​​and ranges are included within the scope of the present invention. Furthermore, all values ​​within these ranges, and any upper or lower limits on the ranges of values, are also contemplated in this application.

[0174] Where used herein, the term “each” is intended to identify individual items within a set of items, but not necessarily all items within the set. Exceptions may be made where explicit disclosure or context clearly indicates otherwise.

[0175] Any use of any examples or illustrative language (e.g., "etc.") provided herein is intended solely to clarify the invention and does not limit the scope of the invention unless otherwise claimed. No language herein should be construed as indicating an unclaimed element essential to the practice of the invention.

[0176] All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless the context clearly contradicts it. With respect to any of the methods provided, the steps of the method may be performed simultaneously or sequentially. If the steps of the method are performed sequentially, the steps may be performed in any order unless otherwise indicated.

[0177] If a method involves a combination of steps, any combination or partial combination of steps is included within the scope of this 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 conflict with this disclosure. The publications disclosed herein are provided only for their disclosure prior to the filing date of the present invention. Nothing described herein should be construed as acknowledging that the present invention has no prior rights to such publications for the sake of prior art. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be verified individually.

[0179] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes are proposed in light of them and should be understood to be included in the spirit and scope of the claims of this application and the appended claims. [Examples]

[0180] Example 1 Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without purification. As described in this document, patient-derived induced pluripotent stem cells (iPSCs) were obtained from the NIH-funded biorepository (www.nimhgenetics.org / available_data / ipsc / ). Both normal, healthy cells and cells from individuals diagnosed with familial ALS and carrying a mutation in SOD1(N139K) were used. A differentiation protocol was established to result in motor neuron culture after 40 days of treatment with defined regimens of growth factors and specialty media (Figure 1). Therapeutic (ASPA) and control (GFP) genes were packaged in AAV vectors, and an initial cohort of cells was treated to evaluate the effects on mitochondrial function. Mitochondria isolated from SOD1-mutated motor neurons were shown to have lower ATP synthesis rates compared to wild-type cells; however, transduction of these mutant cells with AAV-ASPA significantly restored mitochondrial ATP synthesis rates compared to AAV-GFP controls, as assessed by a luminescence-based in situ assay (Figure 2).

[0181] Example 2 Analysis of the enhancement of ATP synthesis in mitochondria isolated from the spinal cord of 16-week-old SOD G93A mice by free aspartate derived from ASPA. Mitochondria were isolated from 16-week-old SOD G93A whole spinal cords using mechanical homogenization and fractional centrifugation. The mitochondria were kept on ice until use in the assay. The rate of ATP synthesis in 30 μg of isolated mitochondria was analyzed 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 were added. The addition of 2 U / ml aspartate aminotransferase and 3 U / ml malate dehydrogenase drives the MAS and initiates ATP synthesis. In this example, free aspartate was used instead of the reaction product produced by incubating lysates from cells overexpressing either wild-type ASPA or a non-functional mutant ASPA with 5 mM NAA, according to the scheme in Figure 8. When the wild-type ASPA reaction product was added to SOD spinal cord mitochondria, the ATP synthesis rate increased significantly, as measured by luminescence over a 3-minute period. However, no increase was observed when the non-functional E285A ASPA mutant reaction product was added (Figure 9). HPLC evaluation of the aspartic acid content of these reaction products (Table 1) showed that the wild-type ASPA reaction product increased aspartic acid by more than 800-fold.

[0182] Table 1 shows the aspartic acid content of the reaction product after incubation of 50 μl lysates from HeLa cells transfected with wild-type ASPA plasmid (WT) or non-functional E285 ASPA (E285A) with 5 mM NAA for 2 hours. The reaction was carried out in 200 μl of a 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 stopped by heating to 95°C for 3 minutes. HPLC analysis of the OPA-derivativeized samples was performed to assay the aspartic acid concentration of 20 μl of the reaction mixture. 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 transduced with 1x10 11 AAV9-CBh-ASPA vector genomes (vg) by intrathecal injection into the lumbar spinal cord. 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). Animals were tested weekly from 9 to 16 weeks of age on an accelerating rotarod (4 - 40 rpm). The mean drop latency over three consecutive 3-minute trials was recorded (30-second rest period between individual trials). Weeks 9 - 11 were designated as training sessions, and the drop latencies at weeks 12, 13, 14, 15, and 16 were compared between the saline-treated group and the AAV9-ASPA-treated group (n = 15 / group). The rotarod analysis was performed by individuals blinded to the treatment group. Mutant mice treated with AAV9-ASPA showed a long-term improvement in rotarod performance (drop latency), which was statistically significant at 15 and 16 weeks of age (p = 0.0218, 0.0271, respectively).

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

[0185] NAA levels are characteristically reduced in association with increased pathological energy deficiency in a wide range of neurodegenerative diseases, including ALS. A central hypothesis in the current study is that this metabolic response is due to an attempt at uncoupling NAA synthesis from mitochondrial oxidative phosphorylation and ATP synthesis, given the common requirement for free aspartate by both synthetic processes. Therefore, as shown in the data presented in Figure 9, the release of aspartate from endogenous NAA by exogenously supplied ASPA is expected to support ATP synthesis by mitochondria 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 fuel for ETC, measured as an increase in available energy currency. A key metric related to this is the function of the ASPA transgene. The function of ASPA delivered via AAV was confirmed in the spinal cord isolated from 16-week-old SOD G93A animals immediately after 16-week-old rotarod analysis. Age-matched C57BL / 6J wild-type (non-SOD) male mice were used as a calibration reference control. NAA, AMP, and ATP content in the spinal cord of AAV9-ASPA and saline-controlled SOD G93A mice were analyzed by HPLC. Flash-frozen whole spinal cords were obtained from 16-week-old animals immediately after 16-week-old rotarod analysis. The whole spinal cords were homogenized in a precipitation solution using mechanical dispersion elements and extracted with chloroform. The thus prepared samples were divided and stored at -80°C for subsequent analysis. Absolute molar concentrations of target metabolites were calculated using standard curves created from purified reference standards. As previously reported for this metabolite in both clinical and animal model populations, 16-week-old saline-treated SOD G93A mice showed reduced spinal cord NAA compared to the wild-type reference (p=0.0015, n=5 / group, Figure 11). The spinal cords of mutant mice treated with physiological saline also showed a decrease in the ATP:AMP ratio (p=0.0134), suggesting that ATP hydrolysis (i.e., utilization) exceeded its synthesis.Spinal cord treated with SOD G93A AAV9-ASPA showed a further 1.7-fold reduction in NAA compared to controls treated with physiological saline (p=0.011, n=5 / group), suggesting the function of the AAV-delivered ASPA transgene associated with a significant increase in the ATP:AMP ratio (p=0.0045), indicating increased ATP production and improved energy status. This suggests that the products of increased NAA catabolism resulting from transduction by AAV9-ASPA support a bioavailable energy currency with relevant benefits for motor function (Figure 10).

[0186] Spinal cord mitochondria treated with AAV9-ASPA show increased ATP synthesis levels compared to aspartate derived from available NAA.

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

[0188] While the present invention has been described with an emphasis on embodiments, it will be apparent to those skilled in the art that variations of compositions and methods may be used, and that the invention may be carried out in ways other than those specifically described herein. Accordingly, the present invention includes all modifications that are encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

1. A method for increasing the amount of neuroaspartic acid in the spinal cord of a subject at risk of developing ALS or who is suffering from ALS, comprising: identifying the subject by screening the subject for ALS; and administering to the subject a therapeutically effective amount of a composition comprising nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75% identical to the sequence of Sequence ID No. 1 supported on a recombinant adeno-associated virus (rAAV) vector.

2. The method according to claim 1, wherein the rAAV vector is AAV9.

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

4. The method according to claim 1 or 2, further comprising administering a second therapeutic agent to the subject.

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

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

7. A method for treating, improving, or reversing at least one symptom of amyotrophic lateral sclerosis (ALS) in subjects who require treatment, improvement, or reversal of at least one symptom of ALS, comprising administering to the subjects a therapeutically effective amount of a composition that increases the level or activity of aspartoacylase (ASPA) in at least one cell of the subjects.

8. The method according to claim 7, wherein the composition comprises a gene therapy composition.

9. The method according to claim 7 or 8, wherein the composition comprises a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence that is at least 75% identical to the sequence of Sequence ID No.

1.

10. The method according to claim 7, wherein the nucleic acid encoding ASPA or a fragment thereof comprises the amino acid sequence of SEQ ID NO:

1.

11. The method according to claim 7 or 8, comprising introducing the nucleic acid into the at least one of the target cells by viral transduction.

12. The method according to claim 11, wherein the composition is provided with a virus or virus-like particles containing the nucleic acid.

13. The method according to claim 12, wherein the nucleic acid is supported on a recombinant adeno-associated virus (rAAV) vector.

14. The method according to claim 13, wherein the rAAV vector is AAV9.

15. The method according to any one of claims 1 to 14, wherein the administered composition increases the protein expression level of ASPA in the target cells.

16. The method according to any one of claims 1 to 15, comprising administering the composition to at least a portion of the spinal cord of the subject.

17. The method according to claim 16, comprising locally administering the composition to a portion of the spinal cord of the subject.

18. The method according to any one of claims 1 to 17, further comprising administering a second therapeutic agent to the subject.

19. The method according to claim 18, wherein the second therapeutic agent is administered to the subject before, after, or simultaneously with the composition.

20. The method according to claim 12 or 13, wherein the second therapeutic agent is riluzole, edaravone, or a salt or solvate thereof, or a combination thereof.

21. The method according to any one of claims 1 to 20, wherein the composition is administered by a route selected from oral, parenteral, transdermal, transpulmonary, intranasal, buccal, intrathecal, and intravenous.

22. The method according to any one of claims 1 to 21, wherein the composition is administered via an intrathecal route.

23. The method according to any one of claims 1 to 22, wherein the subject is a mammal.

24. The method according to claim 23, wherein the mammal is a human.

25. The method according to any one of claims 1 to 24, wherein the at least one cell is located within the spinal cord of the subject.

26. The method according to any one of claims 1 to 25, wherein at least one symptom of ALS is mitochondrial dysfunction.

27. The method according to any one of claims 1 to 26, wherein the administration of the composition enhances the substrates for energy metabolism in the subject.

28. The method according to any one of claims 1 to 27, wherein the administration of the composition increases the survival of cells in the subject.

29. The method according to any one of claims 1 to 28, wherein the administration of the composition increases the survival of motor neurons in the subject.

30. The method according to any one of claims 1 to 29, wherein the administration of the composition extends the average life expectancy of the subject.

31. The method according to any one of claims 1 to 30, wherein the administration of the composition increases the level of NAA catabolism in the mitochondrial region of spinal cord neurons.

32. A kit for increasing the level or activity of ASPA in target cells containing an rAAV vector or virus-like particles, wherein the virus or virus-like particles contain a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75% identical to the sequence of Sequence ID No.

1.

33. The kit according to claim 32, wherein the nucleic acid encoding ASPA or a fragment thereof comprises the amino acid sequence of SEQ ID NO:

1.

34. The kit according to claim 32 or 33, wherein the rAAV vector is AAV9.

35. A method for increasing NAA catabolism in spinal mitochondria of a subject at risk of developing ALS or who is suffering from ALS, comprising: identifying the subject; and administering to the subject a therapeutically effective amount of a composition comprising a nucleic acid encoding ASPA or a functional fragment thereof, having an amino acid sequence at least 75% identical to the sequence of Sequence ID No. 1 supported on a recombinant adeno-associated virus (rAAV) vector.

36. The method according to claim 35, wherein the rAAV vector is AAV9.

37. The method according to claim 36, wherein the administration of the composition increases the survival of motor neurons in the subject.

38. The method according to claim 37, further comprising administering a second therapeutic agent to the subject.

39. The method according to claim 38, wherein the second therapeutic agent is administered to the subject before, after, or simultaneously with the composition.

40. The method according to claim 39, wherein the second therapeutic agent is riluzole, edaravone, or a salt or solvate thereof, or a combination thereof.

Images