Pharmaceutical Compositions for Treating Neurological Conditions

Abstract

Provided are methods and compositions comprising agents that reduce nNOS activity and their use in treating diseases or conditions in which a beneficial clinical effect is achieved by reducing neuronal nitric oxide synthase (nNOS) activity.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 186,389, filed May 10, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Description of sequence listing An ASCII file entitled "91961SequenceListing.txt", created on May 10, 2022, containing 4,096 bytes, and submitted contemporaneously with the filing of this application, is hereby incorporated by reference.

[0003] The present invention, in some embodiments thereof, relates to therapeutic methods, and more particularly to pharmaceutical compositions and methods for treating neurological conditions associated with aberrant activity of neuronal nitric oxide synthase (nNOS), including, but not limited to, autistic spectrum disorders (ASD) and neurally derived cancers.

[0004] Nitric oxide (hereinafter, "NO" (Nitric oxide)) is the smallest signaling molecule. NO is also called a "double-edged sword". A large amount of accumulating evidence suggests that NO is one of the key factors in the development of many brain-related disorders. S-nitrosylation (SNO) (NO-mediated post-translational modification at cysteine) targets a wide range of prominent intracellular proteins, leading to alterations in signaling pathways, which may converge to synaptic, neuronal and behavioral deficits. Thus, NO promotes neurogenesis, but abnormal SNO may be the cause of different neurodevelopmental disorders such as autism spectrum disorder (ASD). This "double-edged sword" function also depends on the concentration of NO. At low concentrations, NO is involved in the regulation of synaptic activity, synaptic plasticity, synaptic activity, synaptic plasticity, and vesicle trafficking. However, at high concentrations, NO may be toxic, leading to phenotypic alterations and cell death. This is due to the formation of superoxide radicals (O2 - ) and ultimately induces damage to DNA, lipids, and proteins during oxidative stress. - ) is formed.

[0005] NO can affect cell signaling through protein S-nitrosylation (SNO), tyrosine nitration, and S-nitrosoglutathione (GSNO) formation. Protein tyrosine nitration (NO overproduction) under stress conditions has been considered a possible marker of nitrosative stress. 3-Nitrotyrosine (Ntyr) is the product of tyrosine nitration mediated by peroxynitrite acting as a nitrating agent.

[0006] SNO is a reversible NO-mediated post-translational modification of cysteine ​​thiol, in which cysteine ​​is converted to nitrosothiol. SNO plays a key role in the localization and activity of a wide range of important enzymes and receptors, resulting in the regulation of many signaling pathways, axonal transport, synaptic plasticity, and protein assembly. Thus, abnormal SNO signaling and increased Ntyr may contribute to the progression of many neurodegenerative, neurodevelopmental, and neuropsychiatric disorders, as well as neurotumor, as summarized below.

[0007] NO cannot be stored intracellularly and therefore requires de novo synthesis to mediate its activity.

[0008] NO is produced by three types of NO synthase, nNOS, iNOS and eNOS. nNOS, which stands for neuronal nitric oxide synthase, is found in neuronal tissue.

[0009] Nitric oxide synthase (EC 1.14.13.39) (NOS) is a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. In mammals, there are three different genes encoding NOS isozymes: neuronal (nNOS or NOS-1), cytokine-inducible (iNOS or NOS-2), and endothelial (eNOS or NOS-3). iNOS and nNOS are soluble and reside primarily in the cytosol, while eNOS is membrane-bound.

[0010] Neuronal nitric oxide synthase (nNOS) produces nitric oxide (NO) in neural tissues of both the central and peripheral nervous systems, whose functions include synaptic plasticity in the central nervous system (CNS), synaptic transmission and axonal outgrowth in the peripheral nervous system and CNS, and aberrant signal transduction leading to multiple neurological disorders.

[0011] nNOS also plays a role in cell-cell communication and is associated with the plasma membrane.

[0012] The intracellular localization of nNOS in skeletal muscle is mediated by its anchoring to dystrophin. nNOS contains an additional N-terminal domain, the PDZ domain. The gene encoding nNOS is located on chromosome 12.

[0013] nNOS has been found in neurons, astrocytes, the adventitia of cerebral blood vessels, and cardiac myocytes. In addition to brain tissue, nNOS has also been found by immunohistochemistry in other tissues. nNOS is known to be specifically inhibited by 7-nitroindazole, which acts as a noncompetitive substrate inhibitor.

[0014] It has been shown that the pathogenesis of ASD may be, at least in part, related to synaptic dysfunction that can lead to functional and cognitive impairment.(1) Previous studies have shown that mutations in genes, such as SHANK3 and CNTNAP2, and others, converge on common synapse-related cellular pathways that are strongly associated with ASD.(2)

[0015] Mutant model NO-mediated post-translational modification (PTM) of cysteine ​​thiols (SNO) in the SHANK3 human gene has been found to result in various neuropsychiatric disorders, including autism spectrum disorder (ASD). Pathway analysis shows that affected processes are increased in ASD. A significant increase in 3-nitrotyrosine was found in cortical regions of adult mutants, signaling both oxidative and nitrosative stress (5). Interestingly, nNOS knockdown (6) shows an ASD-like phenotype in cultured cells.

[0016] Neuroblastoma (NB) refers to a spectrum of neuroblastic tumors derived from neural crest cells during fetal development. Childhood cancers, like NB, account for 97% of all neuroblastic tumors. NB is the most common tumor among children under 1 year of age worldwide.

[0017] Despite extensive efforts, the underlying mechanisms of NB remain largely unclear. One of the essential regulators of carcinogenesis of various tumors, including NB, is nitric oxide (NO). It will be understood that the process of SNO can be related to the development of different cancers, as mentioned above (Mishra et al., 2020). This post-translational modification (PTM) has been reported to confer either tumor-suppressing or tumor-promoting effects and has been described as a process involved in all stages of cancer progression (Sharma et al., 2021). One of the pathways that can be affected by SNO is the mechanistic target of rapamycin (mTOR), a key regulator of cell proliferation, metabolism, and tumorigenesis (Johnsen et al., 2008). In previous studies, using SNO-proteome analysis of mouse brain, it has been found that the mTOR pathway is significantly amplified by SNO when NO levels are elevated (Mencer et al., 2021).

[0018] Further background art includes: Haim-Zada et al.2017.Stable polyanhydride synthesized from sebacic acid and ricinoleic acid.J.Controlled Release,257:156-162, Aquilano K,Filomeni G,Baldelli S,Piccirillo S,De Martino A,Rotilio G,Ciriolo MR (2007)Neuronal nitric oxide synthase protects neuroblastoma cells from oxidative stress mediated by garlic derivatives.J Neurochem 101:1327-1337, Berry T et al.(2012)The ALKF1174L mutation potentiates the oncogenic activity of MYCN in neuroblastoma.Cancer Cell 22:117-130、 Burke AJ,Sullivan FJ,Giles FJ,Glynn SA(2013)The yin and yang of nitric oxide in cancer progression.Carcinogenesis 34:503-512、 Ciani E,Guidi S,Della Valle G,Perini G,Bartesaghi R,Contestabile A(2002)Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein(CREB)activation.J Biol Chem 277:49896-49902、 Corasaniti M,Melino G,Navarra M,Garaci E,Finazzi-Agro A,Nistico G(1995)Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase.Neurodegeneration 4:315-321、 Fujibayashi T,Kurauchi Y,Hisatsune A,Seki T,Shudo K,Katsuki HJJops(2015)Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.129:119-126、 Gao R-N,Levy IG,Woods WG,Coombs BA,Gaudette LA,Hill G(1997)Incidence and mortality of neuroblastoma in Canada compared with other childhood cancers.Cancer Causes Control 8:745-754、 Gordon JL,Hinsen KJ,Reynolds MM,Smith TA,Tucker HO,Brown MA(2021)Anticancer potential of nitric oxide(NO)in neuroblastoma treatment.RSC Adv 11:9112-9120、 Hickok JR,Thomas DD(2010)Nitric oxide and cancer therapy:the emperor has NO clothes.Curr Pharm Des 16:381-391、 Huang Z,Fu J,Zhang Y(2017)Nitric oxide donor-based cancer therapy:advances and prospects.J Med Chem 60:7617-7635、 Kiessling MK,Curioni-Fontecedro A,Samaras P,Lang S,Scharl M,Aguzzi A,Oldrige DA,Maris JM,Rogler G(2016)Targeting the mTOR complex by everolimus in NRAS mutant neuroblastoma.PLoS One 11:e0147682、 Lamant L,Pulford K,Bischof D,Morris SW,Mason DY,Delsol G,Mariame B(2000)Expression of the ALK tyrosine kinase gene in neuroblastoma.Am J Pathol 156:1711-1721、 Lange I,Koster J,Koomoa D-LT(2019)Calcium signaling regulates fundamental processes involved in Neuroblastoma progression.Cell Calcium 82:102052、 Lopez-Rivera E,Jayaraman P,Parikh F,Davies MA,Ekmekcioglu S,Izadmehr S,Milton DR,Chipuk JE,Grimm EA,Estrada YJCr(2014)Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.74:1067-1078、 Ortiz-Ortiz MA, Moran JM, Gonzalez-Polo RA, Niso-Santano M, Soler G, Bravo-San Pedro JM, Fuentes JMJNr (2009) Nitric oxide-mediated toxicity in paraquat-exposed SH-SY5Y cells: a protective role of 7-nitroindazole.16:160-173, Tripathi MK,Kartawy M,Amal H(2020)The role of nitric oxide in brain disorders:Autism spectrum disorder and other psychiatric,neurological,and neurodegenerative disorders.Redox Biol:101567, and Vahora H, Khan MA, Alalami U, Hussain A (2016) The potential role of nitric oxide in halting cancer progression through chemoprevention. J Cancer Prev 21:1. Summary of the Invention

[0019] According to an aspect of some embodiments of the present invention, there is provided a method of treating a disease or condition in which a beneficial clinical effect is achieved by reducing neuronal nitric oxide synthase (nNOS) activity, comprising administering to a subject in need of such treatment an effective amount of a composition comprising an active agent that reduces nNOS activity, as described herein in any of the respective embodiments, thereby treating the disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity.

[0020] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising an active agent that reduces nNOS activity as described herein in any of the respective embodiments, and optionally a pharma- ceutically acceptable carrier, for use in the treatment of a disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity.

[0021] According to some of any of the embodiments described herein, the reduction in nNOS activity is in the central nervous system (CNS).

[0022] According to some of any of the embodiments described herein, the reduction in nNOS activity is selective or preferential in the CNS of the subject as compared to other tissues.

[0023] According to some of any of the embodiments described herein, the reduction is a reduction in nNOS activity in the brain.

[0024] According to some of any of the embodiments described herein, the composition includes an active agent that is nNOS specific.

[0025] According to some of any of the embodiments described herein, the mode of administration achieves a reduction in nNOS activity selectively and preferentially in the CNS.

[0026] According to some of the embodiments described herein, the mode of administration comprises topical administration.

[0027] According to some of any of the embodiments described herein, the composition includes a carrier that delivers the agent to the CNS of a subject.

[0028] According to some of any of the embodiments described herein, the carrier comprises an adeno-associated virus.

[0029] According to some of any of the embodiments described herein, the agent reduces nNOS expression.

[0030] According to some of any of the embodiments described herein, the agent decreases the translation of nNOS.

[0031] According to some of any of the embodiments described herein, the agent inhibits nNOS enzyme activity.

[0032] According to some of any of the embodiments described herein, the agent is selected from the group consisting of a small molecule, a competing peptide and an antibody, or a fragment thereof.

[0033] According to some of any of the embodiments described herein, the agent binds to the active site of nNOS and blocks substrate binding.

[0034] According to some of the embodiments described herein, the agent is N ω -nitroarginine, or any small molecule drug described herein.

[0035] According to some of the embodiments described herein, the agent is represented by formula I:

[0036] [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony R1 is selected from hydrogen, alkyl and cycloalkyl, preferably hydrogen; R2-R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, halo, haloalkyl, hydroxy, alkoxy, aryloxy, thiol, amine, carboxylate, thiocarboxylate, and poly(alkylene glycol) moieties.

[0037] According to some of the embodiments described herein, R1 is hydrogen.

[0038] According to some of any of the embodiments described herein, each of R2-R5 is hydrogen.

[0039] According to some of any of the embodiments described herein, the compound of formula I is in the form of its anionic salt.

[0040] According to some of any of the embodiments described herein, the compound of formula I is in the form of its cationic salt.

[0041] According to some of any of the embodiments described herein, the agent is an amino acid based inhibitor.

[0042] According to some of any of the embodiments described herein, the agent is PIN or nitric oxide synthase interacting protein (NOSIP).

[0043] According to some of any of the embodiments described herein, the agent is a selective nNOS inhibitor.

[0044] According to some of any of the embodiments described herein, the agent reduces levels of NO precursors.

[0045] According to some of any of the embodiments described herein, the agent increases the level of GSNO reductase levels.

[0046] According to some of any of the embodiments described herein, the agent is a nNOS-specific nucleic acid sequence.

[0047] According to some of any of the embodiments described herein, the agent is an antisense or siRNA.

[0048] According to some of any of the embodiments described herein, the agent is a DNA editing agent.

[0049] According to some of any of the embodiments described herein, the agent is CRISPR / Cas9 for selectively reducing expression of nNOS.

[0050] According to some of any of the embodiments described herein, the carrier for the agent is a viral delivery vector.

[0051] According to some of any of the embodiments described herein, the disease or condition is a neurological disease or condition.

[0052] According to some of any of the embodiments described herein, the disease or condition is a brain disorder.

[0053] According to some of the embodiments described herein, the disease or condition is an autism spectrum disorder (ASD).

[0054] According to some of any of the embodiments described herein, the disease or condition is ADD or ADHD.

[0055] According to some of any of the embodiments described herein, the disease or condition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, schizophrenia, addiction, amyotrophic lateral sclerosis (ALS), epilepsy, bipolar disorder, and migraine.

[0056] According to some of any of the embodiments described herein, the disease or condition is a neurodevelopmental disorder, a psychiatric disorder, or a neurodegenerative disease.

[0057] According to some of any of the embodiments described herein, the disease or condition is a cancer of neural or neuroectodermal origin.

[0058] According to some of any of the embodiments described herein, the cancer is selected from the group consisting of neuroblastoma, glioma, ganglioglioma, central neurocytoma, ganglioblastoma, medulloblastoma, and primitive neuroectodermal tumor (PNET).

[0059] According to some of any of the embodiments described herein, the neuroblastoma comprises a mutation in ALK.

[0060] According to some of any of the embodiments described herein, administration is to the striatum and / or cortex of the CNS of the subject.

[0061] According to some of any of the embodiments described herein, the composition comprises a CNS (eg, brain) selective delivery vehicle.

[0062] According to some of any of the embodiments described herein, the brain-selective delivery vehicle comprises a nanoparticle, a liposome, or an exosome.

[0063] According to some of any of the embodiments described herein, the delivery vehicle comprises a ligand or receptor to enhance uptake across the BBB of the subject.

[0064] According to some of any of the embodiments described herein, the composition is administered in conjunction with ultrasound or magnetic stimulation to enhance penetration across the BBB of the subject.

[0065] According to some of any of the embodiments described herein, upon administration of the composition, the level of physiologically available nitric oxide in neural cells of the subject is reduced by at least 20%.

[0066] According to some of any of the embodiments described herein, upon administration of the composition, the concentration of nitro-tyrosine in the subject's plasma is reduced by at least 20%.

[0067] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a therapeutically active agent that reduces neuronal nitric oxide synthase (nNOS) activity as described herein in any of the respective embodiments and a pharma- ceutically acceptable carrier, wherein the composition and the therapeutically active agent are selected such that, upon administration of the composition to a subject, a reduction in nNOS activity is selectively or preferentially achieved in the subject's central nervous system (CNS) or brain relative to other tissues.

[0068] According to some of any of the embodiments described herein, the reduction in nNOS activity in the subject's neurons (e.g., expressing nNOS) or CNS (e.g., brain) is at least 20%, or at least 30%, or at least 40%. According to some embodiments, the reduction in NOS activity in other tissues or organs is 10% or less, or 5% or less, or 2% or less, or 1% or less.

[0069] According to some of any of the embodiments described herein, the reduction in nNOS activity in the subject's neural cells (e.g., expressing nNOS) or CNS (e.g., brain) is at least 20%, or at least 30%, or at least 40% greater compared to other tissues or organs.

[0070] According to some of any of the embodiments described herein, the composition is configured for local administration to the CNS of a subject.

[0071] According to some of any of the embodiments described herein, the composition is configured for local administration to the brain of a subject.

[0072] According to some of any of the embodiments described herein, the composition is configured to selectively deliver a therapeutically active agent to the central nervous system of a subject.

[0073] According to some of any of the embodiments described herein, the composition is configured to selectively deliver a therapeutically active agent to the brain of a subject.

[0074] According to some of any of the embodiments described herein, the therapeutically active agent is selective for nNOS.

[0075] According to some of any of the embodiments described herein, the composition is configured to selectively deliver a therapeutically active agent to the brain or central nervous system for an extended period of time.

[0076] According to some of any of the embodiments described herein, the composition is configured to deliver a sufficient amount of a therapeutically active agent to reduce nNOS activity in the brain for a period of at least one day, or at least one week, or at least 12 days.

[0077] According to some of any of the embodiments described herein, the pharmaceutical composition is formulated for oral, nasal, or buccal delivery or administration.

[0078] According to some of any of the embodiments described herein, the carrier comprises an aqueous solution.

[0079] According to some of any of the embodiments described herein, the carrier comprises a mixture of at least one lipid, at least one surfactant, and a water-miscible organic solvent dispersed in an aqueous solution.

[0080] According to some of any of the embodiments described herein, the pharmaceutical composition is in the form of a tablet, capsule, syrup, solution, spray, aerosol, or dispersion.

[0081] According to some of the embodiments described herein, the pharmaceutical composition is formulated for administration by injection.

[0082] According to some of any of the embodiments described herein, the carrier comprises an aqueous solution.

[0083] According to some of any of the embodiments described herein, the carrier comprises a mixture of at least one lipid, at least one surfactant, and a water-miscible organic solvent, and according to some embodiments, the mixture, upon contact with an aqueous solution, forms lipid nanoparticles dispersed in the aqueous solution.

[0084] According to some of any of the embodiments described herein, the carrier forms a depot for sustained release of the therapeutically active agent.

[0085] According to some of any of the embodiments described herein, the carrier is preferably a polymeric carrier comprising at least one biodegradable polymer.

[0086] According to some of the embodiments described herein, the polymeric carrier comprises poly(sebacic-co-ricinoleic) acid.

[0087] According to some of the embodiments described herein, the polymeric carrier comprises PLGA, PLA, PCL, polycarbonate, or a combination thereof.

[0088] According to some of any of the embodiments described herein, the carrier is an aqueous carrier and the therapeutically active agent is soluble in the aqueous carrier.

[0089] According to an aspect of some embodiments of the invention there is provided a pharmaceutical composition as described herein in any of the respective embodiments for use in the treatment of a disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity as described herein.

[0090] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising an agent that reduces nNOS activity and is nNOS-specific and a pharma- ceutically acceptable carrier, the pharmaceutical composition being formulated for oral delivery or administration.

[0091] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound of formula I, as described herein in any of the respective embodiments, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, the pharmaceutical composition being formulated for oral, nasal, or buccal delivery or administration.

[0092] According to some of any of the embodiments described herein, the carrier comprises an aqueous solution.

[0093] According to some of any of the embodiments described herein, the compound includes a pharma- ceutically acceptable salt thereof that is water soluble or water immiscible.

[0094] According to some of any of the embodiments described herein, the carrier further comprises lipid nanoparticles dispersed in the aqueous solution.

[0095] According to some of the embodiments described herein, the pharmaceutical composition is for oral administration 1 to 4 times per day to a subject in need thereof.

[0096] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising an agent that reduces nNOS activity and is nNOS-specific and a pharma- ceutically acceptable carrier, wherein the carrier comprises an aqueous solution and lipid nanoparticles dispersed in the aqueous solution.

[0097] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound of formula I, or a pharma- ceutically acceptable salt thereof, as described herein in any of the respective embodiments, and a pharma- ceutically acceptable carrier, wherein the carrier comprises an aqueous solution and lipid nanoparticles dispersed in the aqueous solution.

[0098] According to some of these embodiments, the carrier is formed upon contacting the PNL formulation described herein with an aqueous solution.

[0099] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising an agent that reduces nNOS activity and is nNOS-specific and a pharma- ceutically acceptable carrier, wherein the carrier comprises a mixture of at least one lipid, at least one surfactant (preferably hydrophilic), and a water-miscible organic solvent, and upon contact with an aqueous solution or medium (e.g., a physiological medium), forms a dispersion of lipid nanoparticles in the aqueous solution or medium.

[0100] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound of formula I or a pharma- ceutically acceptable salt thereof as described herein in any of the respective embodiments, and a pharma- ceutically acceptable carrier, the carrier comprising a mixture of at least one lipid, at least one surfactant (preferably hydrophilic), and a water-miscible organic solvent, which upon contact with an aqueous solution or medium (e.g., a physiological medium), forms a dispersion of lipid nanoparticles in the aqueous solution or medium.

[0101] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising an agent that reduces nNOS activity as described herein in any of the respective embodiments and a pharma- ceutical acceptable carrier, the pharmaceutical composition being formulated to release a composition of formula I or a salt thereof over a period of at least one day or at least one week.

[0102] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound of formula I, or a pharma- ceutically acceptable salt thereof, as described herein in any of the respective embodiments, and a pharma- ceutically acceptable carrier, the pharmaceutical composition being formulated to release the compound of formula I or a salt thereof over a period of at least one day, or at least one week.

[0103] According to some of the embodiments described herein, the pharmaceutical composition is formulated for administration by injection.

[0104] According to some of any of the embodiments described herein, the carrier is preferably a polymeric carrier comprising at least one biodegradable polymer.

[0105] According to some of the embodiments described herein, the polymeric carrier comprises poly(sebacic-co-ricinoleic) acid.

[0106] According to some of the embodiments described herein, the polymeric carrier comprises PLGA, PLA, PCL, polycarbonate, or a combination thereof.

[0107] According to an aspect of some embodiments of the invention, there is provided a pharmaceutical composition as described herein in any of the respective embodiments for use in the treatment of a medical condition in which a beneficial clinical effect is achieved by reducing neuronal nitric oxide synthase (nNOS) activity.

[0108] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition as described herein in any of the respective embodiments for use in treating a medical condition in which a beneficial effect is achieved by reducing the level of physiologically available nitric oxide in neuronal cells of a subject by at least 20%.

[0109] According to an aspect of some embodiments of the present invention, there is provided a method of treating an autism spectrum disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an active agent that reduces nNOS activity, as described herein in any of the respective embodiments, wherein the reduction in nNOS activity is selective or preferential in the brain of the subject relative to other tissues, thereby treating the autism spectrum disorder.

[0110] According to an aspect of some embodiments of the present invention there is provided a method of treating an autism spectrum disorder in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition, as described herein in any of the respective embodiments, thereby treating the autism spectrum disorder.

[0111] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition as described herein in any of the respective embodiments for use in treating an autism spectrum disorder in a subject in need thereof.

[0112] According to an aspect of some embodiments of the present invention, there is provided a method of treating neural cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising an active agent that reduces nNOS activity, as described herein in any of the respective embodiments, wherein the reduction in nNOS activity is selective or preferential in each neural tissue of the subject relative to other tissues, thereby treating the neural cancer.

[0113] According to an aspect of some embodiments of the present invention, there is provided a method of treating neural cancer in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein in any of the respective embodiments, thereby treating the cancer. According to some of these embodiments, the neural cancer is neuroblastoma.

[0114] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition as described herein in any of the respective embodiments, for use in treating neural cancer in a subject in need thereof.

[0115] According to some of these embodiments, the agent that reduces nNOS activity is a compound of Formula I, or a salt thereof, as described herein in any of the respective embodiments.

[0116] According to some of these embodiments, the neural cancer is neuroblastoma.

[0117] According to some of these embodiments, the agent that reduces nNOS activity is a compound of formula I, or a salt thereof, as described herein in any of the respective embodiments, and the cancer is neuroblastoma.

[0118] According to some of the embodiments described herein, the agent is represented by Formula I, as described herein in any of the respective embodiments.

[0119] According to an aspect of some embodiments of the present invention there is provided a method of producing an experimental animal model of autism spectrum disorder (ASD), the method comprising administering to the experimental animal an effective amount of a composition that increases NO levels in the animal's brain, resulting in an ASD-like phenotype.

[0120] According to some of the embodiments described herein, the composition includes an NO donor.

[0121] According to some of any of the embodiments described herein, the NO donor comprises S-nitroso-N-acetylpenicillamines (SNAP).

[0122] According to some of the embodiments described herein, the administration is systemic.

[0123] According to some of any of the embodiments described herein, administration is directly to the brain of the animal.

[0124] According to some of any of the embodiments described herein, the NO donor is selected from the group consisting of SNAP, nitrate, nitrite, N-nitroso, C-nitroso, S-nitroso, heterocyclic compounds, metal / NO complexes, diazeniumdiolates, S-nitrosothiols, sydnonimines, and sodium nitroprusside (SNP).

[0125] According to an aspect of some embodiments of the present invention there is provided an animal model produced according to the methods described herein in any of the respective embodiments.

[0126] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition for delivering 7-nitroindazole to a mammal.

[0127] According to some embodiments of the present invention, the pharmaceutical compositions described herein are for oral delivery of 7-nitroindazole.

[0128] According to some embodiments of the present invention, the pharmaceutical compositions described herein are for delivery of 7-nitroindazole by injection over an extended period of time.

[0129] According to some embodiments of the present invention, the pharmaceutical compositions described herein are polymeric microparticle compositions.

[0130] According to some embodiments of the invention, the polymer is PLGA.

[0131] According to some embodiments of the present invention, the pharmaceutical compositions described herein are pro-nanodispersion lipid formulations (PNL) or dispersions of lipid particles in aqueous solutions formed therefrom.

[0132] According to some embodiments, the present invention, the pharmaceutical compositions described herein comprise a solution or mixture of surfactants, lipids, and solvents that form nanoparticles upon addition to an aqueous medium.

[0133] According to some embodiments, the pharmaceutical compositions described herein comprise microspheres comprised of biodegradable polymers.

[0134] According to some embodiments, the present invention provides pharmaceutical compositions described herein include compositions administered subcutaneously or intramuscularly having sustained, extended or sustained release administration over an extended period of time, or targeted slow and controlled delivery.

[0135] According to some embodiments of the present invention, the pharmaceutical compositions of the present invention, as described herein, are filled into a soft gelatin capsule for oral administration.

[0136] According to some embodiments of the present invention, the pharmaceutical compositions described herein comprise microspheres dispersed in water for injection.

[0137] According to some embodiments of the present invention, the pharmaceutical compositions described herein comprise an active agent (an agent that reduces nNOS activity as described herein) dispersed in an injectable paste-like biodegradable polymer carrier.

[0138] According to an aspect of some embodiments of the present invention, there are provided 7-nitroindazole salts having divalent and trivalent metal ions or ammonium counterions, as described herein.

[0139] According to an aspect of some embodiments of the present invention there is provided a method for treating autism spectrum disorder (ASD), comprising administering to a subject in need of such treatment an effective amount of a neuronal NO reducing composition.

[0140] According to some embodiments of the invention, the NO-reducing composition comprises one or more (or a combination of several) small molecules, amino acid-based molecules, or nucleic acid-based molecules, as described herein.

[0141] According to some embodiments of the present invention, the reduction of NO may be due to one of the following mechanisms: Decreased expression from the nNOS gene, Inhibition of nNOS activity at the enzyme level, Decreased L-arginine levels, and Increased GSNO reductase levels or activity (decreased levels of GSNO).

[0142] According to an aspect of some embodiments of the present invention there is provided a method for producing an animal model of an autism spectrum disorder comprising administering to an experimental animal an effective amount of a nitric oxide donor.

[0143] According to some embodiments of the invention, the administration is systemic.

[0144] According to some embodiments of the invention, administration is by a CNS targeted delivery system.

[0145] According to some embodiments of the invention, the administration is to the brain.

[0146] According to some embodiments of the invention, administration is to the brain using nanoparticles that are delivered to the olfactory tissue.

[0147] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [Brief description of the drawings]

[0148] Some embodiments of the present invention are hereinbelow described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made specifically to the drawings in detail, stressing that the particulars shown are merely exemplary and are for the purpose of discussing embodiments of the present invention by way of example, and in this regard the description made with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0149] The drawings are as follows: [Figure 1A] Western blot (WB) analysis (FIG. 1A) showing elevated levels of Ntyr in blood samples from children with ASD compared to their typically developing control children. **P<0.01. Controls (n=5) and ASD (n=10). [Figure 1B] A bar graph (FIG. 1B) is shown showing elevated levels of Ntyr in blood samples of children with ASD compared to their typically developing control children. **P<0.01. Controls (n=5) and ASD (n=10). [Figure 1C] Western blot (WB) analysis (FIG. 1C) and bar graphs (FIG. 1C and FIG. 1D) of cortical tissues prepared from wild-type mice (C57BL / 6 mice, WT1) and two ASD mouse models (SHANK3 mouse model (M1) and CNTNAP2 mouse model (M2)). **P<0.01. Control (n=5) and ASD (n=5). [Figure 1D] Western blot (WB) analysis (FIG. 1C) and bar graphs (FIG. 1C and FIG. 1D) of cortical tissues prepared from wild-type mice (C57BL / 6 mice, WT1) and two ASD mouse models (SHANK3 mouse model (M1) and CNTNAP2 mouse model (M2)). **P<0.01. Control (n=5) and ASD (n=5). [Figure 1E]Western blot (WB) analysis (FIG. 1C) and bar graphs (FIG. 1C and FIG. 1D) of cortical tissues prepared from wild-type mice (C57BL / 6 mice, WT1) and two ASD mouse models (SHANK3 mouse model (M1) and CNTNAP2 mouse model (M2)). **P<0.01. Control (n=5) and ASD (n=5). [Figure 2A] Figures 2A-B are bar graphs showing the effect of S-nitro-N-acetylpenicillamine (SNAP, an NO donor) treatment on WT mice as determined in the novel object recognition (NOR) test (Figure 2A) and the three-chamber sociability test (Figure 2B). SNAP alters WT behavior. WT and SNAP mice were significantly more interested in the familiar object (NOR) and the empty cage (sociability test) compared to untreated WT. [Figure 2B] Figures 2A-B are bar graphs showing the effect of S-nitro-N-acetylpenicillamine (SNAP, an NO donor) treatment on WT mice as determined in the novel object recognition (NOR) test (Figure 2A) and the three-chamber sociability test (Figure 2B). SNAP alters WT behavior. WT and SNAP mice were significantly more interested in the familiar object (NOR) and the empty cage (sociability test) compared to untreated WT. [Figure 3A] 3A-3B are bar graphs showing the effect of 7-NI treatment on mutant mice as determined in the NOR (FIG. 3A) and three-chamber sociability (FIG. 3B) tests. 7-NI reversed autistic behavior in mutant mice. [Figure 3B] 3A-3B are bar graphs showing the effect of 7-NI treatment on mutant mice as determined in the NOR (FIG. 3A) and three-chamber sociability (FIG. 3B) tests. 7-NI reversed autistic behavior in mutant mice. [Figure 3C]Western blot (WB) analysis (Figure 3C) and quantitative bar graphs (Figure 3D) of cortical tissues prepared from wild-type mice (WT), SHANK3 model mice (M1), and SHANK3 model mice treated with 7-NI (M1+7-NI) are shown. [Figure 3D] Western blot (WB) analysis (Figure 3C) and quantitative bar graphs (Figure 3D) of cortical tissues prepared from wild-type mice (WT), SHANK3 model mice (M1), and SHANK3 model mice treated with 7-NI (M1+7-NI) are shown. [Figure 3E] Western blot (WB) analysis (Figure 3E) and quantitative bar graphs (Figure 3F) of cortical tissues prepared from wild-type mice (WT), CNTNAP2(- / -) model mice (M2), and CNTNAP2(- / -) model mice treated with 7-NI (M2+7-NI) are shown. [Figure 3F] Western blot (WB) analysis (Figure 3E) and quantitative bar graphs (Figure 3F) of cortical tissues prepared from wild-type mice (WT), CNTNAP2(- / -) model mice (M2), and CNTNAP2(- / -) model mice treated with 7-NI (M2+7-NI) are shown. [Figure 3G] Confocal microscopy images (Figure 3G) and quantitative bar graphs (Figure 3H) showing nitrotyrosine in the cortical regions of neurons in wild-type mice (WT), SHANK3 model mice (M1), and SHANK3 model mice treated with 7-NI (M1+7-NI). [Figure 3H] Confocal microscopy images (Figure 3G) and quantitative bar graphs (Figure 3H) showing nitrotyrosine in the cortical regions of neurons in wild-type mice (WT), SHANK3 model mice (M1), and SHANK3 model mice treated with 7-NI (M1+7-NI). [Figure 4A] Schematics of the novel object recognition test (NOR, FIG. 4A), the three-chamber sociability test (FIG. 4B), the elevated plus maze test (FIG. 4C), and the marble burying test (FIG. 4D) are shown. [Figure 4B]Schematics of the novel object recognition test (NOR, FIG. 4A), the three-chamber sociability test (FIG. 4B), the elevated plus maze test (FIG. 4C), and the marble burying test (FIG. 4D) are shown. [Figure 4C] Schematics of the novel object recognition test (NOR, FIG. 4A), the three-chamber sociability test (FIG. 4B), the elevated plus maze test (FIG. 4C), and the marble burying test (FIG. 4D) are shown. [Figure 4D] Schematics of the novel object recognition test (NOR, FIG. 4A), the three-chamber sociability test (FIG. 4B), the elevated plus maze test (FIG. 4C), and the marble burying test (FIG. 4D) are shown. [Figure 5A] We show that daily administration of SNAP at 20 mg / kg ip injections for 10 days induces an ASD-like phenotype in C57BL / 6 mice, as determined in behavioral test analyses for wild-type (WT) and SNAP-treated mice. These results confirm the results of the experiments shown in Figures 2A-B. Figure 5A is a bar graph showing data obtained in the novel object recognition test shown in Figure 4A, showing object exploration time. WT mice spent significantly more time exploring the novel object than the familiar object (left bar, n=21, **P<0.01). SNAP-treated mice showed no significant preference for either the novel or familiar object, indicating a lack of novelty exploration and interest (right bar, n=19, ns=not significant). [Figure 5B]We show that daily administration of SNAP at 20 mg / kg ip injections for 10 days induces an ASD-like phenotype in C57BL / 6 mice, as determined in behavioral test analyses for wild-type (WT) and SNAP-treated mice. These results confirm the results of the experiments shown in Figures 2A-B. Figure 5B is a bar graph showing data obtained in the three-chamber sociability test shown in Figure 4B, showing the time spent interacting with either a familiar mouse (S1) or a novel mouse (S2). WT mice spent more time interacting with novel mice than familiar mice (left bar, n=31, *P<0.05), whereas SNAP-treated mice showed no significant preference to engage in social interactions with novel mice (right bar, n=32, ns=not significant). [Figure 5C] We show that daily administration of SNAP at 20 mg / kg ip injections for 10 days induces an ASD-like phenotype in C57BL / 6 mice, as determined in behavioral test analyses for wild-type (WT) and SNAP-treated mice. These results confirm the results of the experiments shown in Figures 2A-B. Figure 5C is a bar graph showing data obtained in the elevated plus maze test shown in Figure 4C, showing the time spent in the open arms. SNAP-treated mice (n=16) showed significantly reduced time in the open arms compared to their WT counterparts (n=32, *P<0.05). [Figure 5D]Figure 5 shows that daily administration of SNAP at 20 mg / kg ip injections for 10 days induces an ASD-like phenotype in C57BL / 6 mice, as determined in behavioral test analysis for wild-type (WT) and SNAP-treated mice. These results confirm the results of the experiment shown in Figures 2A-B. Figure 5D is a bar graph showing data obtained in the marble burying test shown in Figure 4D, showing the number of marbles buried. Analysis showed that WT mice buried more marbles than SNAP-treated mice, indicating a lack of novelty-seeking tendency and limited interest (n=20, 17 for WT and WT+SNAP, respectively. ***P<0.001). Data are shown as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 6A] Bar graphs showing data obtained in the novel object recognition test (see FIG. 4A) for wild-type (WT), SHANK3 knockout (M1) and 7-NI-treated (M1+7NI) mice (ip injection of 80 mg / kg daily for 10 days) at 6 weeks (FIG. 6A) and 10 months (FIG. 6B and FIG. 6C). In FIG. 6A, it can be seen that WT mice spent significantly more time exploring the novel object than the familiar object (n=21, **P<0.01). M1 mice showed no significant interest in exploring either the novel or familiar object, indicating a tendency toward novel exploration and lack of interest (n=33, ns=not significant). 7-NI-treated mice (M1+7NI) showed a significant increase in the time spent exploring the novel object than the familiar object (n=41, ***P<0.001). Data are shown as mean±SEM. Two-tailed t-tests were performed. *P<0.05, **P<0.01, ***P<0.001. A similar trend was observed in aged mice, as seen in Figures 6B and 6C. [Figure 6B]Bar graphs showing data obtained in the novel object recognition test (see FIG. 4A) for wild-type (WT), SHANK3 knockout (M1) and 7-NI-treated (M1+7NI) mice (ip injection of 80 mg / kg daily for 10 days) at 6 weeks (FIG. 6A) and 10 months (FIG. 6B and FIG. 6C). In FIG. 6A, it can be seen that WT mice spent significantly more time exploring the novel object than the familiar object (n=21, **P<0.01). M1 mice showed no significant interest in exploring either the novel or familiar object, indicating a tendency toward novel exploration and lack of interest (n=33, ns=not significant). 7-NI-treated mice (M1+7NI) showed a significant increase in the time spent exploring the novel object than the familiar object (n=41, ***P<0.001). Data are shown as mean±SEM. Two-tailed t-tests were performed. *P<0.05, **P<0.01, ***P<0.001. A similar trend was observed in aged mice, as seen in Figures 6B and 6C. [Figure 6C] Bar graphs showing data obtained in the novel object recognition test (see FIG. 4A) for wild-type (WT), SHANK3 knockout (M1) and 7-NI-treated (M1+7NI) mice (ip injection of 80 mg / kg daily for 10 days) at 6 weeks (FIG. 6A) and 10 months (FIG. 6B and FIG. 6C). In FIG. 6A, it can be seen that WT mice spent significantly more time exploring the novel object than the familiar object (n=21, **P<0.01). M1 mice showed no significant interest in exploring either the novel or familiar object, indicating a tendency toward novel exploration and lack of interest (n=33, ns=not significant). 7-NI-treated mice (M1+7NI) showed a significant increase in the time spent exploring the novel object than the familiar object (n=41, ***P<0.001). Data are shown as mean±SEM. Two-tailed t-tests were performed. *P<0.05, **P<0.01, ***P<0.001. A similar trend was observed in aged mice, as seen in Figures 6B and 6C. [Figure 7A]Data are shown for wild-type (WT), CNTNPA2 mutant (M2), and 7-NI-treated mice (M2+7NI, ip injection of 80 mg / kg daily for 10 days) in the three-chamber sociability test (FIG. 7A) and the elevated plus maze test (FIG. 7B). 7-NI reversed the autistic phenotype in both tests. [Figure 7B] Data are shown for wild-type (WT), CNTNPA2 mutant (M2), and 7-NI-treated mice (M2+7NI, ip injection of 80 mg / kg daily for 10 days) in the three-chamber sociability test (FIG. 7A) and the elevated plus maze test (FIG. 7B). 7-NI reversed the autistic phenotype in both tests. [Figure 8A] 8A shows the behavioral test analysis of SHANK3 mutant mice treated with 0, 20, 50, and 80 mg / kg 7-NI. FIG. 8A shows the average speed observed in the open field test. No significant difference was observed between 80 mg / kg treated mice and 20 and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8B shows the distance traveled observed in the open field test. No significant difference was observed in the total distance traveled between 80, 20, and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8C shows the time to interact with stranger mice in the sociability test, first session. 80 mg / kg treated mice spent more time interacting with stranger mice compared to mice treated with low doses of 7-NI. One-way ANOVA was performed. *P<0.05, **P<0.01. N=5. Figure 8D shows the time of interaction with a novel stranger mouse (S2) in the sociability test. Mice treated with 80 mg / kg spent more time interacting with the novel mouse compared to their counterparts treated with low doses of 7-NI. One-way ANOVA was performed ***P<0.001. N=5. [Figure 8B]8A shows the behavioral test analysis of SHANK3 mutant mice treated with 0, 20, 50, and 80 mg / kg 7-NI. FIG. 8A shows the average speed observed in the open field test. No significant difference was observed between 80 mg / kg treated mice and 20 and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8B shows the distance traveled observed in the open field test. No significant difference was observed in the total distance traveled between 80, 20, and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8C shows the time to interact with stranger mice in the sociability test, first session. 80 mg / kg treated mice spent more time interacting with stranger mice compared to mice treated with low doses of 7-NI. One-way ANOVA was performed. *P<0.05, **P<0.01. N=5. Figure 8D shows the time of interaction with a novel stranger mouse (S2) in the sociability test. Mice treated with 80 mg / kg spent more time interacting with the novel mouse compared to their counterparts treated with low doses of 7-NI. One-way ANOVA was performed ***P<0.001. N=5. [Figure 8C]8A shows the behavioral test analysis of SHANK3 mutant mice treated with 0, 20, 50, and 80 mg / kg 7-NI. FIG. 8A shows the average speed observed in the open field test. No significant difference was observed between 80 mg / kg treated mice and 20 and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8B shows the distance traveled observed in the open field test. No significant difference was observed in the total distance traveled between 80, 20, and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8C shows the time to interact with stranger mice in the sociability test, first session. 80 mg / kg treated mice spent more time interacting with stranger mice compared to mice treated with low doses of 7-NI. One-way ANOVA was performed. *P<0.05, **P<0.01. N=5. Figure 8D shows the time of interaction with a novel stranger mouse (S2) in the sociability test. Mice treated with 80 mg / kg spent more time interacting with the novel mouse compared to their counterparts treated with low doses of 7-NI. One-way ANOVA was performed ***P<0.001. N=5. [Figure 8D]8A shows the behavioral test analysis of SHANK3 mutant mice treated with 0, 20, 50, and 80 mg / kg 7-NI. FIG. 8A shows the average speed observed in the open field test. No significant difference was observed between 80 mg / kg treated mice and 20 and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8B shows the distance traveled observed in the open field test. No significant difference was observed in the total distance traveled between 80, 20, and 50 mg / kg treated mice. One-way ANOVA was performed. ns=not significant. N=5. FIG. 8C shows the time to interact with stranger mice in the sociability test, first session. 80 mg / kg treated mice spent more time interacting with stranger mice compared to mice treated with low doses of 7-NI. One-way ANOVA was performed. *P<0.05, **P<0.01. N=5. Figure 8D shows the time of interaction with a novel stranger mouse (S2) in the sociability test. Mice treated with 80 mg / kg spent more time interacting with the novel mouse compared to their counterparts treated with low doses of 7-NI. One-way ANOVA was performed ***P<0.001. N=5. [Figure 9A] Comparative immunofluorescence analysis of synaptophysin and 3-nitrotyrosine in differentiated SH-SY-5Y cells, SH-SY-5Y+SHANK3 siRNA treatment, and SH-SY-5Y cells with SHANK3 siRNA+nNOS si-RNA groups. Rabbit MAP2+primary mouse monoclonal 3-nitrotyrosine (Figure 9A-9B). Primary rabbit monoclonal synaptophysin+primary mouse Map2 (Figure 9C-9D). Cells were incubated with anti-mouse secondary Alexa fluor488 (green) / 594 (red), and anti-rabbit secondary Alexa fluor488 (green) / 594 (red), conjugated secondary antibody DAPI (blue). Images shown in Figure 9A and Figure 9C were acquired using a confocal microscope. Data shown in the bar graphs in Figure 9B and Figure 9D are mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test was performed. *P<0.05, **P<0.01, ***P<0.001. [Figure 9B] Comparative immunofluorescence analysis of synaptophysin and 3-nitrotyrosine in differentiated SH-SY-5Y cells, SH-SY-5Y+SHANK3 siRNA treatment, and SH-SY-5Y cells with SHANK3 siRNA+nNOS si-RNA groups. Rabbit MAP2+primary mouse monoclonal 3-nitrotyrosine (Figure 9A-9B). Primary rabbit monoclonal synaptophysin+primary mouse Map2 (Figure 9C-9D). Cells were incubated with anti-mouse secondary Alexa fluor488 (green) / 594 (red), and anti-rabbit secondary Alexa fluor488 (green) / 594 (red), conjugated secondary antibody DAPI (blue). Images shown in Figure 9A and Figure 9C were acquired using a confocal microscope. Data shown in the bar graphs in Figure 9B and Figure 9D are mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test was performed. *P<0.05, **P<0.01, ***P<0.001. [Figure 9C] Comparative immunofluorescence analysis of synaptophysin and 3-nitrotyrosine in differentiated SH-SY-5Y cells, SH-SY-5Y+SHANK3 siRNA treatment, and SH-SY-5Y cells with SHANK3 siRNA+nNOS si-RNA groups. Rabbit MAP2+primary mouse monoclonal 3-nitrotyrosine (Figure 9A-9B). Primary rabbit monoclonal synaptophysin+primary mouse Map2 (Figure 9C-9D). Cells were incubated with anti-mouse secondary Alexa fluor488 (green) / 594 (red), and anti-rabbit secondary Alexa fluor488 (green) / 594 (red), conjugated secondary antibody DAPI (blue). Images shown in Figure 9A and Figure 9C were acquired using a confocal microscope. Data shown in the bar graphs in Figure 9B and Figure 9D are mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test was performed. *P<0.05, **P<0.01, ***P<0.001. [Figure 9D]Comparative immunofluorescence analysis of synaptophysin and 3-nitrotyrosine in differentiated SH-SY-5Y cells, SH-SY-5Y+SHANK3 siRNA treatment, and SH-SY-5Y cells with SHANK3 siRNA+nNOS si-RNA groups. Rabbit MAP2+primary mouse monoclonal 3-nitrotyrosine (Figure 9A-9B). Primary rabbit monoclonal synaptophysin+primary mouse Map2 (Figure 9C-9D). Cells were incubated with anti-mouse secondary Alexa fluor488 (green) / 594 (red), and anti-rabbit secondary Alexa fluor488 (green) / 594 (red), conjugated secondary antibody DAPI (blue). Images shown in Figure 9A and Figure 9C were acquired using a confocal microscope. Data shown in the bar graphs in Figure 9B and Figure 9D are mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test was performed. *P<0.05, **P<0.01, ***P<0.001. [Figure 10A] Photographs of vials containing exemplary pre-nanoparticle lipid (PNL) formulations without (MA-9-61-stock) and with (MA-9-61-A) 2% w / v 7-NI are shown (FIG. 10A), photographs of vials containing aqueous dispersions of each formulation (FIG. 10B), and DLS particle size data for each formulation (FIG. 10C). [Figure 10B] Photographs of vials containing exemplary pre-nanoparticle lipid (PNL) formulations without (MA-9-61-stock) and with (MA-9-61-A) 2% w / v 7-NI are shown (FIG. 10A), photographs of vials containing aqueous dispersions of each formulation (FIG. 10B), and DLS particle size data for each formulation (FIG. 10C). [Figure 10C] Photographs of vials containing exemplary pre-nanoparticle lipid (PNL) formulations without (MA-9-61-stock) and with (MA-9-61-A) 2% w / v 7-NI are shown (FIG. 10A), photographs of vials containing aqueous dispersions of each formulation (FIG. 10B), and DLS particle size data for each formulation (FIG. 10C). [Figure 11A]Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 11B] Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 11C] Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 11D] Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 11E] Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 11F] Exemplary synthetic routes for preparing exemplary cationic salts of 7-NI (FIGS. 11A-11E) and exemplary anionic salts of 7-NI (FIG. 11F) are shown. [Figure 12A] Shown is a representative UV spectrum of 7-NI and the generated standard curve in methanol at 355 nm (FIG. 12A), as well as a plot showing the release of 7-NI from a PSARA gel formulation (MA-8-75-A) in phosphate buffer solution (PBS) at 37° C. The drug content in the formulation is 5% w / w. [Figure 12B] Shown is a representative UV spectrum of 7-NI and the generated standard curve in methanol at 355 nm (FIG. 12A), as well as a plot showing the release of 7-NI from a PSARA gel formulation (MA-8-75-A) in phosphate buffer solution (PBS) at 37° C. The drug content in the formulation is 5% w / w. [Figure 13A]Behavioral testing analysis for wild type (control), SHANK3 mutant (SHANK3) and 7-NI treated SHANK3 mice (single dose-treatment) (SHANK3- / -, ip injection of 5% w / w 7-NI PSARA gel formulation described in Example 12 herein) is shown. Experiments were started 4 days after injection. Figure 13A shows data obtained in the open field test-locomotor activity (day 4 after single dose). Figures 13B-C show data obtained in the 3-chamber sociability test on day 1 (day 9 after single dose) (Figure 13B) and day 2 (day 10 after single dose) (Figure 13C). SHANK3 mice did not show a significant change in the time to interact with stranger 1 mice compared to control mice. 7-NI-treated mice significantly increased the time interacting with stranger 1 mice compared to untreated SHANK3 mice (Figure 13B, n=4, 4, 4 for control, SHANK3, and treated mice, respectively. ns=not significant, **P<0.01). SHANK3 mice spent significantly less time interacting with stranger 2 mice compared to control mice. 7-NI-treated mice spent significantly more time interacting with stranger 2 mice compared to untreated SHANK3 mice (Figure 13C, n=4, 4, 4 for control, SHANK3, and treated, respectively. ***P<0.001, **P<0.01). Data from the marble burying test are shown in Figure 13D, where the number of buried marbles is shown 7 days after a single dose. Analysis showed that SHANK3 mice buried significantly fewer marbles than control mice, indicating a lack of novelty-seeking tendency and limited interest. 7-NI-treated mice buried more marbles compared to SHANK3 mice (n=4, 4, 4 for control, SHANK3, and treated mice, respectively, ***P<0.001, ***P<0.001). Data obtained in the elevated plus maze test are shown in FIG. 13E (12 days after a single dose). SHANK3 mice showed no significant change in the time spent in the open arms compared to their control counterparts. 7-NI-treated mice spent significantly more time in the open arms compared to their untreated SHANK3 littermates.(n=4, 4, 4 for control, SHANK3 and treated, respectively. ns=not significant, ***P<0.05). Data are presented as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 13B]Behavioral testing analysis for wild type (control), SHANK3 mutant (SHANK3) and 7-NI treated SHANK3 mice (single dose-treatment) (SHANK3- / -, ip injection of 5% w / w 7-NI PSARA gel formulation described in Example 12 herein) is shown. Experiments were started 4 days after injection. Figure 13A shows data obtained in the open field test-locomotor activity (day 4 after single dose). Figures 13B-C show data obtained in the 3-chamber sociability test on day 1 (day 9 after single dose) (Figure 13B) and day 2 (day 10 after single dose) (Figure 13C). SHANK3 mice did not show a significant change in the time to interact with stranger 1 mice compared to control mice. 7-NI-treated mice significantly increased the time interacting with stranger 1 mice compared to untreated SHANK3 mice (Figure 13B, n=4, 4, 4 for control, SHANK3, and treated mice, respectively. ns=not significant, **P<0.01). SHANK3 mice spent significantly less time interacting with stranger 2 mice compared to control mice. 7-NI-treated mice spent significantly more time interacting with stranger 2 mice compared to untreated SHANK3 mice (Figure 13C, n=4, 4, 4 for control, SHANK3, and treated, respectively. ***P<0.001, **P<0.01). Data from the marble burying test are shown in Figure 13D, where the number of buried marbles is shown 7 days after a single dose. Analysis showed that SHANK3 mice buried significantly fewer marbles than control mice, indicating a lack of novelty-seeking tendency and limited interest. 7-NI-treated mice buried more marbles compared to SHANK3 mice (n=4, 4, 4 for control, SHANK3, and treated mice, respectively, ***P<0.001, ***P<0.001). Data obtained in the elevated plus maze test are shown in FIG. 13E (12 days after a single dose). SHANK3 mice showed no significant change in the time spent in the open arms compared to their control counterparts. 7-NI-treated mice spent significantly more time in the open arms compared to their untreated SHANK3 littermates.(n=4, 4, 4 for control, SHANK3 and treated, respectively. ns=not significant, ***P<0.05). Data are presented as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 13C]Behavioral testing analysis for wild type (control), SHANK3 mutant (SHANK3) and 7-NI treated SHANK3 mice (single dose-treatment) (SHANK3- / -, ip injection of 5% w / w 7-NI PSARA gel formulation described in Example 12 herein) is shown. Experiments were started 4 days after injection. Figure 13A shows data obtained in the open field test-locomotor activity (day 4 after single dose). Figures 13B-C show data obtained in the 3-chamber sociability test on day 1 (day 9 after single dose) (Figure 13B) and day 2 (day 10 after single dose) (Figure 13C). SHANK3 mice did not show a significant change in the time to interact with stranger 1 mice compared to control mice. 7-NI-treated mice significantly increased the time interacting with stranger 1 mice compared to untreated SHANK3 mice (Figure 13B, n=4, 4, 4 for control, SHANK3, and treated mice, respectively. ns=not significant, **P<0.01). SHANK3 mice spent significantly less time interacting with stranger 2 mice compared to control mice. 7-NI-treated mice spent significantly more time interacting with stranger 2 mice compared to untreated SHANK3 mice (Figure 13C, n=4, 4, 4 for control, SHANK3, and treated, respectively. ***P<0.001, **P<0.01). Data from the marble burying test are shown in Figure 13D, where the number of buried marbles is shown 7 days after a single dose. Analysis showed that SHANK3 mice buried significantly fewer marbles than control mice, indicating a lack of novelty-seeking tendency and limited interest. 7-NI-treated mice buried more marbles compared to SHANK3 mice (n=4, 4, 4 for control, SHANK3, and treated mice, respectively, ***P<0.001, ***P<0.001). Data obtained in the elevated plus maze test are shown in FIG. 13E (12 days after a single dose). SHANK3 mice showed no significant change in the time spent in the open arms compared to their control counterparts. 7-NI-treated mice spent significantly more time in the open arms compared to their untreated SHANK3 littermates.(n=4, 4, 4 for control, SHANK3 and treated, respectively. ns=not significant, ***P<0.05). Data are presented as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 13D]Behavioral testing analysis for wild type (control), SHANK3 mutant (SHANK3) and 7-NI treated SHANK3 mice (single dose-treatment) (SHANK3- / -, ip injection of 5% w / w 7-NI PSARA gel formulation described in Example 12 herein) is shown. Experiments were started 4 days after injection. Figure 13A shows data obtained in the open field test-locomotor activity (day 4 after single dose). Figures 13B-C show data obtained in the 3-chamber sociability test on day 1 (day 9 after single dose) (Figure 13B) and day 2 (day 10 after single dose) (Figure 13C). SHANK3 mice did not show a significant change in the time to interact with stranger 1 mice compared to control mice. 7-NI-treated mice significantly increased the time interacting with stranger 1 mice compared to untreated SHANK3 mice (Figure 13B, n=4, 4, 4 for control, SHANK3, and treated mice, respectively. ns=not significant, **P<0.01). SHANK3 mice spent significantly less time interacting with stranger 2 mice compared to control mice. 7-NI-treated mice spent significantly more time interacting with stranger 2 mice compared to untreated SHANK3 mice (Figure 13C, n=4, 4, 4 for control, SHANK3, and treated, respectively. ***P<0.001, **P<0.01). Data from the marble burying test are shown in Figure 13D, where the number of buried marbles is shown 7 days after a single dose. Analysis showed that SHANK3 mice buried significantly fewer marbles than control mice, indicating a lack of novelty-seeking tendency and limited interest. 7-NI-treated mice buried more marbles compared to SHANK3 mice (n=4, 4, 4 for control, SHANK3, and treated mice, respectively, ***P<0.001, ***P<0.001). Data obtained in the elevated plus maze test are shown in FIG. 13E (12 days after a single dose). SHANK3 mice showed no significant change in the time spent in the open arms compared to their control counterparts. 7-NI-treated mice spent significantly more time in the open arms compared to their untreated SHANK3 littermates.(n=4, 4, 4 for control, SHANK3 and treated, respectively. ns=not significant, ***P<0.05). Data are presented as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 13E]Behavioral testing analysis for wild type (control), SHANK3 mutant (SHANK3) and 7-NI treated SHANK3 mice (single dose-treatment) (SHANK3- / -, ip injection of 5% w / w 7-NI PSARA gel formulation described in Example 12 herein) is shown. Experiments were started 4 days after injection. Figure 13A shows data obtained in the open field test-locomotor activity (day 4 after single dose). Figures 13B-C show data obtained in the 3-chamber sociability test on day 1 (day 9 after single dose) (Figure 13B) and day 2 (day 10 after single dose) (Figure 13C). SHANK3 mice did not show a significant change in the time to interact with stranger 1 mice compared to control mice. 7-NI-treated mice significantly increased the time interacting with stranger 1 mice compared to untreated SHANK3 mice (Figure 13B, n=4, 4, 4 for control, SHANK3, and treated mice, respectively. ns=not significant, **P<0.01). SHANK3 mice spent significantly less time interacting with stranger 2 mice compared to control mice. 7-NI-treated mice spent significantly more time interacting with stranger 2 mice compared to untreated SHANK3 mice (Figure 13C, n=4, 4, 4 for control, SHANK3, and treated, respectively. ***P<0.001, **P<0.01). Data from the marble burying test are shown in Figure 13D, where the number of buried marbles is shown 7 days after a single dose. Analysis showed that SHANK3 mice buried significantly fewer marbles than control mice, indicating a lack of novelty-seeking tendency and limited interest. 7-NI-treated mice buried more marbles compared to SHANK3 mice (n=4, 4, 4 for control, SHANK3, and treated mice, respectively, ***P<0.001, ***P<0.001). Data obtained in the elevated plus maze test are shown in FIG. 13E (12 days after a single dose). SHANK3 mice showed no significant change in the time spent in the open arms compared to their control counterparts. 7-NI-treated mice spent significantly more time in the open arms compared to their untreated SHANK3 littermates.(n=4, 4, 4 for control, SHANK3 and treated, respectively. ns=not significant, ***P<0.05). Data are presented as mean±SEM. Two-tailed t-tests were performed. **P<0.05. [Figure 14A] The optimal concentration of 7-NI (Figure 14A) and the successful knockdown of nNOS by siRNA (Figures 14B-E) are shown. Figure 14A shows a comparative plot showing the cell viability of SH-SY5Y as measured by MTT (dose-response curve). The concentration of 7-NI for cell treatment was 100 μM. Data is represented as 100%. [Figure 14B] The optimal concentration of 7-NI (FIG. 14A) and successful knockdown of nNOS by siRNA (FIGS. 14B-E) are shown. FIG. 14B is a representative blot of nNOS. 1: SH-SY5Y; 2: SH-SY5Y+negative control (NC); and 3: SH-SY5Y+si-nNos. [Figure 14C] The optimal concentration of 7-NI (Figure 14A) and successful knockdown of nNOS by siRNA (Figures 14B-E) are shown. Figure 14C shows representative fluorescent images of nNOS immunofluorescence. Blue indicates DAPI (a marker for nuclei), green indicates NeuN (a marker for neurons), and red indicates nNOS. [Figure 14D] The optimal concentration of 7-NI (FIG. 14A) and successful knockdown of nNOS by siRNA (FIGS. 14B-E) are shown. FIG. 14D is a bar graph showing the relative abundance of nNOS. [Figure 14E] The optimal concentration of 7-NI (FIG. 14A) and successful knockdown of nNOS by siRNA (FIGS. 14B-E) are shown. FIG. 14E is a bar graph showing the relative fluorescence intensity of nNOS. [Figure 15A] 15A and 15B are bar graphs showing the effect of 7-NI and nNOS silencing on nNOS activity and NO levels in SH-SY5Y cells. FIG. 15A shows NADPH-diaphorase activity in cell lysates. Absorbance at 585 nm was normalized for total protein. Data are expressed as mean±SEM (n=3), *P<0.05, **P<0.01. [Figure 15B] 15A and 15B are bar graphs showing the effect of 7-NI and nNOS silencing on nNOS activity and NO levels in SH-SY5Y cells. FIG. 15B shows nitrite levels (a marker of NO levels) measured by Griess assay. Data are expressed as mean±SEM (n=3), *P<0.05, **P<0.01. [Figure 16A] The effect of 7-NI and nNOS silencing on SH-SY5Y cell proliferation (clonogenic proliferation assay) is shown. Figure 16A shows representative images of plates with untreated SH-SY5Y cells and SH-SY5Y cells treated with 7-NI, si-nNOS RNA (silenced nNOS), and vehicle (negative control, NC). [Figure 16B] Figure 16 shows the effect of 7-NI and nNOS silencing on SH-SY5Y cell proliferation (clonogenic proliferation assay). Figure 16B shows a bar graph showing the number of cell colonies. Data are expressed as mean ± SEM (n=3), *P<0.05, **P<0.01. [Figure 17A] Data are shown from Western blot analysis of 3-nitrotyrosine, components of the mTOR signaling pathway, and synaptophysin levels. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS (nNOS knockdown); n=9 for each group. Figure 17A shows representative WBs of proteins prepared from cell lysates of SH-SY5Y (1), SH-SY5Y+7-NI (2), and SH-SY5Y+si-nNOS (3). [Figure 17B] Data from Western blot analysis of 3-nitrotyrosine, components of the mTOR signaling pathway, and synaptophysin levels are shown. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS (nNOS knockdown); n=9 for each group. Figure 17B is a bar graph showing the relative abundance of 3-Ntyr in the three groups of cells. [Figure 17C]Data from Western blot analysis of 3-nitrotyrosine, components of the mTOR signaling pathway, and synaptophysin levels are shown. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS (nNOS knockdown); n=9 for each group. Figure 17C shows representative Western blotting (WB) of proteins (p-mTOR, mTOR, TSC2, pAKT, AKT, pRPS6, RPS6, and Syp) prepared from cell lysates of the three groups. [Figure 17D] Data are shown from Western blot analysis of 3-nitrotyrosine, components of the mTOR signaling pathway, and synaptophysin levels. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS (nNOS knockdown); n=9 for each group. FIG. 17D is a bar graph showing the relative abundance of p-mTOR, mTOR, TSC2, pAKT, AKT, pRPS6, RPS6, and Syp. Data were normalized to β-actin and presented as mean±SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test was performed. *P<0.05, **P<0.01. [Figure 18A] Immunofluorescence of synaptophysin and 3-nitrotyrosine in SH-SY5Y cells with inhibited nNOS activity. Cells (with or without treatment) were post-fixed in 4% paraformaldehyde and then incubated with either primary rabbit monoclonal Syp (red) and primary mouse monoclonal NeuN (green) or primary mouse monoclonal 3-Ntyr (green) antibodies in separate cell groups. Cells were incubated with respective secondary antibodies such as anti-mouse Alexa fluor488 or anti-rabbit Alexa fluor594 and then mounted with DAPI (blue) in both cell groups. Images were acquired using a confocal microscope. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS. Each group consisted of three independent experimental sets, performed in triplicate. Initial cell seeding was approximately 106 / cm2. Figure 18A shows representative images of Syp and 3-Ntyr immunofluorescence. [Figure 18B] Immunofluorescence of synaptophysin and 3-nitrotyrosine in SH-SY5Y cells with inhibited nNOS activity. Cells (with or without treatment) were post-fixed in 4% paraformaldehyde and then incubated with either primary rabbit monoclonal Syp (red) and primary mouse monoclonal NeuN (green) or primary mouse monoclonal 3-Ntyr (green) antibodies in separate cell groups. Cells were incubated with respective secondary antibodies such as anti-mouse Alexa fluor488 or anti-rabbit Alexa fluor594 and then mounted with DAPI (blue) in both cell groups. Images were acquired using a confocal microscope. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS. Each group consisted of three independent experimental sets, performed in triplicate. Initial cell seeding was approximately 106 / cm2. Figures 18B-C are bar graphs showing the relative fluorescence intensity of Syp (Figure 18B) and N-Tyr (Figure 18C) immunofluorescence in the three groups of cells. Data are shown as mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test. *P<0.05, **P<0.01, ***P<0.001. Scale bars are 100 μm. [Figure 18C]Immunofluorescence of synaptophysin and 3-nitrotyrosine in SH-SY5Y cells with inhibited nNOS activity. Cells (with or without treatment) were post-fixed in 4% paraformaldehyde and then incubated with either primary rabbit monoclonal Syp (red) and primary mouse monoclonal NeuN (green) or primary mouse monoclonal 3-Ntyr (green) antibodies in separate cell groups. Cells were incubated with respective secondary antibodies such as anti-mouse Alexa fluor488 or anti-rabbit Alexa fluor594 and then mounted with DAPI (blue) in both cell groups. Images were acquired using a confocal microscope. Cell groups: SH-SY5Y, SH-SY5Y+7-NI, and SH-SY5Y+si-nNOS. Each group consisted of three independent experimental sets, performed in triplicate. Initial cell seeding was approximately 106 / cm2. Figures 18B-C are bar graphs showing the relative fluorescence intensity of Syp (Figure 18B) and N-Tyr (Figure 18C) immunofluorescence in the three groups of cells. Data are shown as mean ± SEM. One-way ANOVA followed by Fisher's LSD multiple comparison post-hoc test. *P<0.05, **P<0.01, ***P<0.001. Scale bars are 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0150] The present invention, in some embodiments thereof, relates to therapeutic methods, and more particularly to pharmaceutical compositions and methods for treating neurological conditions associated with aberrant activity of neuronal nitric oxide synthase (nNOS), including, but not limited to, autism spectrum disorder (ASD) and neurally derived cancers.

[0151] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0152] The inventors have recognized that neurological conditions such as, for example, ASD, Alzheimer's disease, and neuroblastoma are characterized by significant elevations in nitric oxide (NO) (e.g., free, physiologically available NO), particularly in neurons, and have surprisingly demonstrated that treatment with an nNOS inhibitor results in reversal of the diseased phenotype, thereby demonstrating a role for inhibition or reduction of nNOS activity in treating neurological conditions associated with elevated NO levels.

[0153] The present inventors have designed pharmaceutical compositions that can be used in the inhibition of nNOS activity and in the treatment of neurological conditions associated with elevated NO levels and / or abnormal nNOS activity.

[0154] Embodiments of the present invention relate to methods for reducing the level (eg, amount or concentration) of physiologically available nitric oxide and / or reducing the activity of nNOS in a subject in need thereof.

[0155] Embodiments of the present invention relate to methods of treating diseases or conditions in which a beneficial clinical effect is achieved by reducing nNOS activity, such as, but not limited to, ASD or cancer of neural origin (cancer of neural origin or neural cancer).

[0156] Embodiments of the present invention further relate to pharmaceutical compositions that can be used in any of the above methods.

[0157] Some embodiments of the present invention relate to pharmaceutical compositions and / or methods in which an active agent that reduces the activity of nNOS selectively or preferentially reduces nNOS activity in the CNS (eg, brain) of a subject.

[0158] Some embodiments of the present invention relate to pharmaceutical compositions comprising an agent that is a selective nNOS inhibitor.

[0159] Methods and Use: According to an aspect of some embodiments of the present invention there is provided a method of treating a disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity, comprising administering to a subject in need of such treatment an effective amount of a composition comprising an active agent that reduces nNOS activity, thereby treating the disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity.

[0160] According to an aspect of some embodiments of the present invention there is provided a composition comprising an active agent that reduces nNOS activity, and optionally a pharma- ceutically acceptable carrier, for use in the treatment of a disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity.

[0161] According to some of any of the embodiments described herein, the methods and uses described herein are for reducing the level (e.g., amount) of physiologically available nitric oxide (NO) in neural cells of a subject in need thereof. According to some embodiments, the reduction is at least 20%, or at least 30%, or at least 40%, or at least 50%.

[0162] According to some of any of the embodiments described herein, the methods and uses described herein are for reducing the level (e.g., amount) of physiologically available nitric oxide (NO) in the CNS (e.g., brain) of a subject in need thereof. According to some embodiments, the reduction is at least 20%, or at least 30%, or at least 40%, or at least 50%.

[0163] According to an aspect of some embodiments of the present invention, there is provided a method for treating a disease or condition in which a beneficial clinical effect is achieved by reducing the amount of physiologically available nitric oxide in an affected tissue, comprising administering to a subject in need thereof an effective amount of a composition comprising an active agent that reduces nNOS activity, thereby treating the disease or condition. According to some embodiments, the effective amount of the composition is an amount that reduces the amount of physiologically available nitric oxide by at least 20%, or at least 30%, or at least 50%, or at least 60%, or at least 70%. According to some embodiments, the affected tissue comprises a neuronal cell. According to some embodiments, the affected tissue is in the CNS (e.g., brain) of a subject.

[0164] Reducing the activity of nNOS or reducing the amount of physiologically available nitric oxide can be measured by determining the amount of Ntyr in the subject's plasma, or by any other method known in the art, as discussed in more detail below.

[0165] According to some of any of the embodiments described herein, the methods and uses described herein are for reducing nNOS activity in the CNS or brain.

[0166] According to some of any of the embodiments described herein, the methods and uses described herein are for reducing the amount of physiologically available nitric oxide in neural cells, e.g., in the CNS or brain.

[0167] According to an aspect of some embodiments of the present invention, there is provided a method of treating a disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity in the brain, comprising administering to a subject in need of such treatment an effective amount of a composition comprising an active agent that reduces nNOS activity, wherein the reduction in nNOS activity is selective or preferential in the subject's brain compared to other tissues, whereby a beneficial clinical effect is achieved by reducing nNOS activity in the brain.

[0168] Alternatively or additionally, there is provided a composition comprising an active agent that reduces nNOS activity, for treating a disease or condition in a subject in need thereof in which a beneficial clinical effect is achieved by reducing nNOS activity in the brain, wherein the composition is formulated such that the reduction in nNOS activity is selective or preferential in the subject's brain compared to other tissues.

[0169] As used herein, the terms "treating" and "treatment" include arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0170] According to certain embodiments, the term "treatment" can refer to reducing the severity or duration of an undesirable behavior, increasing the duration between boosts of an undesirable behavior, preventing a behavior before it occurs, improving development, and improving the overall condition.

[0171] According to certain embodiments, the terms "treatment" and "treating" exclude prevention.

[0172] As used herein, a "disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity" refers to a medical condition in which elevated levels of nitric oxide (NO) are evident as compared to NO levels in control subjects not suffering from the medical disease or condition. According to certain embodiments, the control subjects belong to a control population of the same sex, developmental stage and ethnic group as the subject being treated.

[0173] As used herein, a "disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity in the brain" refers to a medical condition in which elevated levels of nitric oxide (NO) are evident in a portion of the central nervous system (CNS) as compared to NO levels in control subjects not suffering from the medical condition in the CNS. According to certain embodiments, the control subjects belong to a control population of the same sex, developmental stage and ethnic group as the subject being treated.

[0174] As used herein, "nNOS" refers to the protein product of the NOS1 gene located on human chromosome 12, 12q24.22, BRENDA 1.14.13.39. It is also known as NOS1, the enzyme encoded by the NOS1 gene in humans. The nNOS gene is designated in humans as Accession Number P29475.

[0175] Orthologs from other non-human animals are also contemplated herein.Nitric oxide synthase (EC 1.14.13.39) (NOS) is a family of synthases that catalyze the production of nitric oxide (NO) from L-arginine.NO is a chemical messenger with diverse functions throughout the body depending on its enzyme source and tissue localization.In the brain and peripheral nervous system, where NOS1 is primarily located, NO exhibits many properties of a neurotransmitter and can be involved in long-term potentiation.

[0176] Neuronal NOS (NOS1), endothelial NOS (NOS3), and inducible NOS macrophage NOS are distinct isoforms. Both the neuronal and macrophage forms are unusual among oxidative enzymes, requiring several electron donors (cofactors), including, for example, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), NADPH, and tetrahydrobiopterin. All nitric oxide synthase isoforms are calcium-calmodulin regulated. For example, nNOS is activated at calcium concentrations above 100 nM.

[0177] As used herein, unless otherwise indicated, "nNOS activity" refers to the production of free (physiologically available) nitric oxide (NO) in neuronal cells or nerve tissue (neuronal tissue), including the peripheral nervous system and / or the central nervous system.

[0178] "nNOS activity" may also refer to the production of free (physiologically available) nitric oxide (NO) by the nNOS enzyme in neural tissue (CNS, e.g., brain) or peripheral organs that contain neurons that express nNOS.

[0179] The production of NO by NOS is achieved by catalyzing the five-electron oxidation of the nitrogen atom of the guanidine group of L-arginine. The oxidation of L-Arg to L-citrulline is ω The oxidation of NO occurs via two successive monooxygenation reactions that generate 1,2-hydroxy-L-arginine (NOHLA) as an intermediate. Two moles of O2 and 1.5 moles of NADPH are consumed per mole of NO formed.

[0180] nNOS activity can be effected (increased or decreased) by interfering with the production of free NO, for example by affecting the level of one or more cofactors involved in the monooxygenation reaction and / or the level of its substrate (L-arginine), and / or by interfering with its level of binding to L-arginine, for example by a substrate-competitive inhibitor.

[0181] Unregulated production of free NO by nNOS (increased compared to healthy conditions) can induce ASD-like symptoms in model animals as described below and in the Examples section below.

[0182] Reduction of nNOS activity can be achieved by downregulating the levels of the nNOS protein product, its encoding DNA or mRNA, or by inhibiting the activity of the enzyme, by interfering with the cellular localization of the enzyme, inhibiting its intrinsic activity, or inhibiting the activity of its activators or effectors, each alternative being considered a separate embodiment of the invention.

[0183] For example, nNOS activity can be inhibited by interfering with the production of free NO, e.g., by reducing the level of one or more cofactors involved in the monooxygenation reaction, and / or by reducing the level of its substrate (L-arginine) and / or by interfering with its binding to L-arginine, e.g., by substrate-competitive inhibitors.

[0184] Selective inhibition of nNOS, relative to inhibition of other NOS isoforms, can be achieved by disrupting the levels of cofactors involved in nNOS-catalyzed NO production but not NO production catalyzed by other isoforms, and / or by using substrate-competitive inhibitors that have a higher affinity for nNOS relative to their affinity for other NOS isoforms. In some embodiments, a selective nNOS inhibitor has an affinity for nNOS, as determined by its dissociation constant (Ki) for nNOS, that is at least 10-fold, or at least 100-fold, or at least 1,000-fold higher than its dissociation constant (Ki) for eNOS or iNOS.

[0185] Nitric oxide synthase activity assays are commercially available. These assays include, for example, NOS activity assay kits by Abcam, e.g., ab211083. Activity parameters can be tyrosine nitration, NO2, NO3, etc.

[0186] Alternatively, or additionally, nNOS levels can be determined at the protein or mRNA level.

[0187] nNOS protein levels can be determined using immunological assays, such as Western blotting, ELISA, for example by using an ELISA assay kit such as those available from Cosmo Bio USA or LSBio, e.g. LS-F4243.

[0188] Dotsch et al. Int J Cancer. 2000 Oct 15;88(2):172-5 teaches the determination of nNOS at the mRNA level.

[0189] As used herein, a "disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity" refers to a medical condition whose onset or progression depends on overproduction of nitric oxide (NO) in neuronal cells (compared to its levels in normal / healthy CNS tissue), which is typically achieved by dysregulated (increased) activity in nNOS compared to healthy neural tissue (peripheral or central nervous system).

[0190] In some embodiments, a "disease or condition in which a beneficial clinical effect is achieved by reducing nNOS activity" refers to a medical condition whose onset or progression depends on overproduction of nitric oxide (NO) in the CNS (e.g., brain) (compared to its levels in normal / healthy CNS tissue), which is typically achieved by dysregulated (increased) activity in nNOS compared to a healthy CNS.

[0191] The beneficial effect relates to a reduction in at least one pathological symptom.

[0192] As used herein, "reduction in at least one symptom associated with a disease or condition" refers to a statistically significant reduction, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even complete elimination of the symptom.

[0193] One of ordinary skill in the art would know how to determine the level or presence or absence of a symptom.

[0194] According to certain embodiments, the reduction / decrease in nNOS activity (or expression) is optimally to that level or levels of NO in normal / healthy tissues of the CNS, or to a level that improves the medical condition of the patient.

[0195] Exemplary diseases or conditions according to embodiments of the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, schizophrenia, addiction, ALS, epilepsy, bipolar disease, migraine, as well as all types of neurodevelopmental disorders and neurodegenerative diseases associated with imbalanced NO levels. Further examples include, but are not limited to, depression, ADD, ADHD, and hypoxic and ischemic conditions of the CNS, such as neonatal trauma during or after surgery, hypoxic-ischemic encephalopathy, etc. Metabolic diseases involving the central nervous system, such as sphingolipid metabolic disorders (i.e., GM1-gangliosidosis, GM2-gangliosidosis, Tay-Sachs disease, Sandhoff disease, AB variant of GM2-gangliosidosis, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease, types A, B, C, or Farber disease). See Tripathi MK, Kartawy M and Amal H. The role of nitric oxide in brain disorders: Autism spectrum disorder and other psychiatric, neurological, and neurodegenerative disorders. Redox Biol. 2020:101567, incorporated by reference to the same extent as if fully set forth herein.

[0196] As shown in the Examples section below, the inventors have demonstrated alleviation of ASD-like symptoms in a mouse model treated with an nNOS inhibitor.

[0197] According to certain embodiments, the disease or condition is a brain disorder.

[0198] According to certain embodiments, the disease or condition is a memory disorder or a mood disorder.

[0199] In certain embodiments, the disease or condition is a neuropsychiatric disorder.

[0200] According to certain embodiments, the disease or condition is an autism spectrum disorder (ASD).

[0201] According to certain embodiments, the term "autism spectrum disorder" refers to conditions previously diagnosed as autism (autistic disorder), including high-functioning pervasive developmental disorder, Asperger's syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), and childhood disintegrative disorder.

[0202] The term "autism spectrum disorder" or ASD also encompasses other diseases or medical conditions that include ASD symptoms (e.g., communication and social interaction disorders), e.g., as defined below, including, for example, ADD, ADHD, ADNP (Helsmoortel-van der Aa) syndrome, and NAP (Davnetide) syndrome, and conditions related thereto (e.g., Alzheimer's disease).

[0203] The term "treatment of ASD" refers to the improvement of at least one undesirable parameter, characteristic or behavior associated with ASD, including problems with social communication and social interaction; the presence of restricted and repetitive patterns of behavior, interests, or activities; self-injurious behaviors (SIB); abnormal responses to the senses, including sight, sound, touch, and smell; problems maintaining a consistent speech rhythm; developmental problems manifested in sensory disorders, developmental, associational, speech and language, and motor disorders, including memory deficits and other cognitive dysfunctions.

[0204] According to certain embodiments, ASD is defined according to DSM-5, as provided below. A. Persistent deficits in social communication and social interaction across multiple settings, currently or in the past, manifested by (examples are illustrative and not exhaustive): 1. Deficits in social-emotional interactions, e.g. abnormal social approaches and problems in normal conversational exchanges, inability to share interests, feelings, or emotions, failure to initiate or respond to social interactions. 2. Deficits in non-verbal communication behaviour used for social interaction, such as poorly integrated verbal and non-verbal communication, abnormalities in eye contact and body language, or inability to understand and use gestures, complete lack of facial expressions and non-verbal communication. 3. Deficits in the ability to develop, maintain and understand relationships, e.g. inability to adjust behavior to fit different social situations, difficulties in sharing creative play or making friends, lack of interest in peers.

[0205] Severity is based on repetitive patterns of social-communicative impairment and restricted behavior (see Table 1 below). B. A restricted, repetitive pattern of behavior, interests, or activities, currently or in the past, manifested by at least two of the following (examples are illustrative and not exhaustive): 1. Stereotyped or repetitive motor movements, object use, or speech (e.g., simple motor stereotypies, lining up toys or tipping over objects, echolalia, idiosyncratic phrases). 2. Preoccupation with sameness, inflexibility in adhering to habits, or ritualized patterns or verbal and nonverbal behaviors (e.g., extreme distress over small changes, difficulty making transitions, rigid thought patterns, greeting rituals, need to take the same route or eat the same foods every day). 3. Highly restricted, fixed interests that are abnormal in intensity or focus (e.g., intense fixation on unusual objects or preoccupation with unusual objects, excessively localized or perseverative interest). 4. Hyper- or hypo-responsiveness to sensory input or unusual interest in sensory aspects of the environment (e.g., apparent indifference to pain / temperature, adverse reactions to particular sounds or textures, excessive smell or touch of objects, visual fascination with light or movement).

[0206] Severity is based on repetitive patterns of social-communicative impairment and restricted behavior (see Table A below). C. Symptoms must be present during early development (but may not become fully manifest until social demands exceed limited capabilities, or may be masked by learned strategies later in life). D. The symptoms cause clinically significant impairment in social, occupational, or other important areas of current functioning. E. The disorders are not fully explained by intellectual disability (intellectual developmental disorder) or global developmental delay. Intellectual disability and autism spectrum disorder frequently co-occur, and to make a comorbid diagnosis of autism spectrum disorder and intellectual disability, social communication should be less than expected for a typical developmental level.

[0207] Individuals with a well-established DSM-IV diagnosis of autistic disorder, Asperger's disorder, or pervasive developmental disorder not otherwise specified should be given a diagnosis of autism spectrum disorder. Individuals with significant deficits in social communication, but whose symptoms do not otherwise meet criteria for an autism spectrum disorder, should be evaluated for social (pragmatic) communication disorder.

[0208] The following should be identified: With or without intellectual disability, With or without speech impediment, (Coding Note: Use additional codes to identify associated medical or genetic conditions.) Associated with another neurodevelopmental, psychiatric, or behavioral disorder; (Coding note: Use additional codes to identify associated neurodevelopmental, psychiatric, or behavioral disorders.) Is it accompanied by catatonia? Associated with a known medical or genetic condition or environmental factor;

[0209] [Table 1]

[0210] According to certain embodiments, the condition is a social communication disorder. The diagnostic criteria according to DSM-5 are summarized below: A. Persistent difficulty in the social use of verbal and non-verbal communication as manifested by all of the following: 1. Deficits in using communication for social purposes, such as greetings and sharing information, in a manner appropriate to the social situation. 2. Impaired ability to adapt communication to fit the situation or needs of the listener, such as speaking differently in the classroom than on the playground, speaking differently to children than to adults, and avoiding the use of overly formal language. 3. Difficulty following the rules of conversation and eloquence, such as taking turns in a conversation, rephrasing when misunderstood, and knowing how to use verbal and non-verbal signals to regulate the interaction. 4. Has difficulty understanding what is not explicitly stated (e.g., making inferences) and the non-literal or ambiguous meaning of language (e.g., idioms, humor, metaphors, multiple meanings that depend on context for interpretation). B. The deficits, individually or in combination, result in functional limitations in effective communication, social participation, social relationships, academic achievement, or occupational performance. C. The onset of symptoms is early in development (although deficits may not become fully manifest until social-communicative demands exceed limited abilities). D. The symptoms are not attributable to another medical or neurological condition or to poor ability in an area or in word structure and grammar and are not better explained by autism spectrum disorder, intellectual disability (intellectual developmental disorder), global developmental delay, or another psychiatric disorder.

[0211] Each of ASD and SCD is contemplated herein as a co-morbidity or alternative embodiment.

[0212] The following co-occurrences are also contemplated, each of which is considered an alternative embodiment:

[0213] Genetic disorders. Approximately 10-15% of autism cases have an identifiable Mendelian (single-gene) condition, chromosomal abnormality, or other genetic syndrome, and ASD is associated with several genetic disorders [Zafeiriou DI, Ververi A, Vargiami E (2007). "Childhood autism and associated comorbidities". Brain Dev. 29(5):257-272. doi:10.1016 / j.braindev.2006.09.003].

[0214] Intellectual disability. The percentage of autistic individuals who also meet criteria for intellectual disability has been reported to range anywhere from 25% to 70% [Dawson et al. Learning and Memory: A Comprehensive Reference. Vol. 2. Elsevier. pp. 759-772. doi:10.1016 / B978-012370509-9.00152-2. ISBN 978-0-12-370504-4. OCLC 775005136].

[0215] Anxiety disorders are common among children with ASD, with prevalence rates ranging from 11% to 84% [White et al. 2009 Clin Psychol Rev. 29(3):216-229].

[0216] Epilepsy is associated with variation in risk of epilepsy due to age, cognitive level, and type of language disorder [Spence et al. ediatr Res. 65(6):599-606. doi:10.1203 / PDR.0b013e31819e7168].

[0217] Some metabolic disorders, such as phenylketonuria, are associated with autism symptoms [Manzi et al. J Child Neurol. 23(3):307-314. doi:10.1177 / 0883073807308698].

[0218] Attention deficit hyperactivity disorder (ADHD), tic disorders, attention deficit disorder (ADD) and others of these conditions are often present and are increasingly accepted as co-occurring conditions.

[0219] Sleep disorders affect approximately two-thirds of individuals with ASD at some point during childhood. These most commonly include symptoms of insomnia, such as difficulty falling asleep, frequent nighttime awakenings, and early morning awakenings.

[0220] According to certain embodiments, the disease or condition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, schizophrenia, addiction, amyotrophic lateral sclerosis (ALS), epilepsy, bipolar disorder and migraine.

[0221] In certain embodiments, the disease or condition is a neurodevelopmental disorder, a psychiatric disorder, or a neurodegenerative disease.

[0222] NO is involved in the development of cancer. We have shown that inhibition of NO production in neuroblastoma (NB) cells can suppress NB malignancy by pharmacological intervention using the selective nNOS inhibitor 7-NI or by genetic ablation using specific gene silencing tools to silence the expression of nNOS. This effect was manifested by reduced proliferation, a significant decrease in the levels of markers of NB Syp (MIETTINEN and RAPOLA, 1987), and nitrosative stress seen by a decrease in the levels of 3-Ntyr. We further showed for the first time that the cancer-promoting effect of nNOS is mediated by the activation of the mTOR signaling pathway, and that the selective nNOS inhibitor 7-NI can significantly inhibit both the activation of mTOR signaling and the malignancy of human NB cells. These results suggest that the nNOS-mTOR axis may serve as a novel potential target for NB treatment.

[0223] According to certain embodiments, the disease or condition is a cancer of neural or neuroectodermal origin, also referred to herein as neural cancer or neurally derived cancer.

[0224] According to certain embodiments, the cancer is selected from the group consisting of neuroblastoma, glioma, ganglioglioma, central neurocytoma, ganglioblastoma, medulloblastoma, and primitive neuroectodermal tumor (PNET).

[0225] According to certain embodiments, the cancer is neuroblastoma.

[0226] According to certain embodiments, the neuroblastoma comprises a mutation in ALK. The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) can be aberrantly activated in neuroblastoma, and somatic ALK mutations occur in 6%-10% of patients.

[0227] According to certain embodiments, the tumor is a primary tumor.

[0228] According to certain embodiments, the tumor is a metastatic tumor.

[0229] According to certain embodiments, the tumor is a tumor metastasis.

[0230] According to certain embodiments, the tumor is a recurrent tumor.

[0231] According to certain embodiments, the tumor is resistant to first-line (eg, chemotherapy) treatment.

[0232] Active agents (agents that reduce nNOS activity): Regardless of the medical condition being treated, embodiments of the invention relate to the administration of a composition comprising an effective amount of an active agent that reduces nNOS activity, as defined herein. In some embodiments, the active agent reduces the amount of NO physiologically available in neuronal cells, as described herein.

[0233] According to certain embodiments, the reduction in nNOS activity is selective or preferential in neural cells or neural tissue of the subject as compared to other tissues.

[0234] According to certain embodiments, the reduction in nNOS activity is selective or preferential in the peripheral and / or central nervous system of the subject as compared to other tissues.

[0235] According to certain embodiments, the reduction in nNOS activity is selective or preferential in the subject's brain compared to other tissues.

[0236] Alternatively or additionally, there is provided a pharmaceutical composition comprising an active agent that reduces nNOS activity and a pharma- ceutically acceptable carrier, wherein the composition and active agent are such that, upon administration to a subject, a reduction in nNOS activity is selectively or preferentially achieved in neural tissue of the subject relative to other tissues, as described herein.

[0237] Such compositions are also referred to herein as "nNOS-reducing compositions" or alternatively, "NO-reducing compositions."

[0238] As used herein, "active agent that decreases nNOS activity" refers to a substance or physical condition that decreases the activity of nNOS by any one of its intrinsic catalytic activity, its ability to interact with other cellular proteins or factors, by affecting its cellular localization, and / or by affecting its levels or levels of NO within a cell.

[0239] As noted, in some embodiments, the agents (also referred to as "nNOS inhibitors" or "neuronal NO reducing compositions") act to selectively or preferentially achieve a reduction in nNOS activity in the neuronal cells or neural tissue being treated, e.g., the CNS (e.g., brain) of a subject, relative to other tissues. nNOS is expressed in a variety of tissues, e.g., the brain, retina, frontal cortex, heart, colon, colonic muscle, rectum, prostate, and pancreas.

[0240] Alternatively, or in addition, according to some embodiments of the invention, the agent is active against nNOS and does not affect the activity of other NOS enzymes, such as NOS2 and / or NOS3, as described hereinabove with respect to nNOS selectivity.

[0241] Any of these embodiments, individually or in combination, ensure activity of the agent only at the affected site (or predisposing site), without acting on other NOS (e.g., iNOS) enzymes or other tissues in which nNOS is expressed.

[0242] According to one embodiment, the agent reduces nNOS expression.

[0243] According to one embodiment, the agent decreases nNOS translation.

[0244] According to one embodiment, the agent inhibits nNOS enzyme activity.

[0245] According to certain embodiments, the agent affects nNOS interaction with other proteins or its dimerization. For example, the protein acts as a homodimer. It interacts with DLG4. The interaction is probably prevented by the association between NOS1 and CAPON. It forms a ternary complex with CAPON and RASD1. It forms a ternary complex with CAPON and SYN1. It interacts with ZDHHC23. It interacts with NOSIP. This may impair its synaptic location (by similarity). It interacts with HTR4. It interacts with VAC14 (by similarity). It interacts with SLC6A4 (by similarity). It interacts with DLG4 (through the N-terminal domain) (through the N-terminal tandem pair of PDZ domains).

[0246] According to some of any of the embodiments described herein, the active agent in the composition may be a small molecule, an amino acid-based molecule (peptide, protein, antibody or RNA encoding same), or a nucleic acid-based molecule (e.g., RNA:DNA, including antisense and inhibitor RNA) or a combination of several types of molecules.

[0247] It is understood that when RNA encoding a claimed proteinaceous drug is used, measures will be taken to improve its stability and bioavailability, e.g., as described in U.S. Patent Application Publication No. 2015 / 0030576.

[0248] As mentioned above, the reduction in the amount of free NO may be due to one of the following mechanisms: 1. Reducing the expression of nNOS from its gene (at the transcriptional or translational level), or at the DNA level, as achieved by genome editing. 2. Inhibition of nNOS activity at the enzyme level (by small molecules, competitive peptides, antibodies, and antibody fragments) 3. Decreased levels of L-arginine, a precursor of NO. Using targeted mass spectrometry of small molecules (Triple quad), precise concentrations of arginine can be measured. 4. Increase in GSNO reductase levels or activity (decrease in the levels of GSNO). By measuring the protein expression using WB and its activity using ELISA, we can make conclusions about the levels and activity.

[0249] As used herein, the phrases "reduce expression or activity" or "downregulate expression or activity" (used interchangeably) refer to downregulating the expression of a protein (e.g., nNOS) at the genomic level (e.g., homologous recombination and site-specific endonucleases) and / or transcriptional level using various molecules that interfere with transcription and / or translation (e.g., RNA silencing agents), or at the protein level (e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave polypeptides, antibodies, etc.).

[0250] According to a particular embodiment, the nNOS gene is designated by the accession number P29475. According to one embodiment of the invention, expression from the gene may be selectively reduced at the transcriptional or translational level.

[0251] For the same culture conditions, expression or activity is generally expressed relative to expression or activity in cells of the same species, but not contacted with an agent or with a vehicle control (also referred to as a control).

[0252] Downregulation of expression can be either temporary or permanent.

[0253] According to certain embodiments, downregulating expression refers to the absence of mRNA and / or protein, as detected by RT-PCR or Western blot, respectively.

[0254] Primers for detecting nNOS expression are well within the capabilities of one of ordinary skill in the art, some of which are described hereinafter in the Examples section below.

[0255] According to other specific embodiments, downregulating expression refers to a decrease in mRNA and / or protein levels as detected by RT-PCR or Western blot, respectively. The decrease can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0256] Non-limiting examples of agents capable of downregulating nNOS expression are described in detail below.

[0257] Nucleic acid agents: Downregulation at the nucleic acid level is typically achieved using a nucleic acid agent having a nucleic acid backbone, DNA, RNA, a mimic thereof, or a combination thereof. The nucleic acid agent can be encoded from a DNA molecule or can be provided in the cell itself.

[0258] According to certain embodiments, the down-regulating agent is a polynucleotide.

[0259] According to certain embodiments, the downregulatory agent is a polynucleotide capable of hybridizing to the gene or mRNA encoding nNOS.

[0260] According to certain embodiments, the downregulatory agent interacts directly with nNOS.

[0261] According to certain embodiments, the agent binds directly to nNOS.

[0262] In certain embodiments, the agent indirectly binds to nNOS (e.g., binds to the effector of nNOS). More specifically, nNOS inhibitors (e.g., 7-NI) cause denitrosylation of TSC2, which leads to a decrease in mTOR signaling.

[0263] According to certain embodiments, the down-regulatory agent is an RNA silencing agent or a genome editing agent.

[0264] Therefore, the downregulation of nNOS can be achieved by RNA silencing.As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms mediated by RNA molecules that cause the inhibition or "silencing" of the expression of corresponding protein-coding genes (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational suppression).RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0265] As used herein, the term "RNA silencing agent" refers to an RNA that can specifically inhibit or "silence" the expression of a target gene. In certain embodiments, an RNA silencing agent can prevent complete processing (e.g., complete translation and / or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, such as RNA duplexes that include paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs, such as siRNAs, miRNAs, and shRNAs.

[0266] In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

[0267] In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0268] According to one embodiment of the invention, the RNA silencing agent is specific for the target RNA (e.g., nNOS) and does not cross-inhibit or silence other targets (e.g., (iNOS or eNOS) or splice variants exhibiting 99% or less overall homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% overall homology to the target gene (as determined by PCR, Western blot, immunohistochemistry and / or flow cytometry).

[0269] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNA (siRNA).

[0270] Below is a detailed description of RNA silencing agents that can be used in accordance with certain embodiments of the present invention.

[0271] dsRNA, siRNA, and shRNA - The presence of long dsRNA in cells stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer is involved in the processing of dsRNA into short pieces of dsRNA known as small interfering RNAs (siRNAs). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides long and contain duplexes of about 19 base pairs. The RNAi response is also characterized by an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNAs that have sequences complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0272] Thus, some embodiments of the present invention contemplate the use of dsRNA to downregulate protein expression from mRNA.

[0273] According to one embodiment, dsRNA longer than 30bp is used.Various studies have demonstrated that long dsRNA can be used to silence gene expression without inducing stress response or causing significant off-target effects (see, for example, Strat et al., Nucleic Acids Research, 2006, Vol.34, No.13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003; 13:381-392; Paddison PJ, et al., Proc. Natl Acad. Sci. USA. 2002; 99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134).

[0274] According to some embodiments of the invention, dsRNA is provided in cells in which the interferon pathway is not activated (see, e.g., Billy et al., PNAS 2001, Vol 98, pages 14428-14433, and Diallo et al, Oligonucleotides, October 1, 2003, 13(5):381-392. doi:10.1089 / 154545703322617069).

[0275] According to an embodiment of the present invention, long dsRNA is specifically designed not to induce interferon and PKR pathways to down-regulate gene expression.For example, Shinagwa and Ishii [Genes & Dev.17(11):1340-1345,2003] developed a vector named pDECAP to express long double-stranded RNA from RNA polymerase II (Pol II) promoter.Since the transcript from pDECAP lacks both 5'-cap structure and 3'-poly(A) tail that facilitates the transport of ds-RNA into cytoplasm, the long ds-RNA from pDECAP does not induce interferon response.

[0276] Another method to circumvent the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.

[0277] The term "siRNA" refers to small inhibitory RNA duplexes (generally 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21-mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the ends, but it has recently been described that chemically synthesized RNA duplexes of 25-30 bases in length can have as much as a 100-fold increase in potency compared to 21-mers at the same positions. It is suggested that the observed increase in potency obtained using longer RNAs in inducing RNAi is due to providing substrate (27-mer) to Dicer instead of product (21-mer), and that this improves the rate or efficiency of entry of the siRNA duplex into RISC. Exemplary siRNAs that can be used according to the present teachings are provided in the Examples section below and in SEQ ID NOs: 3-8.

[0278] The location of the 3'-overhang influences the potency of siRNA, with asymmetric duplexes with a 3'-overhang on the antisense strand generally being found to be more potent than those with a 3'-overhang on the sense strand (Rose et al., 2005). This may be due to asymmetric strand loading into RISC, as the opposite efficacy pattern is observed when targeting antisense transcripts.

[0279] The strands of double-stranded interfering RNA (e.g., siRNA) can be connected to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as described above, the RNA silencing agent of some embodiments of the present invention can be a small hairpin RNA (shRNA).

[0280] Exemplary siRNAs that can be used in accordance with the present teachings are provided in the Examples section below and in SEQ ID NOs:9-14.

[0281] The term "shRNA" as used herein refers to an RNA agent having a stem-loop structure that includes first and second regions of complementary sequence, the degree of complementarity and orientation of the regions being sufficient for base pairing to occur between the regions, the first and second regions being connected by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) in the loop region. The number of nucleotides in the loop is between 3-23, or 5-15, or 7-13, or 4-9, or 9-11. Some of the nucleotides in the loop may participate in base pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that may be used to form the loop include 5'-CAAGAGA-3' (SEQ ID NO: 1) and 5'-UUACAA-3' (SEQ ID NO: 2) (International Patent Applications Nos. 2013 / 126963 and 2014 / 107763). It will be appreciated by those skilled in the art that the resulting single stranded oligonucleotide forms a stem loop or hairpin structure that contains a double stranded region that can interact with the RNAi machinery.

[0282] Synthesis of RNA silencing agents suitable for use in some embodiments of the present invention can be performed as follows: First, nNOS mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. The occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as a potential siRNA target site. Preferably, siRNA target sites are selected from the open reading frame, since untranslated regions (UTRs) are more rich in regulatory protein binding sites. UTR binding proteins and / or translation initiation complexes may interfere with the binding of siRNA endonuclease complexes [Tuschl ChemBiochem.2:239-245]. However, it will be appreciated that siRNAs directed to untranslated regions may also be effective, as demonstrated for GAPDH, where siRNAs directed to the 5'UTR mediated a reduction of approximately 90% of cellular GAPDH mRNA and completely abolished protein levels (www(dot)ambion(dot)com / techlib / tn / 91 / 912.html).

[0283] Second, potential target sites are compared to the appropriate genome database (e.g., human, mouse, rat, etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov / BLAST / ). Putative target sites that show significant homology to other coding sequences are filtered out.

[0284] Select a suitable target sequence as a template for siRNA synthesis. The preferred sequence is one that contains low G / C content, since it has been proven to be more effective in mediating gene silencing compared to sequences with G / C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNA, it is preferable to use a negative control in combination. The negative control siRNA preferably contains the same nucleotide composition as the siRNA, but lacks significant homology to the genome. Therefore, a scrambled nucleotide sequence of siRNA is preferably used, provided that it does not show any significant homology to any other gene.

[0285] As noted above, it will be understood that the RNA silencing agents of some embodiments of the present invention need not be limited to molecules containing only RNA, but further encompass chemically modified nucleotides and non-nucleotides.

[0286] miRNAs and miRNA mimics - According to another embodiment, the RNA silencing agent may be a miRNA.

[0287] In certain embodiments, the miRNA is miR-146a or a synthetic version thereof. Other contemplated miRS include hsa-miR-146a-5p (MIRT735124), hsa-miR-1273f (MIRT783312), hsa-miR-143-3p (MIRT783811), hsa-miR-147a (MIRT783856), hsa-miR-3622a-5p (MIRT784976), hsa-miR-3911 (MIRT785428), hsa-miR-4269 (MIRT785560), hsa-miR-4708-5p (MIRT786482), hsa-miR-4710 (MIRT786499), hsa-miR-4770 (MIRT786499), and hsa-miR-4770 (MIRT786499). These include, but are not limited to, hsa-miR-4792 (MIRT786788), hsa-miR-574-5p (MIRT787873), hsa-miR-6088 (MIRT788038), hsa-miR-644a (MIRT788184), hsa-miR-6715b-5p (MIRT788450), hsa-miR-6768-5p (MIRT788631), hsa-miR-6867-5p (MIRT789213), hsa-miR-7855-5p (MIRT789601), and hsa-miR-8485 (MIRT789865).

[0288] The terms "microRNA", "miRNA", and "miR" are synonymous and refer to a collection of non-coding, single-stranded RNA molecules approximately 19-28 nucleotides in length that regulate gene expression. miRNAs are found in a wide range of organisms (viruses to humans) and have been shown to play roles in development, homeostasis, and disease pathogenesis.

[0289] Below is a brief description of the mechanism of miRNA activity.

[0290] Genes encoding miRNAs are transcribed, resulting in the production of miRNA precursors known as pri-miRNAs. Pri-miRNAs are typically part of a polycistronic RNA that contains multiple pri-miRNAs. Pri-miRNAs can form hairpins with stems and loops. The stems can contain mismatched bases.

[0291] The hairpin structure of the pri-miRNA is recognized by Drosha, an RNase III endonuclease. Drosha typically recognizes a terminal loop in the pri-miRNA and cleaves approximately two helical turns into the stem to generate a 60-70 nucleotide precursor known as the pre-miRNA. Drosha cleaves the pri-miRNA with a staggered cut typical of RNase III endonucleases to yield a pre-miRNA stem loop with a 5' phosphate and a ∼2-nucleotide 3' overhang. Approximately one turn of the stem (∼10 nucleotides) extending beyond the Drosha cleavage site is presumed to be essential for efficient processing. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by Ran-GTP and the transport receptor Ex-portin-5.

[0292] The double-stranded stem of the pre-miRNA is then recognized by Dicer, which is also an RNase III endonuclease. Dicer may also recognize a 5' phosphate at the base of the stem-loop and a 3' overhang. Dicer then cleaves the terminal loops of the two helical turns away from the base of the stem-loop, leaving an additional 5' phosphate and a 3' overhang of approximately 2 nucleotides. The resulting siRNA-like duplex, which may contain mismatches, is then synthesized into the mature miRNA and the miRNA. * miRNA and miRNA fragments of similar size known as * can be derived from opposing arms of the pri-miRNA and the pre-miRNA. *The sequence may be found in a library of cloned miRNAs, but typically at a lower frequency than the miRNAs.

[0293] miRNA was originally * miRNAs exist as double-stranded species with the nucleotide sequence β-terminally linked to the miRNA nucleotide sequence, but are ultimately incorporated as single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). A variety of proteins can form RISC, which in turn catalyzes the synthesis of miRNAs / miRNAs. * Duplex specificity, binding site of target gene, activity of miRNA (repression or activation), and miRNA / miRNA * This allows for variation in which strand of the duplex is loaded into RISC.

[0294] miRNA:miRNA * When the double-stranded miRNA strand is loaded into RISC, the miRNA * The strand of the miRNA:miRNA duplex that is loaded into RISC is the one that is less tightly paired at the 5' end. miRNA:miRNA * If both ends of the miRNA have approximately equal 5' pairing, the miRNA and the miRNA * Both may have gene silencing activity.

[0295] RISC identifies target nucleic acids based on the high level of complementarity between the miRNA and the mRNA, specifically by nucleotides 2-7 of the miRNA.

[0296] Many studies have examined the base pairing requirements between miRNAs and their mRNA targets to achieve efficient inhibition of translation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells, the first 8 nucleotides of the miRNA may be important (Doench & Sharp 2004 GenesDev 2004-504). However, other parts of the microRNA may also be involved in mRNA binding. Furthermore, sufficient base pairing at 3' can compensate for insufficient pairing at 5' (Brennecke et al, 2005 PLoS 3-e85). Computational studies analyzing miRNA binding to the whole genome suggested a specific role for bases 2-7 on the 5' side of the miRNA in target binding, but also recognized a role for the first nucleotide, usually found to be an "A" (Lewis et at 2005 Cell 120-15). Similarly, targets were identified and validated using nucleotides 1-7 or 2-8 by Krek et al. (2005, Nat Genet 37-495).

[0297] The target site in mRNA can be in 5'UTR, 3'UTR, or coding region. Interestingly, multiple miRNAs can regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translation inhibition.

[0298] miRNAs can instruct RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. If the mRNA has some degree of complementarity to the miRNA, the miRNA can specify the cleavage of the mRNA. If the miRNA guides the cleavage, the cleavage is typically between the nucleotides that pair with residues 10 and 11 of the miRNA. Alternatively, if the miRNA does not have the required degree of complementarity to the miRNA, the miRNA can repress translation. Translational repression may be more prevalent in animals, since animals may have a lower degree of complementarity between the miRNA and the binding site.

[0299] miRNA and miRNA * It should be noted that there may be variability at the 5' and 3' ends of any pair of miRNAs. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the cleavage site. * Variability at the 5' and 3' ends of the miRNAs may also result from mismatches in the stem structures of the pri-miRNA and pre-miRNA. Mismatches in the stem strands may result in a population of different hairpin structures. Variability in the stem structure may also result in variability in the cleavage products by Drosha and Dicer.

[0300] The term "microRNA mimic" or "miRNA mimic" refers to synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression. miRNA mimics mimic the function of endogenous miRNAs and can be designed as mature double-stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be composed of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNA or 2'-O,4'-C-ethylene bridged nucleic acid (ENA)). For mature double-stranded miRNA mimics, the length of the duplex region can vary between 13-33, 18-24, or 21-23 nucleotides. The miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.

[0301] Preparation of miRNA mimics can be by any method known in the art, such as chemical synthesis or recombinant methods.

[0302] It will be understood from the explanation provided herein above that contacting a cell with a miRNA can be achieved, for example, by transfecting the cell with the mature double-stranded miRNA, pre-miRNA or pri-miRNA.

[0303] The pre-miRNA sequence may comprise 45-90, 60-80, or 60-70 nucleotides.

[0304] The pri-miRNA sequence may contain 45-30,000, 50-25,000, 100-20,000, 1,000-1,500, or 80-100 nucleotides.

[0305] Antisense-Antisense is a single stranded RNA designed to prevent or inhibit the expression of a gene by specifically hybridizing to its mRNA. Downregulation of nNOS can be achieved using antisense polynucleotides capable of specifically hybridizing to the mRNA transcripts encoding nNOS.

[0306] The design of antisense molecules that can be used to efficiently downregulate nNOS must be done while taking into account two aspects that are important for the antisense approach: the first aspect is the delivery of the oligonucleotide to the cytoplasm of the appropriate cells, while the second aspect is the design of an oligonucleotide that specifically binds to a designated mRNA in the cell in a manner that inhibits its translation.

[0307] The prior art teaches several delivery strategies that can be used to efficiently deliver oligonucleotides to a wide variety of cell types [e.g., Jaaskelainen et al. Cell Mol Biol Lett. (2002) 7(2): 236-7; Gait, Cell Mol Life Sci. (2003) 60(5): 844-53; Martino et al. J Biomed Biotechnol. (2009) 2009: 410260; Grijalvo et al. Expert Opin Ther Pat. (2014) 24(7): 801-19; Falzarano et al, Nucleic Acid Ther. (2014) 24(1): 87-100; Shilakari et al. Biomed Res Int. (2014) 2014: 526391; Prakash et al. Nucleic Acids Res. (2014) 42(13):8796-807 and Asseline et al. J Gene Med. (2014) 16(7-8):157-65].

[0308] Furthermore, algorithms are available for identifying sequences with the highest predicted binding affinity to their target mRNAs based on thermodynamic cycles that describe the energetics of structural changes in both the target mRNA and the oligonucleotide [see, e.g., Walton et al. Biotechnol Bioeng 65:1-9 (1999)]. Such algorithms have been successfully used to implement antisense approaches in cells.

[0309] Additionally, several approaches for designing and predicting the efficacy of specific oligonucleotides using in vitro systems have also been published (Matveeva et al., Nature Biotechnology 16:1374-1375 (1998)).

[0310] Thus, the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems enables one of skill in the art to design and implement suitable antisense approaches to downregulate the expression of known sequences without having to resort to undue trial and error experimentation.

[0311] For example, a suitable antisense oligonucleotide targeted to nNOS mRNA (which encodes the nNOS protein) is of the sequence:

[0312] Korneev et al. [Scientific Reports volume 5, Article number: 11815 (2015)] report the long NAT contemplated herein (Mm-antiNos1RNA) as well as a synthetic version thereof.

[0313] Nucleic acid agents can also act at the DNA level, as summarized below.

[0314] Downregulation of nNOS can also be achieved by inactivating genes (eg, NOS1) through the introduction of targeted mutations, including loss-of-function changes in gene structure (eg, point mutations, deletions and insertions).

[0315] As used herein, the phrase "loss-of-function modification" refers to any mutation in the DNA sequence of a gene (e.g., NOS1) that results in downregulation of the expression level and / or activity of the expression product (i.e., mRNA transcript and / or translated protein). Non-limiting examples of such loss-of-function changes include: missense mutations, i.e., mutations that change an amino acid residue in a protein to another amino acid residue, thereby abolishing the enzymatic activity of the protein; nonsense mutations, i.e., mutations that introduce a stop codon into a protein, e.g., an early stop codon resulting in a shorter protein lacking enzymatic activity; frameshift mutations, i.e., mutations, usually deletions or insertions, of a nucleic acid that change the reading frame of a protein and can result in premature termination by introducing a stop codon into the reading frame (e.g., a truncated protein, lacking enzymatic activity) or can affect the secondary or tertiary structure of a protein, resulting in a longer amino acid sequence resulting in a non-functional protein lacking the enzymatic activity of the non-mutated polypeptide; read-through mutations due to frameshift mutations or altered stop codon mutations with destroyed enzymatic activity (i.e., when a stop codon is mutated to an amino acid codon); promoter Mutations, i.e., mutations in promoter sequences, usually 5' to the transcription start site of a gene, that result in downregulation of a particular gene product; regulatory mutations, i.e., mutations in regions upstream or downstream of a gene or within a gene that affect expression of the gene product; deletion mutations, i.e., mutations that delete coding nucleic acid in a gene sequence, which may result in a frameshift mutation or an in-frame mutation (deletion of one or more amino acid codons within the coding sequence); insertion mutations, i.e., mutations that insert coding or non-coding nucleic acid into a gene sequence, which may result in a frameshift mutation or an in-frame insertion of one or more amino acid codons; inversions, i.e., mutations that result in an inverted coding or non-coding sequence; splice mutations, i.e., mutations that result in aberrant or inefficient splicing; and duplication mutations, i.e., mutations that result in a duplicated coding or non-coding sequence, which may be in-frame or may cause a frameshift.

[0316] According to certain embodiments, the loss-of-function modification of a gene may involve at least one allele of the gene.

[0317] The term "allele" as used herein refers to any of one or more alternative forms of a genetic locus, all of which alleles are associated with a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0318] According to other specific embodiments, the loss-of-function alteration of a gene includes both alleles of the gene.In such a case, for example, NOS1 can be homozygous or heterozygous.According to this embodiment, homozygosity is the state in which both alleles at, for example, the NOS1 locus are characterized by the same nucleotide sequence.Heterozygosity refers to the different states of a gene at, for example, the NOS1 locus.

[0319] Methods for introducing nucleic acid modifications into a gene of interest are well known in the art (see, for example, Genesis (2013) 51:-618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci, the contents of which are incorporated herein by reference). (See USA (2008) 105: 5809-5814, International Patent Application Nos. 2014 / 085593, 2009 / 071334 and 2011 / 146121, U.S. Patent Nos. 8771945, 8586526, 6774279 and U.S. Patent Application Publication Nos. 2003 / 0232410, 2005 / 0026157 and 2006 / 0014264), which include targeted homologous recombination, site-specific recombinase, PB transposase and engineered nuclease-based genome editing. Agents for introducing nucleic acid modifications into genes of interest can be designed from publicly available sources or can be obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

[0320] Below are descriptions of various exemplary methods used to introduce nucleic acid modifications into a gene of interest, as well as agents for effecting the nucleic acid modifications that may be used according to certain embodiments of the invention.

[0321] Genome editing using engineered endonucleases - this approach refers to a reverse genetic method in which artificially engineered nucleases are used to cut and create specific double-stranded breaks at desired locations in the genome, which are then repaired by cell-intrinsic processes such as homology directed repair (HDR) and non-homologous end joining (NFfEJ). NFfEJ directly joins DNA ends at double-stranded breaks, while HDR utilizes homologous sequences as templates to regenerate lost DNA sequences at the break points. In order to introduce specific nucleotide modifications into genomic DNA, a DNA repair template containing the desired sequence must be present in HDR. Genome editing cannot be performed using conventional restriction endonucleases because most restriction enzymes recognize a few base pairs on DNA as their targets, and there is a very high probability that the combination of recognized base pairs will be found at many locations throughout the genome, resulting in multiple breaks that are not limited to the desired location. To overcome this challenge and create site-specific single-stranded or double-stranded breaks, several different classes of nucleases have been discovered and bioengineered to date. These include meganucleases, zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR / Cas systems.

[0322] Meganucleases-Meganucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from each other in terms of conserved structural elements and therefore DNA recognition sequence specificity and catalytic activity. Meganucleases are commonly found in microbial species and have the unique property of having very long recognition sequences (>14bp), thus making them naturally highly specific for cleavage at desired positions. This can be utilized to perform site-specific double-strand breaks in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to generate hybrid enzymes that recognize new sequences. Alternatively, the DNA-interacting amino acids of meganucleases can be modified to design sequence-specific meganucleases (see, for example, U.S. Patent No. 8,021,867). Meganucleases can be designed using methods described, for example, in Certo, MT et al. Nature Methods (2012) 9:073-975, U.S. Pat. Nos. 8,304,222, 8,021,867, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015, 8,143,016, 8,148,098, or 8,163,514, the contents of each of which are incorporated herein by reference in their entirety.Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technologies, for example Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0323] ZFNs and TALENs - Two different classes of engineered nucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven effective in generating targeted double-stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0324] Essentially, ZFN and TALEN restriction endonuclease technologies utilize a non-specific DNA cleavage enzyme linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme is selected whose DNA recognition and cleavage sites are distant from each other. The cleavage portion is separated and then linked to a DNA binding domain, thereby resulting in an endonuclease with very high specificity for the desired sequence. An exemplary restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage that it requires dimerization to have nuclease activity, which means that the specificity is dramatically increased because each nuclease partner recognizes a unique DNA sequence. To enhance this effect, FokI nucleases have been developed that can only function as heterodimers and have increased catalytic activity. Heterodimeric functioning nucleases avoid the possibility of undesired homodimer activity, thus increasing the specificity of double-stranded cleavage.

[0325] Thus, for example, to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. When transiently expressed in cells, the nucleases bind to their target site and the FokI domains heterodimerize to generate double-stranded breaks. Repair of these double-stranded breaks via the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Because each repair made by NHEJ is unique, the use of a single nuclease pair can generate an allelic series with a range of different deletions at the target site. Deletions typically range anywhere from a few base pairs to hundreds of base pairs in length, although larger deletions have been successfully generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). Additionally, if a fragment of DNA with homology to the target region is introduced in conjunction with a nuclease pair, the double-stranded break can be repaired via homology-directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0326] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 zinc fingers are typically found in repeats spaced 3 bp apart, and are found in a variety of combinations in various nucleic acid interacting proteins. On the other hand, TALEs are found in repeats with a one-to-one recognition ratio between the amino acid and the recognized nucleotide pair. Because both zinc fingers and TALEs occur in a repeating pattern, different combinations can be tried to create a wide variety of sequence specificities. Approaches for generating site-specific zinc finger endonucleases include, among others, for example, modular assembly (where zinc fingers correlating with triplet sequences are joined in a line to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides, followed by high stringency selection of peptide combinations versus the final target in a bacterial system), and bacterial one-hybrid screening of zinc finger libraries. ZFNs can be designed or obtained commercially, for example, from Sangamo Biosciences™ (Richmond, Calif.).

[0327] Methods for designing and obtaining TALENs are described, for example, in Reyon et al. Nature Biotechnology 2012 May;30(5):460-5, Miller et al. Nat Biotechnol.(2011)29:143-148, Cermak et al. Nucleic Acids Research(2011)39(12):e82 and Zhang et al. Nature Biotechnology(2011)29(2):149-53. A recently developed web-based program called Mojo Hand (accessible via www(dot)talendesign(dot)org) was introduced by Mayo Clinic to design TAL and TALEN constructs for genome editing applications. TALENs can be designed or obtained commercially, for example, from Sangamo Biosciences™ (Richmond, CA).

[0328] ZFIN (among other vendors) has commercialized the nNOS-specific Talen editing tool (ZDB-TALEN-181114-1).

[0329] CRISPR-Cas systems - Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade the nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeats (CRISPR) genes that produce an RNA component, and CRISPR-associated (Cas) genes that code for a protein component. CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade complementary nucleic acids of the corresponding pathogens. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that three components: the Cas9 nuclease, the crRNA that contains 20 base pairs of homology to the target sequence, and the trans-activating crRNA (tracrRNA) form an RNA / protein complex that together is sufficient for sequence-specific nuclease activity (Jinek et al. Science (2012) 337:816-821.). It was further demonstrated that synthetic chimeric guide RNAs (gRNAs) composed of a fusion between a crRNA and a tracrRNA can guide Cas9 to cleave DNA targets complementary to the crRNA in vitro, and that transient expression of Cas9 in combination with synthetic gRNAs can be used to produce targeted double-stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013; Mali et al., 2013).

[0330] The CRISPR / Cas system for genome editing contains two distinct components: a gRNA and an endonuclease, e.g., Cas9.

[0331] gRNAs are typically 20 nucleotide sequences that code for a combination of a target homologous sequence (crRNA) and an endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence by base pairing between the gRNA sequence and complementary genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas9 complex localizes Cas9 to the genomic target sequence such that Cas9 can cleave both strands of DNA to create a double-stranded break. Similar to ZFNs and TALENs, the double-stranded brakes produced by CRISPR / Cas can undergo homologous recombination or NHEJ.

[0332] The Cas9 nuclease has two functional domains: RuvC and HNH, each of which cleaves a different DNA strand. When both of these domains are active, Cas9 creates a double-stranded break in genomic DNA.

[0333] A key advantage of CRISPR / Cas is that the high efficiency of the system combined with the ability to easily generate synthetic gRNAs allows multiple genes to be targeted simultaneously. Moreover, the majority of cells carrying mutations display biallelic mutations in the targeted genes.

[0334] However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cleaved by Cas9.

[0335] Modified versions of Cas9 that contain a single inactive catalytic domain, either RuvC- or HNH-, are called "nickases." With only one active nuclease domain, Cas9 nickases cleave only one strand of the target DNA, generating a single-strand break or "nick." Single-strand breaks or nicks are usually rapidly repaired via the HDR pathway, using the intact complementary DNA strand as a template. However, two proximal, opposite-strand nicks introduced by Cas9 nickases are treated as double-strand breaks, often referred to as "double-nick" CRISPR systems. Double nicks can be repaired by either NHEJ or HDR, depending on the desired effect on the gene target. Thus, when specificity and reduced off-target effects are important, off-target effects are reduced by using Cas9 nickases to design two gRNAs with target sequences on adjacent and opposite strands of genomic DNA, since either gRNA alone will generate a nick that does not alter the genomic DNA.

[0336] A modified version of Cas9 enzyme containing two inactive catalytic domains (deadCas9 or dCas9) does not have nuclease activity, but can still bind to DNA based on gRNA specificity. dCas9 can be used as a platform for DNA transcription regulators to activate or suppress gene expression by fusing the inactive enzyme to a known regulatory domain. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

[0337] There are many publicly available tools available to aid in the selection and / or design of target sequences, such as Target Finder from the Feng Zhang Laboratory, Target Finder (E-CRISP) from the Michael Boutros Laboratory, RGEN Tools: Cas-OFFinder, CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes, and CRISPR Optimal Target Finder, as well as lists of bioinformatically determined unique gRNAs for different genes in different species.

[0338] The following vendors offer CRISPR-based products for NOS1 editing: Applied Biological Materials (abm):CRISPR clones against NOS1. NOS1 CRISPR sgRNA+Cas9 as a ready-to-use knockout vector or virus: Cas9 protein, Cas9 nuclease vectors / viruses, and Cas9-expressing cell lines are also available. OriGene CRISPR knockout for NOS1 GA103232 GA202933 Synthego CRISPR Products for NOS1: DIY CRISPR Kits: Gene Knockout Kits, Synthetic sgRNA, Cas9 / Engineered Cells: Immortalized KO Pools, KO Clones, iPSC KO, iPSC KI / Free Bioinformatics Tools: CRISPR Knockout Design Tools, CRISPR Analysis Tools VectorBuilder CRISPR Vectors for NOS1 (i.e., knockout, knockin, CRISPRRa, CRISPRi) VectorBuilder viral packaging for NOS1 CRISPR vectors (i.e., lentivirus, AAV, adenovirus) Santa Cruz Biotechnology (SCBT) CRISPR for NOS1 NOS1 CRISPR / Cas9 KO Plasmid (h) NOS1 CRISPR Activation Plasmid (h) NOS1 ZCRISPR plasmids for human targeting (h) are available from Santa-Cruz and other suppliers.

[0339] To use the CRISPR system, both gRNA and Cas9 should be expressed in the target cell. The insertion vector can contain both cassettes on a single plasmid, or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available, such as the px330 plasmid from Addgene.

[0340] "Hit-and-run" or "in-out" involves a two-step recombination procedure. In the first step, an insertion vector containing a dual positive / negative selectable marker cassette is used to introduce the desired sequence modification. The insertion vector contains a single continuous region of homology to the target locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at one site within the region of homology, electroporated into cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain local duplications separated by intervening vector sequences that contain the selection cassette. In the second step, the targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the recombination site, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without retaining any exogenous sequences.

[0341] The "double replacement" or "tag and exchange" strategy involves a two-step selection procedure similar to the hit-and-run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a double positive / negative selectable cassette near the position where the mutation is to be introduced. After electroporation and positive selection, homologously targeted clones are identified. A second targeting vector containing a region homologous to the desired mutation is then electroporated into the targeted clone, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating the undesired exogenous sequence.

[0342] Site-specific recombinase-Cre recombinase from P1 bacteriophage and Flp recombinase from yeast Saccharomyces cerevisiae are site-specific DNA recombinases (referred to as "Lox" and "FRT", respectively) that recognize a unique 34 base pair DNA sequence and sequences flanking either the Lox or FRT sites can be easily removed via site-specific recombination upon expression of Cre recombinase or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric 8 base pair spacer region flanked by a 13 base pair inverted repeat. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeat sequence and catalyzing strand breaks and religation within the spacer region. The staggered DNA breaks made by Cre in the spacer region are separated by 6 base pairs, providing overlapping regions that act as homology sensors, ensuring that only recombination sites with the same overlapping region recombine.

[0343] Essentially, the site-specific recombinase system provides a means for the removal of the selection cassette after homologous recombination. This system also allows for the generation of conditionally modified alleles that can be inactivated or activated in a time- or tissue-specific manner. Notably, Cre and Flp recombinases leave a 34-base pair Lox or FRT "scar". The remaining Lox or FRT sites are typically left in introns or 3'UTRs of the modified locus, and current evidence suggests that these sites do not usually significantly interfere with gene function.

[0344] Thus, Cre / Lox and Flp / FRT recombination involves the introduction of a targeting vector with 3' and 5' homology arms that contain the mutation of interest, two Lox or FRT sequences, and a selectable cassette typically located between the two Lox or FRT sequences. Positive selection is applied to identify homologous recombinants that contain the targeted mutation. Transient expression of Cre or Flp combined with negative selection results in excision of the selection cassette and selects for cells in which the cassette has been lost. The final targeted allele contains the exogenous sequence Lox or FRT scar.

[0345] Transposase - As used herein, the term "transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.

[0346] As used herein, the term "transposon" refers to a mobile genetic element that contains a nucleotide sequence that can move to different locations within the genome of a single cell. In the process, transposons can cause mutations and / or change the amount of DNA in the genome of a cell.

[0347] Several transposon systems capable of transposing in cells, including vertebrates, have been isolated and engineered, such as Sleeping Beauty [Izsvak and Ivics Molecular Therapy (2004) 9, 147-156], piggyBac [Wilson et al. Molecular Therapy (2007) 15, 139-145], Tol2 [Kawakami et al. PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al. Nucleic Acids Res. Dec 1, (2003) 31 (23): 6873-6881]. In general, DNA transposons move from one DNA site to another in a simple cut-and-paste fashion. Each of these elements has its own advantages, for example, Sleeping Beauty is particularly useful in region-directed mutagenesis, while Tol2 has the highest tendency to integrate into expressed genes. Highly active systems are available for Sleeping Beauty and piggyBac. Most importantly, these transposons have different target site preferences and can therefore introduce sequence changes in overlapping but distinct sets of genes. Therefore, the use of two or more elements is particularly preferred to achieve the best possible coverage of genes. The basic mechanism is shared between the different transposases, therefore, we describe piggyBac (PB) as an example.

[0348] PB is a 2.5 kb insect transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The PB transposon consists of asymmetric terminal repeat sequences flanked by the transposase PBase. PBase recognizes the terminal repeats and directs transposition via a "cut-and-paste" based mechanism, preferentially transposing into the host genome at the tetranucleotide sequence TTAA. Upon insertion, the TTAA target site is duplicated such that the PB transposon is flanked by this tetranucleotide sequence. Upon mobilization, PB typically precisely excises itself to re-establish a single TTAA site, thereby restoring the host sequence to its pre-transposon state. After excision, PB can either transpose to a new location or be permanently lost from the genome.

[0349] Typically, the transposase system provides an alternative means to remove the selection cassette after completion of homologous recombination, similar to the use of Cre / Lox or Flp / FRT. Thus, for example, the PB transposase system involves the introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two PB terminal repeats at the site of the endogenous TTAA sequence, and a selection cassette located between the PB terminal repeats. Positive selection is applied to identify homologous recombinants containing the target mutation. Transient expression of PBase removal combined with negative selection results in excision of the selection cassette, selecting for cells in which the cassette has been lost. The final target allele contains the introduced mutation without the exogenous sequence.

[0350] For PB to be useful for introducing sequence changes, there must be a naturally occurring TTAA site relatively close to the position where the particular mutation is to be inserted.

[0351] Genome editing using recombinant adeno-associated virus (rAAV) platform - This genome editing platform is based on rAAV vectors that allow the insertion, deletion or replacement of DNA sequences in the genome of living mammalian cells. The rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule of either positive or negative sense that is approximately 4.7 kb long. These single-stranded DNA viral vectors have the unique property of having high transduction rates and stimulating endogenous homologous recombination in the absence of double-stranded DNA breaks in the genome. Those skilled in the art can design rAAV vectors to target desired genomic loci and perform both global and / or minor endogenous genetic modifications in cells. rAAV genome editing has the advantage that it targets a single allele and does not result in any off-target genome modifications. rAAV genome editing technology is commercially available, for example the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).

[0352] It will be appreciated that the agent may be a mutagen that induces random mutations and cells that show downregulation of nNOS expression levels and / or activity may be selected.

[0353] The mutagen may be, but is not limited to, a genetic material, a chemical, or a radioactive material. For example, the mutagen may be, but is not limited to, ionizing radiation, such as, but not limited to, ultraviolet light, gamma rays, or alpha particles. Other mutagens may include, but are not limited to, base analogs (which may cause copy errors), deaminating agents such as nitrous acid, intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., bromouracil), transposons, natural and synthetic alkaloids, bromine and its derivatives, sodium azide, psoralens (e.g., in combination with ultraviolet light). The mutagen may be a chemical mutagen, such as, but not limited to, ICR191, 1,2,7,8-diepoxyoctane (DEO), 5-azaC, N-methyl-N-nitrosoguanidine (MNNG), or ethyl methane sulfonate (EMS).

[0354] Methods for assessing efficacy and detecting sequence variations are well known in the art and include, but are not limited to, DNA sequencing, electrophoresis, enzyme-based mismatch detection assays and hybridization assays (e.g., PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot, Northern blot and dot blot analysis).

[0355] Sequence variations in specific genes can also be determined at the protein level using, for example, chromatography, electrophoresis, immunodetection assays (eg, ELISA and Western blot analysis), and immunohistochemistry.

[0356] Furthermore, one skilled in the art can easily design knock-in / knock-out constructs that include positive and / or negative selection markers to efficiently select transformed cells that have undergone a homologous recombination event with the construct. Positive selection provides a means to enrich the population of clones that have incorporated foreign DNA. Non-limiting examples of such positive markers include glutamine synthetase, dihydrofolate reductase (DHFR), markers that confer antibiotic resistance (e.g., neomycin, hygromycin, puromycin, and blasticidin S resistance cassettes). Negative selection markers are necessary to select against random integration and / or elimination of the marker sequence (e.g., the positive marker). Non-limiting examples of such negative markers include herpes simplex-thymidine kinase (HSV-TK), which converts ganciclovir (GCV) into a cytotoxic nucleoside analog, hypoxanthine phosphoribosyltransferase (HPRT), and adenine phosphoribosyltransferase (ARPT).

[0357] Modified nucleic acids, including modified DNA or RNA molecules, can be used in place of naturally occurring nucleic acids in the polynucleotides described herein. Modified nucleic acids can improve the half-life, stability, specificity, delivery, solubility, and nuclease resistance of the polynucleotides described herein. For example, siRNA agents can be partially or completely composed of nucleotide analogs that confer the above-mentioned beneficial properties. As described in Elmen et al. (Nucleic Acids Res.33:439-447,2005), synthetic RNA-like nucleotide analogs (e.g., locked nucleic acid (LNA)) can be used to construct siRNA molecules that exhibit silencing activity against target gene products.

[0358] Modified nucleic acids include molecules in which one or more of the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those found in nature, preferably different from those found in the human body. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone).

[0359] Modifications can be incorporated into any double-stranded RNA (e.g., any RNAi agent (e.g., siRNA, shRNA, dsRNA, or miRNA), RNA-like molecules, DNA molecules, and DNA-like molecules. It may be desirable to modify one or both of the antisense and sense strands of a polynucleotide. Because polynucleotides are polymers of subunits or monomers, many of the modifications described below occur at positions that are repeated within the nucleic acid (e.g., modification of a base, or a phosphate moiety, or a non-linked O of a phosphate moiety). In some cases, modifications occur at all of the positions of interest in the nucleic acid, but often, and in most cases, this is not the case. For example, modifications may occur only at the 3' or 5' terminal positions, or only at terminal regions, e.g., positions on the terminal nucleotides or at the end of the strand. It may occur only at 2, 3, 4, 5, or 10 nucleotides. The modification may occur in the double-stranded region, the single-stranded region, or both. For example, phosphorothioate modification at non-linked O position may occur only at one or both ends, or only at the terminal region, e.g., at the position on the terminal nucleotide or the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may occur at the double-stranded and single-stranded regions, especially at the ends. Similarly, the modification may occur in the sense strand, the antisense strand, or both. In some cases, the sense strand and the antisense strand have the same modification or the same class of modification, while in other cases, the sense strand and the antisense strand have different modifications, for example, in some cases, it may be desired to modify only one strand (e.g., the sense strand).

[0360] Two major objectives for the introduction of modifications into the polynucleotides described herein are to aid in their protection from degradation in biological environments and to improve pharmacological properties (e.g., pharmacodynamic properties), which are discussed further below. Other suitable modifications to the sugar, base, or backbone of polynucleotides are described in PCT Publication No. 2004 / 064737, which is incorporated herein by reference. Polynucleotides can include non-naturally occurring bases, such as those described in PCT Publication No. 2004 / 094345, which is incorporated herein by reference. Polynucleotides can include non-naturally occurring sugars (e.g., non-carbohydrate cyclic carrier molecules). Exemplary features of non-naturally occurring sugars for use in the polynucleotides described herein are described in PCT Publication No. 2004 / 094595, which is incorporated herein by reference.

[0361] Any of the polynucleotides described herein can include internucleotide linkages (e.g., chiral phosphorothioate linkages) that are useful for increasing nuclease resistance. Additionally or alternatively, the polynucleotide can include ribose mimics to increase nuclease resistance. Exemplary internucleotide linkages and ribose mimics for increasing nuclease resistance are described in U.S. Patent Application Publication No. 2005 / 0164235.

[0362] Any of the polynucleotides described herein can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325.

[0363] Any polynucleotide may have a ZXY structure as described in US Patent Application Publication No. 2005 / 0164235.

[0364] Any polynucleotide can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with RNAi agents are described in U.S. Patent Application Publication No. 2005 / 0164235.

[0365] Another possibility is to deliver to the subject a DNA sequence encoding antisense or iRNA under a suitable control element (promoter) promoter. According to one option, the expression control element is a universal promoter, such as CMV and U1snRNA. According to an embodiment of the present invention, the expression control element is a neuron-specific promoter, so that expression occurs only in neurons, making the sequence express (antisense) selectively for neurons. Non-limiting examples of such neuron-specific promoters are synapsin I promoter CamkII, MeCP2, NSE and Hb9.

[0366] Alternatively, the sequences may be delivered to the CNS by delivery to the brain (see below), preferably by use of a viral delivery system. A preferred embodiment is by adeno-associated virus (AAV), in particular by adeno-associated virus serotype 9 (AAV9) delivery, which has an increased ability to cross the blood-brain barrier compared to other AAV serotypes, making it a preferred vector for CNS delivery. Further viral delivery systems are lentiviruses and herpes simplex viruses.

[0367] Drugs that act at the protein level: antibody: According to certain embodiments, the agent capable of downregulating nNOS is an antibody or antibody fragment capable of specifically binding to nNOS.Preferably, the antibody specifically binds to at least one epitope of nNOS.As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.Epitopes usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0368] Since nNOS is located intracellularly, the antibody or antibody fragment capable of specifically binding to nNOS is typically an intracellular antibody.

[0369] Intracellular antibodies (also called intrabodies) are antibodies that are produced within a cell and bind to an antigen within the same cell.

[0370] Importantly, full-length antibodies are not functional in the cytosol prior to secretion due to their reducing conditions that affect protein folding and intramolecular disulfide bonds required to maintain antibody conformation and stability. The complementarity determining regions that confer antibodies their exceptional target specificity are located in the variable regions of both the heavy and light chains. It is therefore possible to use antibody fragments that incorporate the specificity-providing regions into single-chain variable fragments (scFvs), which can be further engineered for cytosolic stability, to target intracellular antigens (in this case nNOS). scFvs are single polypeptides, an advantageous feature for in vivo expression, and have been investigated as therapeutics for viral infections and cancer, among other diseases.

[0371] Furthermore, variable (V) region domains can be used alone to form domain antibodies or Dabs. These can be engineered from traditional human Ig or from those derived from camelids (camel or llama) and cartilaginous fish (ray or nurse shark), whose immune systems have been found to have evolved high affinity V-like domains fused to a conserved framework that reflects the constant Fc region found in human Ig. It has been reported that single heavy or light chain V regions can be expressed intracellularly. These are called intracellular domain antibodies, which do not require intramolecular disulfide bonds for stability and therefore represent the smallest format of antibodies that retain target specificity while minimizing size (a key factor for intracellular targeting).

[0372] According to certain embodiments, intrabodies include signals for ER targeting leading to degradation of the target protein, antibody-antigen interaction dependent apoptosis used to induce programmed cell death via activation of caspases, and suicide intrabody technology leading to proteolytic degradation of the target protein.

[0373] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0374] nNOS-specific antibodies are known in the art and can be used to prepare antibody fragments and intracellular antibodies to act in brain cells (e.g., neurons).

[0375] Examples of such antibodies include, but are not limited to, 37-2800 from Thermo Fisher, EP1855Y and EPR 24351-6 from abcam.

[0376] These may be modified in length and sequence to act intracellularly and / or for human use.

[0377] Aptamers: Another agent that can be used with some embodiments of the present invention to downregulate nNOS is an aptamer. As used herein, the term "aptamer" refers to a double-stranded or single-stranded RNA molecule that binds to a specific molecular target, such as a protein. Various methods are known in the art that can be used to design protein-specific aptamers. For efficient selection, one skilled in the art can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403). Creative Biolabs provides aptamer development services by SELEX.

[0378] Drugs that affect nNOS stability: Another contemplated agent that can be used to downregulate any of the above proteins includes protein degradation targeting chimera (PROTAC). Such agents are heterobifunctional, comprising a ligand that binds to ubiquitin ligase (e.g., E3 ubiquitin ligase) and a ligand for one of the above proteins (nNOS), and optionally a linker that connects the two ligands. The binding of PROTAC to target protein leads to ubiquitination of exposed lysine on target protein, followed by ubiquitin proteasome system (UPS)-mediated protein degradation.

[0379] Dominant negative nNOS and competitive inhibitors: It will be appreciated that a non-functional analog of at least the catalytic or binding portion of nNOS may also be used as an agent to downregulate nNOS, since it acts as a homodimer.

[0380] According to some embodiments, the drug is an amino acid based drug, also called an amino acid based inhibitor.

[0381] According to some embodiments, the agent is PIN or nitric oxide synthase interacting protein (NOSIP).

[0382] It has been shown that the N-terminus of nNOS can bind to a protein called PIN, which can inhibit nNOS activity. PIN has been found to destabilize nNOS dimers and inhibit nNOS activity. Recently, it has been demonstrated that PIN inhibits the production of NO and O2, but not nNOS dimerization.

[0383] Another protein that inhibits NO production is nitric oxide synthase interacting protein (NOSIP). NOSIP and nNOS are co-localized in different regions of the central and peripheral nervous systems. NOSIP negatively affects nNOS activity in neuroepithelioma cell lines that stably express nNOS. In addition, overexpression of NOSIP in cultured primary neurons limits nNOS transport to terminal dendrites and targets nNOS to the cell body. These findings suggest that NOSIP regulates NO production in the nervous system by regulating the activity and localization of nNOS. NOSIP upregulation by neuronal activity may prevent NO production in neurons.

[0384] Alternatively, or additionally, small molecules or peptides that interfere with nNOS protein function (eg, catalysis or interaction) can be used.

[0385] Additional agents that can be used with some embodiments of the present invention to downregulate nNOS are molecules that prevent nNOS activation or substrate binding.

[0386] According to certain embodiments, the agent binds to the active site of nNOS and blocks substrate binding. Various amino acids define the active site of nNOS. These include, for example, phenylalanine 589, tryptophan 592, tyrosine 593, glutamic acid 597, aspartic acid 602, arginine 601, methionine 341, histidine 342, and tyrosine 711.

[0387] Small molecule inhibitors: According to certain embodiments, small molecules that are inhibitors of neuronal NO synthase can be used as active agents. ω -nitroarginine, which is a potent competitive inhibitor of nNOS that binds to the active site of the enzyme and blocks substrate binding, but is known to exhibit little selectivity for eNOS.

[0388] For example, arginine analogs can be used as competitive nNOS inhibitors.

[0389] nNOS inhibitors that can be used in accordance with the present invention include 7-nitro-thiocitrulline (7-NI), LN G -Methyl-L-arginine (L-NMMA), LN G- Propyl-L-arginine (N-PLA), LN G -Nitroarginine (L-NNA), LN G -nitroarginine methyl ester (L-NAME), L-thiocitrulline, S-methyl-L-indazole, S-methyl-L-thiocitrulline, ethyl-L-NIO, vinyl-L-NIO, 7-NI-Br (3-bromo-7-nitroindazole), and methylene blue.

[0390] Inhibitors available from Tocris and other suppliers include ARL 17477 dihydrochloride, 3-bromo-7-nitroindazole, IC 87201 (an nNOS-PSD95 protein-protein interaction inhibitor), L-NIO dihydrochloride, and N ωExamples of suitable anti-inflammatory drugs include, but are not limited to, -propyl-L-arginine hydrochloride.

[0391] Inhibitors available from ApexBio as well as other sources include, but are not limited to, (S)-methylisothiourea sulfate, 1400W dihydrochloride, 2,4-diamino-6-hydroxypyrimidine, 2-iminopiperidine hydrochloride and 3-bromo-7-nitroindazole. In certain embodiments, the agent (or selective nNOS inhibitor) is JI-8 (cis-N 1 -[4-6-amino-(4-methylpyridin-2-ylmethyl)pyrrolidin-3-yl]-N 2 -[2-(3″-fluorophenyl)ethyl]ethane-1,2-diamine) and structural analogs described, for example, in Yu et al., Dev Neurosci. 2011 Oct;33(3-4):312-319, Ji et al., Ann Neurol. 2009b;65:209-217, and Ji et al., J Am Chem Soc. 2008;130:3900-3914, which are incorporated by reference as if fully set forth herein. According to certain embodiments, the agent is N ω -nitroarginine.

[0392] According to certain embodiments, the agent reduces the levels of NO precursors.

[0393] According to certain embodiments, the agent increases the level of GSNO reductase levels, thereby decreasing the level of NO precursors.

[0394] U.S. Patent Application Publication No. 2021 / 0239400, which is incorporated by reference as if fully set forth herein, discloses nNOS inhibitors that can be used in accordance with the present teachings.

[0395] US Patent Application Publication No. 2020 / 0222714 discloses phototherapy for the inhibition of nNOS activity.

[0396] According to certain embodiments, the nNOS inhibitor (drug) is 7-NI, including structural analogs thereof.

[0397] According to some of the embodiments described herein, the structural analogs of 7-NI are collectively represented by formula I:

[0398] [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony R1 is selected from hydrogen, alkyl and cycloalkyl, preferably hydrogen; R2-R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, halo, haloalkyl, hydroxy, alkoxy, aryloxy, thiol, amine, carboxylate, thiocarboxylate, poly(alkylene glycol moiety), monosaccharide, disaccharide, and oligosaccharide.

[0399] According to certain embodiments, one of R2-R5 is halo, including fluoro, chloro, bromo, and iodo, preferably bromo. According to certain embodiments, R2 is halo, preferably bromo. Such compounds are known as Br-7-NI.

[0400] According to certain embodiments, each of R2-R5 is hydrogen. Such compounds are known and are referred to herein as 7-nitro-indazole (7-NI).

[0401] According to some embodiments, the compounds represented by formula I act as nNOS inhibitors, preferably selective nNOS inhibitors, as defined herein.

[0402] According to some embodiments, the compound represented by formula I is in the form of a pharma- ceutically acceptable salt thereof.

[0403] As used herein, the phrase "pharmaceutically acceptable salt" refers to a charged species of the parent compound and its counterion, which is typically used to modify the solubility properties of the parent compound and / or reduce any significant irritation to an organism by the parent compound, but does not abolish the biological activity and properties of the administered compound. Alternatively, pharmaceutically acceptable salts of the compounds described herein can be formed during the synthesis of the compounds, for example, during the process of isolating the compounds from a reaction mixture or during the process of recrystallizing the compounds.

[0404] In some contexts of the present embodiments, pharma- ceutically acceptable salts of the compounds described herein may optionally be acid addition salts and / or base addition salts.

[0405] An acid addition salt comprises at least one basic (e.g., amine and / or guanidinyl) group of the compound in positively charged form (e.g., the basic group is protonated) in combination with at least one counterion derived from a selected acid to form a pharma- ceutically acceptable salt. Thus, an acid addition salt of a compound described herein can be a complex formed between one or more basic groups of the compound and one or more equivalents of an acid.

[0406] A base addition salt comprises at least one acid group of the compound in a negatively charged form (e.g., the acid group is deprotonated) in combination with at least one counterion derived from a selected base to form a pharma- ceutically acceptable salt. Thus, a base addition salt of a compound described herein may be a complex formed between one or more acid groups of the compound and one or more equivalents of a base.

[0407] Depending on the stoichiometric ratio between the charged groups in the compound and the counterions in the salt, the acid and / or base addition salts can be either mono- or polyaddition salts.

[0408] The phrase "mono-addition salt," as used herein, refers to a salt in which the stoichiometric ratio of counterion to charged form of the compound is 1:1, such that the addition salt contains one molar equivalent of counterion per one molar equivalent of the compound.

[0409] The phrase "polyaddition salt," as used herein, refers to a salt in which the stoichiometric ratio of counterion to charged form of the compound is greater than 1:1, e.g., 2:1, 3:1, 4:1, etc., such that the addition salt contains two or more molar equivalents of counterion per molar equivalent of compound.

[0410] Non-limiting examples of pharma- ceutically acceptable salts would be ammonium cations and their acid addition salts, also referred to herein as anionic salts, and / or deprotonated amines and their base addition salts, also referred to herein as cationic salts.

[0411] Base addition salts can include cationic counterions such as sodium, potassium, ammonium, calcium, magnesium, etc., which form pharma- ceutically acceptable salts. Exemplary such cationic salts are described in further detail below and in the Examples section below.

[0412] Acid addition salts may include, but are not limited to, various organic and inorganic acids such as hydrochloric acid to give hydrochloric acid addition salts, hydrobromic acid to give hydrobromic acid addition salts, acetic acid to give acetic acid addition salts, ascorbic acid to give ascorbic acid addition salts, benzenesulfonic acid to give besylic acid addition salts, camphorsulfonic acid to give camphorsulfonic acid addition salts, citric acid to give citric acid addition salts, maleic acid to give maleic acid addition salts, malic acid to give malic acid addition salts, methanesulfonic acid to give methanesulfonic acid (mesylate) addition salts, naphthalenesulfonic acid to give naphthalenesulfonic acid addition salts, oxalic acid to give oxalic acid addition salts, phosphoric acid to give phosphoric acid addition salts, toluenesulfonic acid to give p-toluenesulfonic acid addition salts, succinic acid to give succinic acid addition salts, sulfuric acid to give sulfuric acid addition salts, tartaric acid to give tartrate addition salts, and trifluoroacetic acid to give trifluoroacetic acid addition salts. Each of these acid addition salts may be either mono- or polyaddition salts, as these terms are defined herein.

[0413] Exemplary such anionic salts are described in further detail below and in the Examples section which follows.

[0414] Pharmaceutical Compositions: In any of the respective embodiments, the agents described herein (e.g., nNOS inhibitors) can be administered to an organism by themselves or in a pharmaceutical composition mixed with a suitable carrier or excipient.

[0415] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0416] It will be understood that the present teachings contemplate pharmaceutical compositions regardless of their use, and that each embodiment should be considered individually or in combination with the other embodiments.

[0417] As used herein, the term "active ingredient" refers to the agent responsible for the biological effect.

[0418] The terms "active ingredient," "active agent," and "therapeutically active agent" are used interchangeably herein.

[0419] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound. Adjuvants are included in these terms.

[0420] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0421] Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0422] Suitable routes of administration may include, for example, oral, rectal, mucosal, particularly nasal, intestinal or parenteral delivery (including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac (e.g., injection into the right or left ventricular cavity, injection into a common coronary artery), intravenous, intraperitoneal, intranasal or intraocular injections).

[0423] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular engineering of drugs to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing transport peptides with affinity for endothelial cell surface molecules in combination with drugs that cannot themselves cross the BBB); pharmacological strategies designed to increase the lipid solubility of drugs (e.g., conjugation of water-soluble drugs to lipid or cholesterol carriers); temporary disruption of the integrity of the BBB by hyperosmotic disruption (resulting from injection of mannitol solutions into the carotid artery or use of biologically active agents such as angiotensin peptides). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgical procedures, size limitations imposed by the limitations inherent in endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and possible risks of brain damage in regions of the brain where the BBB is disrupted, which makes none an optimal delivery method.

[0424] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, by injecting the pharmaceutical composition directly into the tissue area of ​​the patient.

[0425] The term "tissue" refers to a part of an organism that is made up of cells designed to perform one or more functions, including, but not limited to, brain tissue.

[0426] The pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, powdering, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0427] Thus, pharmaceutical compositions for use according to some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the active ingredients into pharma- ceutically usable preparations. Appropriate formulations depend on the route of administration selected.

[0428] As mentioned, the present teachings also contemplate the use of delivery vehicles that depend on the chemical structure of the active agent (ie, the nNOS inhibitor).

[0429] Delivery vehicles, ie, carriers such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, can be used to deliver the nucleic acid molecules described herein.

[0430] The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-life have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0431] Liposomes have been used successfully with many cell types that are normally resistant to transfection by other procedures. Furthermore, liposomes are not subject to the DNA length constraints typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. Furthermore, several successful clinical trials have been completed testing the efficacy of liposome-mediated drug delivery.

[0432] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å, which contain an aqueous solution in their core.

[0433] Alternatively, nanocapsules or nanoparticle formulations may be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. Nanoparticles can be used to transport drugs through the BBB when administered intravenously, as well as to transport factors that affect their transport.

[0434] NPs are colloidal carriers that can be of natural or synthetic origin and can vary in size from 1 to 1000 nm. Synthetic NPs can be prepared from polymeric materials such as poly(ethyleneimine) (PEI), poly(alkyl cyanoacrylates), poly(amidoamine) dendrimers (PAMAM), poly(ε-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), polyesters (poly(lactic acid) (PLA), or from inorganic materials such as gold, silicon dioxide (silica). These carriers can transport drugs by adsorbing, entrapment or covalently binding to them. Natural NPs are generated from natural polymers such as polysaccharides (chitosan and alginate), amino acids (poly(lysine), poly(aspartic acid) (PASA)), or proteins (gelatin and albumin). Natural NPs have the advantage of providing biological signals that interact with specific receptors / transporters expressed by endothelial cells.

[0435] To facilitate BBB penetration, several ligands have been conjugated to NPs. Such molecules can be classified into four different types: (i) ligands that directly interact with BBB receptors or transporters and mediate the adsorption of proteins from the bloodstream; (ii) ligands that themselves directly interact with BBB receptors or transporters; (iii) ligands that increase charge and hydrophobicity; and (iv) Ligands that improve blood circulation time (e.g., PEG).

[0436] Other methods to assist NPs in crossing the blood-brain barrier include, but are not limited to, receptor-mediated transport, transporter-mediated transport, absorptive-mediated transport, and cell-permeable transport.

[0437] Another method is the use of retrograde tracers based on axonal transport from the periphery, as disclosed in Filler et al. 2010 BMC Neuroscience volume 11, Article number: 8 "Tri-partite complex for axonal transport drug delivery achieves pharmacological effect". According to one embodiment, the tripartite molecular structure concept includes an axonal transport facilitating molecule, a polymer linker, and a retrograde tracer.

[0438] As discussed herein and known in the art, axonal transport can be achieved by neuronal tracers, also called histochemical tracers, which are compounds typically used to reveal cell location and trace neuronal projections. Retrograde tracers are taken up and transported to the cell body at the terminal or along the axon or other neuronal processes, whereas anterograde tracers move away from the cell body of the neuron. The term "retrograde tracer" refers to a molecule that is characterized by its ability to achieve axonal transport from the periphery to the DRG. Non-limiting examples of retrograde tracers are described, for example, in Xiangmin Xu.et al., Neuron, 2020, 107(6), 1029-1047; Christine, S.et al., Frontiers in Neuroscience, 2019, 13, 897; Kumar, P., Mater Methods 2019; 9:2713; Lanciego, J Let al., Brain Structure and Function, 2020, 225, 1193-1224; and Yao, F.et al., PLoS ONE, 13(10), e0205133, the contents of which are incorporated herein by reference.

[0439] Exemplary retrograde tracers include horseradish peroxidase (HRP), dextran, isolectin, isolectin B4 (IB4), cholera toxin subunit B, wheat germ agglutinin (WGA), hydroxystilbamidine (a fluorescent dye), viral-based tracers such as RABV, and any known axonal retrograde transport agent or tracer.

[0440] In the context of the present invention, the terms "retrograde tracer residue" or "remaining of a retrograde tracer" interchangeably refer to the portion of a conjugate that confers or enables axonal transport of the conjugate from the site of administration of the conjugate to the perikarya region.

[0441] Mammalian viral vectors that can be used to deliver RNA include oncoretroviral vectors, adenoviral vectors, herpes simplex viral vectors, and lentiviruses.

[0442] In particular, HSV vectors have a tropism for the central nervous system (CNS) and are able to establish lifelong latent infections in neurons.

[0443] AAV can be delivered to a subject in a composition according to any suitable method known in the art.AAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), can be administered to a subject, for example, a human, a mouse, a rat, a cat, a dog, a sheep, a rabbit, a horse, a cow, a goat, a pig, a guinea pig, a hamster, a chicken, a turkey, or a non-human primate.In certain embodiments, a composition can comprise AAV alone or in combination with one or more other viruses (e.g., a second AAV coding with one or more different transgenes).

[0444] Suitable carriers can be easily selected by those skilled in the art, taking into consideration the indications for which AAV is intended.For example, one suitable carrier includes physiological saline, which can be formulated with various buffers (e.g., phosphate-buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.The selection of carriers is not a limitation of the present invention.

[0445] If necessary, the composition of the present invention may contain other conventional pharmaceutical ingredients (e.g., preservatives, chemical stabilizers) in addition to AAV and carrier.Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, paraben, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

[0446] The dose of AAV virions (e.g., units of vector genome / dose per kilogram of body weight (vg / kg)) required to achieve a desired effect or "therapeutic effect" will vary based on several factors, including, but not limited to, the route of AAV administration, the level of gene or RNA expression required to achieve a therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine the dose range of AAV virions for treating a subject with a particular disease or disorder based on the aforementioned factors, as well as other factors well known in the art. An effective amount of AAV is generally about 10 9 ~10 16 A solution containing genome copies is in the range of about 10 ul to about 100 mL. Other volumes of solution may be used. The volume used typically depends on, among other things, the size of the subject, the dose of AAV, and the route of administration. For example, for intrathecal or intracerebral administration, volumes in the range of 1 ul to 10 ul or 10 ul to 100 ul may be used. For intravenous administration, volumes in the range of 10 ul to 100 ul, 100 ul to 1 mL, 1 mL to 10 mL, or more may be used. In some cases, about 10 per subject may be used. 10 ~10 12 A dose of 10 AAV genome copies per subject is appropriate. In certain embodiments, 12 AAV genome copies are effective for targeting CNS tissue. In some embodiments, AAV is administered at 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the AAV is administered at a dose of 10 genome copies per kg. 10 , 10 11 , 10 12 , 10 13 , or 10 14 It is administered in genome copy doses.

[0447] In some embodiments, the AAV composition is formulated to reduce aggregation of AAV particles in the composition, especially when high AAV concentrations (e.g., about 10 GC / ml or more) are present. Methods for reducing AAV aggregation are well known in the art and include, for example, the addition of detergents, pH adjustment, salt concentration adjustment, etc. (e.g., Wright et al. (2005) Molecular Therapy 12:171-178).

[0448] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for using the particular compositions described herein in various treatment regimens. Typically, these formulations will contain at least about 0.1% or more of active ingredient, although the percentage of active ingredient may of course vary and may conveniently be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of active ingredient in each therapeutically useful composition may be prepared so as to obtain an appropriate dosage in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0449] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0450] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that may be used are known to those skilled in the art. For example, one dose may be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition). Some variation in dosage will necessarily occur depending on the condition of the host. The person skilled in the art of administration will, in any event, determine the appropriate dose for the individual host.

[0451] Sterile injectable solutions are prepared by incorporating the required amount of active AAV in a suitable solvent, together with various other ingredients as listed herein, as necessary, followed by filtration sterilization.In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques, which produce powders of active ingredients and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0452] The compositions disclosed herein may also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like). Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

[0453] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to a host.

[0454] Liposomes, nanocapsules, microparticles, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the vector delivery components can be formulated for delivery encapsulated in either lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0455] In addition to the above delivery methods, the following techniques are also contemplated as alternative methods for delivering compositions to a host. Sonophoresis (i.e., ultrasound) has been used as a device to enhance the rate and efficacy of drug penetration into and through the circulatory system, and is described in U.S. Patent No. 5,656,016. Other contemplated drug delivery alternatives are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations, transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0456] To prepare a pharmaceutical composition of the present invention, the conjugate, vector, lipid, nanoparticle, liposome, adjuvant or diluent may be further mixed with a pharma- ceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984).

[0457] Formulations of the therapeutic agent can be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution or a suspension, by mixing with an acceptable carrier, excipient or stabilizer (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0458] Toxicity and therapeutic efficacy of a therapeutic composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index (LD50 / ED50). In certain embodiments, therapeutic compositions that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosage can vary within this range depending on the dosage form used and the route of administration.

[0459] The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial.

[0460] In certain embodiments, the composition or therapeutic agent may be administered by an invasive route, such as injection. In further embodiments of the invention, the composition, therapeutic agent, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intraarticularly (e.g., in an arthritic joint), intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally, e.g., pills, capsules, or tablets) is also within the scope of the invention.

[0461] To overcome the problem of pharmacological agents crossing the blood / brain barrier, intrathecal administration is the more preferred form of administration. Intrathecal administration involves injecting a drug into the spinal canal, more specifically the subarachnoid space, so that the drug reaches the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and analgesics. Intrathecal administration can be performed by lumbar puncture (bolus injection) or port-catheter system (bolus or infusion). The catheter is most commonly inserted between the lumbar lamina and the tip is threaded into the thecal space to the desired level (generally L3-L4). Intrathecal formulations most commonly use water and saline as excipients, although EDTA and lipids have been used as well.

[0462] Compositions can be administered using medical devices known in the art, for example, pharmaceutical compositions of the invention can be administered by injection with a hypodermic needle, including, for example, a pre-filled syringe or an autoinjector.

[0463] The pharmaceutical compositions of the invention may also be administered using a needleless hypodermic injection device, such as those disclosed in U.S. Pat. Nos. 6,620,135, 6,096,002, 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.

[0464] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, via direct injection into the desired target site, often in a depot or sustained release formulation.Furthermore, the composition may be administered in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody, for example, to target the brain.The liposome is targeted to, and taken up selectively by, the desired tissue.

[0465] The dosing regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic composition, the level of symptoms, and the accessibility of the target cells in the biological matrix. Preferably, the dosing regimen delivers sufficient therapeutic composition to result in improvement of the target disease state while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered depends in part on the particular therapeutic composition and the severity of the condition being treated.

[0466] The determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the dose is started at a dose somewhat less than the optimal dose, and then increased by small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, measures of symptoms of inflammation, or the level of inflammatory cytokines produced. Generally, it is desirable that the biologic used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent.

[0467] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the subject nucleic acid molecule, variant protein / polypeptide and / or modulator that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or in the progression of such disease or condition. A therapeutically effective dose further refers to an amount of an agent sufficient to cause at least a partial improvement of a symptom, e.g., treatment, cure, prevention or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent results in an improvement of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% of a diagnostic measure or parameter. An effective amount may also result in an improvement in a subjective measure when a subjective measure is used to assess disease severity. The agent / composition may prevent or delay the onset or amelioration of symptoms of a condition in a subject, or the achievement of a desired biological outcome, such as the correction of a neuropathology, e.g., a cellular pathology associated with a motor neuron disease.

[0468] For example, antisense RNA or iRNA can be delivered as an RNA molecule in a suitable carrier that is complementary to a sequence in the nNOS gene or nNOS mRNA and can hybridize thereto and reduce its expression. Typically, the antisense is chemically modified to increase its stability. In some embodiments, the chemical modification is a modification of the backbone of the oligonucleotide. In some embodiments, the chemical modification is a modification of the sugar of the oligonucleotide. In some embodiments, the chemical modification is a modification of the nucleobase of the oligonucleotide. In some embodiments, the chemical modification increases the intracellular stability of the oligonucleotide. In some embodiments, the chemical modification increases the stability of the oligonucleotide in vivo, for example, by increasing resistance to proteases. In some embodiments, the chemical modification increases the ability of the oligonucleotide to enter a cell. In some embodiments, the chemical modification improves the ability to bind to a target RNA. In some embodiments, the chemical modification increases the half-life of the oligonucleotide or improves another relevant pharmacokinetic property. In some embodiments, the chemical modification inhibits polymerase extension from the 3' end of the oligonucleotide. In some embodiments, the chemical modification inhibits the recognition of the oligonucleotide by a polymerase. In some embodiments, the chemical modification inhibits double-strand induced degradation. In some embodiments, the chemical modification inhibits RISC-mediated degradation. In some embodiments, the chemical modification inhibits RISC-mediated degradation or any parallel nucleic acid degradation pathway.

[0469] In certain embodiments, the consecutive nucleotide bases are linked by a backbone selected from the group consisting of a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a 2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide backbone, a 2-methoxyethyl phosphorothioate backbone, an alternating locked nucleic acid backbone, a constrained ethyl backbone, and a phosphorothioate backbone, an N3'-P5' phosphoramidate, a 2'-deoxy 2'-fluoro-β-d arabino nucleic acid, a cyclohexene nucleic acid backbone, a tricycloDNA (tcDNA) nucleic acid backbone, a ligand-linked antisense, and combinations thereof.

[0470] The composition of some embodiments of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if necessary. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be contained with a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the agency of the form of the composition or its administration to humans or animals. Such notice may, for example, be that of a label approved by the U.S. Food and Drug Administration for prescription drugs, or that of an approved product insert.

[0471] Exemplary Compositions: A pharmaceutical composition, also referred to herein as a "formulation", is comprised of a pharma- ceutically acceptable carrier and at least one active agent, which may be a small molecule, an amino acid-based compound (peptide, protein, antibody or antibody fragment), or a nucleic acid-based compound (RNA, including antisense, and iRNA, and DNA), as described herein in any of the respective embodiments.

[0472] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising a therapeutically active agent that reduces nNOS activity as described herein in any of the individual embodiments and any combination thereof, and a pharma- ceutically acceptable carrier, wherein the composition and the therapeutically active agent are selected such that, upon administration of the composition to a subject, a selective or preferential reduction in nNOS activity relative to other NOS isoforms is provided.

[0473] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising a therapeutically active agent that reduces nNOS activity as described herein in any of the individual embodiments and any combination thereof, and a pharma- ceutically acceptable carrier, wherein the composition and the therapeutically active agent are selected such that, upon administration of the composition to a subject, a reduction in nNOS activity is achieved selectively or preferentially in neural cells or neural tissue of the subject relative to other tissues.

[0474] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising a therapeutically active agent that reduces nNOS activity as described herein in any of the individual embodiments and any combination thereof, and a pharma- ceutically acceptable carrier, wherein the composition and the therapeutically active agent are selected such that, upon administration of the composition to a subject, a reduction in nNOS activity is selectively or preferentially achieved in the peripheral or central nervous system of the subject relative to other tissues.

[0475] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a therapeutically active agent that reduces nNOS activity as described herein in any of the individual embodiments and any combination thereof, and a pharma- ceutically acceptable carrier, wherein the composition and the therapeutically active agent are selected such that, upon administration of the composition to a subject, a reduction in nNOS activity is selectively or preferentially achieved in the CNS (e.g., brain) of the subject relative to other tissues.

[0476] Since NO is produced in the body and is involved in many physiological processes, it is desirable to selectively reduce the amount of NO in desired tissues (e.g., nervous tissue, brain, etc.). This can be done, for example, by using an active agent that is selective for reducing NO in the brain, and / or an inhibitor of NO synthase that is specific / selective to neuronal NO synthase. Neuronal NO synthase inhibitors can be small molecules specific to the neuronal NO synthase enzyme, antisense and iRNA specific to the mRNA of the enzyme, DNA that can express these antisense and iRNA, or combination systems, such as CRISPR CAS, to selectively reduce the expression of neuronal NO synthase. Such agents are described herein above.

[0477] Alternatively, or in combination, selectivity of NO reduction in neural tissue (e.g., brain) may be caused by the properties of a carrier that is selectively delivered to neural tissue (e.g., CNS), or a delivery system that has an element that targets delivery to neural tissue (e.g., CNS) (delivering more to neural tissue or the CNS than to the body). When selectivity is due to a delivery / targeting system, the active agent (e.g., a non-selective NO synthase inhibitor) does not need to be neuron-specific, since the delivery (and carrier, described below) is what confers neural tissue (e.g., CNS) selectivity.

[0478] Alternatively, or in combination, selectivity for neural tissue (e.g., CNS, brain) is demonstrated by a mode of administration (particularly relevant for administration of nucleic acid-based agents) whereby it is administered locally to the desired tissue (e.g., brain).

[0479] Brain selectivity in reducing NO can be achieved by one of the following mechanisms: Reducing the expression of nNOS by using agents selective for the gene / mRNA (not active against the eNOS and iNOS genes) as described herein; A selective inhibitor of nNOS that does not inhibit iNOS and eNOS, as described herein; using reducing agents that are not selective for nNOS but can reduce all NOS (general inhibitors of all NOS (eNOS, iNOS, nNOS)), or agents that reduce the expression of the eNOS (NOS3), iNOS (NOS2), nNOS (NOS1) genes delivered by a targeted delivery system (such as a carrier) to selectively deliver their cargo to the CNS, as described herein; and / or Using agents that may not be selective for nNOS, but are selectively delivered to the brain by intracerebral administration or by a brain-targeted delivery vehicle as described herein.

[0480] According to certain embodiments, the compositions include active agents that are nNOS-specific (nNOS-selective).

[0481] According to certain embodiments, the mode of administration achieves a reduction in nNOS activity in the CNS.

[0482] According to certain embodiments, the mode of administration of the composition comprises topical administration.

[0483] Methods for determining the activity of nNOS or its expression levels / localization are known in the art and described throughout the specification. These methods validate the selectivity of the methods for nNOS or brain-expressed NOS.

[0484] According to certain embodiments, the agent is a selective nNOS inhibitor.

[0485] An nNOS inhibitor is considered to be selective if it reduces nNOS activity to a degree at least 20%, or at least 30%, or at least 40%, or at least 50% greater than other enzymes, particularly other nitric oxide synthase enzymes.

[0486] An nNOS inhibitor is considered to be selective if its dissociation constant (Ki) for nNOS is at least 10-fold, or at least 100-fold, or at least 1,000-fold lower compared to its Ki for other NOS isoforms.

[0487] An nNOS inhibitor is considered to be selective if its IC50 against nNOS is at least 2-fold, or at least 5-fold, or at least 10-fold lower compared to its IC50 against other NOS isoforms.

[0488] According to certain embodiments, an nNOS reducing composition refers to any pharmaceutical composition that includes one or more active agents capable of reducing the amount of physiologically available NO in the brain in a carrier. "Neuronal" specificity can be a property of an agent that specifically reduces nNOS and does not substantially reduce any other NOS enzymes, as described herein, or a property of a carrier that transports a general NO reducing agent to neurons (by any of the delivery systems or modes of administration described herein).

[0489] Without wishing to be bound by theory, it is suggested that upregulation of nNOS activity / expression (as opposed to iNOS and eNOS) is associated with the onset or progression of the disease. Thus, treatment with inhibitors that are not necessarily selective for nNOS would still achieve selective inhibition.

[0490] According to some of any of the embodiments described herein, the pharmaceutical composition comprises an active agent as described herein in any of the respective embodiments in an effective amount to reduce the amount of physiologically available nitric oxide in a desired tissue as described herein (e.g., neural tissue as described herein, e.g., brain) or neuronal cells, preferably in a selective manner as described herein.

[0491] The pharmaceutical compositions according to these embodiments may be designed for oral, buccal, sublingual, parenteral, nasal, transdermal, or interstitial administration, particularly for repeated administration or continuous, extended or sustained release administration over an extended period of time or for targeted slow and controlled delivery.

[0492] Pharmaceutical compositions according to these embodiments can be designed to provide high systemic bioavailability and optionally sustained release.

[0493] The pharmaceutical compositions according to these embodiments can be applied for repeated or sustained administration to release therapeutically effective concentrations of the active agent over an extended period of time, or for targeted slow and controlled delivery. Formulations include stable aqueous solutions, liposomes, emulsions, and nano- and micro-particle dispersions for subcutaneous or intramuscular injection. Formulations can be in the form of ointments, creams, and gels for topical and transdermal delivery of the agent.

[0494] As used herein, sustained release or controlled release refers to maintaining active levels of an active agent in the blood or in a designated tissue (e.g., neural tissue as described herein) for at least 48 hours from implant or injection SC, IM or tissue.

[0495] In an exemplary embodiment, sustained or controlled release refers to maintaining active levels of the active agent in the blood for at least two hours after oral or intranasal administration.

[0496] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising an active agent that selectively reduces the activity of nNOS as described herein in any of the respective embodiments.

[0497] According to an exemplary embodiment of the present invention, the active agent is JI-8 or a structural analog thereof, as described herein.

[0498] According to an exemplary embodiment of the present invention, the active agent is 7-NI, or a compound represented by Formula I as described herein in any of the individual embodiments, and any combination thereof.

[0499] 7-NI is water-insoluble and has low ability to pass biological membranes. Its target site is the receptor in the nervous system, for example, the brain. Therefore, it is necessary to deliver 7-NI to desired nervous tissue (for example, brain, peripheral and / or central nervous system).

[0500] According to some embodiments, this is possible using nanoparticles delivered via IV injection or by nasal spray from the nose to the brain.

[0501] In addition, the formulations described herein can protect 7-NI from premature degradation or metabolism while allowing for high blood levels over extended periods of time, depending on the type of delivery system used.

[0502] According to some embodiments, the pharmaceutical compositions described herein comprise an active agent described herein, preferably a low molecular weight agent such as a low molecular weight nNOS inhibitor (e.g., 7-NI or a compound of formula I described herein, or JI-8 or a structural analog thereof described herein) in any of the respective embodiments and any combination thereof.

[0503] According to some embodiments, the active agent is 7-NI or a compound of formula I described herein.

[0504] Pre-nanodispersed lipid (PNL) composition: According to some embodiments, the pharmaceutical composition or formulation comprises a carrier pre-nanodispersed carrier that forms a nanodispersion upon contact with an aqueous solution. In an exemplary embodiment, the composition or formulation is a pre-nanodispersed lipid (PNL) composition or formulation. In an exemplary embodiment, the composition comprises an aqueous carrier in which nanoparticles formed from the pre-nanodispersed lipid formulation are dispersed.

[0505] Such formulations increase the bioavailability of the active agent (eg, 7-NI) following oral ingestion.

[0506] PNL formulations typically include a carrier comprising a mixture of at least one lipid, at least one surfactant, a water-miscible solvent (e.g., an edible solvent), and optionally further including emulsifiers, dispersants, and other ingredients. The mixture of surfactant, lipid, and solvent forms nanoparticles upon contact with an aqueous medium (e.g., a physiological medium) or an aqueous solution.

[0507] According to certain embodiments, the carrier of the PNL formulation comprises ingredients that are considered GRAS.

[0508] Exemplary lipids suitable for use in connection with embodiments relating to PNL formulations include solid and liquid lipids such as, but not limited to, one or more of fatty acids, fatty alcohols, triglycerides, hydrogenated vegetable oils, miglyols, vegetable oils, and waxes, specific examples of which are listed in the Examples section below.

[0509] Exemplary surfactants suitable for use in connection with embodiments relating to PNL formulations include, but are not limited to, hydrophilic or amphiphilic surfactants or dispersants, such as, but not limited to, one or more of the Tween® family of surfactants, the Span® family of surfactants, PEG-lipids, and PEG-hydrogenated castor oil, specific examples of which are listed in the Examples section below.

[0510] Exemplary water-immiscible solvents suitable for use in the context of embodiments relating to PNL formulations include, but are not limited to, ethanol, isopropanol, ethyl lactate, ethyl acetate, and propylene glycol, specific examples of which are listed in the Examples section below.

[0511] In some embodiments, the carrier in a PNL formulation is a transparent liquid in which the active agent (e.g., 7-NI or a compound of formula I) is soluble and which upon contact with an aqueous medium spontaneously disperses into nanoparticles having an average size (e.g., diameter) of 200 nm or less, preferably less than 100 nm, and preferably less than 50 nm.

[0512] Pro-nanodispersed lipid (PNL)-based formulations are suitable for oral administration per se, for example as a liquid formulation filled into a soft gelatin capsule whose liquid contents upon swallowing interact with gastric fluids to form a nanodispersion that allows the liquid contents to pass through the GI tract surfaces and enter the bloodstream.

[0513] The PNL formulation acts as a solubilizer for the active agent (e.g., 7-NI of the compound of Formula I as described herein) and a nanodispersion for crossing biological membranes. The smaller the particle size, the better the bioavailability.

[0514] Other forms of pharmaceutical compositions for oral administration as described herein are also contemplated for the PNL formulation.

[0515] The PNL formulation may be in liquid form and can be dispersed in a glass of water or a soft drink and consumed as a drink.

[0516] A PNL formulation can include an aqueous solution in which the pro-nanodispersion mixture forms a nanodispersion (nanoparticles dispersed in an aqueous solution) for injection, oral, nasal administration (e.g., as a nasal spray) or ophthalmic administration (e.g., as eye drops).

[0517] The selection of the inactive ingredients (which constitute the carrier) should take into account the safety of the final formulation for the type of delivery and mode of administration.

[0518] Injectable PNL compositions should contain ingredients approved for subcutaneous (SC), intramuscular (IM) or intravenous (IV) administration and should not cause local or systemic toxicity or irritation. Similarly, compositions for eye drops and nasal sprays should take into account the sensitivity of these tissue surfaces to certain ingredients.

[0519] The PNL formulations described herein can be used for oral, ocular, nasal, transdermal, subcutaneous, and buccal delivery that provides an effective dose to the subject being treated.

[0520] The dispersions in aqueous media can be prepared just prior to administration or can be prepared in advance.

[0521] According to some embodiments, the PNL compositions or formulations described herein in any of the respective embodiments comprise, as an active agent, 7-NI or a compound of formula I described herein, or a pharma- ceutically acceptable salt thereof, e.g., a 7-NI lipid salt described herein.

[0522] Controlled / sustained release compositions: According to some embodiments, the pharmaceutical composition or formulation is configured for controlled or sustained release of the active agent.

[0523] According to some of these embodiments, such formulations are injectable and implantable formulations that increase the bioavailability of the active agent (e.g., 7-NI or a compound of formula I or a salt thereof as described herein) to the surrounding tissue at a controlled rate, for example, over a period of days to months.

[0524] Injectable controlled release and sustained release formulations may include polymer or liquid-based nanoparticles, microparticles, inserts, injectable anhydrous paste formulations, polymer liquid formulations that gel in the body to form in situ implants (e.g., depots) that release the active agent (e.g., 7-NI or a compound of formula I) over a period of one day to several weeks or months. According to some embodiments, such formulations can be prepared from different forms and structures of biodegradable polymers. Exemplary biodegradable polymers include those already used in humans, including, but not limited to, polyesters made from lactide, glycolide, caprolactone, and ethylene / propylene carbonate, and sebacic acid-based polyanhydrides. Protein and polysaccharide-based natural polymer carriers are also contemplated, including, but not limited to, gelatin, collagen, hyaluronic acid, oxidized cellulose or amylose, chitosan, and the like.

[0525] In an exemplary embodiment, a polymer implant (e.g., a depot) in the form of a wedge or rod or fiber is inserted into the body using a trocar or 16-21 G needle. The implant is prepared by forming a homogenous molten mixture of the polymer and the active agent (e.g., 7-NI or a compound of formula I described herein), which is molded into the desired shape suitable for insertion into the body. Alternatively, the device is prepared by compression molding of a polymer-active agent homogenous powder.

[0526] Polymeric injectable sustained release compositions can be configured, for example, for intramuscular or subcutaneous administration. These compositions release the drug continuously over a period of several days to several months. In situ depot-forming systems for parenteral controlled drug delivery can be in the form of aqueous dispersions of microparticles, polymeric liquids or pastes with a wide range of viscosities. Such systems usually contain a biodegradable carrier dissolved or dispersed in a solvent / cosolvent system, while the drug is dispersed or dissolved in the liquid phase of the delivery system. Upon subcutaneous or intramuscular injection, a solid depot is formed at the injection site. Administration of such systems is much less invasive and less costly than the surgical procedures often required for implantation.

[0527] Exemplary injectable formulations for sustained release of active agents have been designed and exemplified in the Examples section below.

[0528] According to an exemplary embodiment, the sustained release formulation is made from ricinoleic acid and castor oil containing polyesters and polyanhydrides adapted for use as carriers, for example as described in US Patent Application Publication Nos. 2004 / 0161464 and 2004 / 0161464 (Domb). The biodegradable carrier is synthesized from ricinoleic acid oligoesters and aliphatic molecules having at least one carboxylic acid and at least one hydroxyl or carboxylic acid group that are liquid or paste at temperatures below 37° C. In an exemplary embodiment, the composition is made from a copolymer of ricinoleic acid and sebacic acid in a 6:4 to 8:2 w / w ratio that is paste-like and injectable at room temperature. These polymers increase their viscosity when immersed in aqueous media or injected into tissues.

[0529] Injectable microparticles and nanoparticles prepared from PLGA are also contemplated for controlled / sustained release. Microspheres and nanospheres can be prepared by dissolving the polymer with an active agent (e.g., 7-NI or a compound of formula I) in an organic solvent such as chloroform, DMSO, and adding an anti-solvent containing a surfactant to form droplets of polymer-drug solution, and then forming microspheres and nanospheres by solvent evaporation (in the case of water as anti-solvent) or diffusion from the droplets into the organic anti-solvent. The size of the particles can be controlled by the concentration of the polymer-drug solution, anti-solvent ratio, surfactant content, solvent evaporation or leaching, and preparation conditions (including temperature, solvent used, mixing speed, and evaporation aid).

[0530] granular composition According to some embodiments, the pharmaceutical compositions described herein are formulated for injection (e.g., by intravenous administration) and comprise lipid or polymeric nanoparticles or liposomes in which the active agent (7-NI or a compound of formula I or a salt thereof) is entrapped or encapsulated.

[0531] According to some embodiments, the active agent is incorporated into a biodegradable polymer or lipid carrier.

[0532] According to some embodiments, the carrier is in the form of a microparticle or nanoparticle.

[0533] According to some embodiments, the micro- or nanoparticle compositions are formulated for administration via oral administration, oral inhalation, nasal spray, IV, IM or SC injection, or transdermal delivery. According to some embodiments of the invention, administration is to the brain using nanoparticles delivered to olfactory tissue.

[0534] Exemplary biodegradable polymers suitable for use in the context of these embodiments include, but are not limited to, lactide homo- and copolymers with glycolide or caprolactone, polycaprolactone and its copolymers, and other biodegradable polymers in the form of injectable particles dispersed in water, in the form of injectable polymer pastes, or in the form of inserts. Liposomal or liposphere-type dispersions are also contemplated.

[0535] The encapsulated active agent may feature hydrodynamic surfaces, such as PEG chains, that reduce the ability of the RAS to clear the active agent from the bloodstream.

[0536] In an exemplary embodiment, the active agent is a compound of Formula I, where one or more of R2-R5 is a poly(alkylene glycol) moiety, as defined herein.

[0537] Methods for making nanoparticles and microparticles include precipitation of a solution or dispersion of an active agent (e.g., 7-NI or a compound of formula I or its salt) in a polymer solution, which is carried out in an anti-solvent under controlled conditions to form nanoparticles and microparticles entrapping the active agent. For example, a solution of PLGA 75:25 in dichloromethane is mixed with a solution of the active agent (e.g., 7-NI or a compound of formula I or its salt) in ethanol to form a clear solution containing a 100:1 to 65:35 w / w ratio of polymer:7NI. This solution is added dropwise into stirred heptane containing a surfactant as an anti-solvent to form particles upon precipitation. Alternatively, the polymer / active agent solution is added to a stirred aqueous solution containing a surfactant such as polyvinyl alcohol or a mixture of Tween® and / or Span® surfactants, and the solvent is slowly evaporated to form nanoparticles and microparticles. The size of the particles can be controlled and adjusted by the ratio of solvent:anti-solvent, the concentration of polymer and 7NI in the solution, the mixing speed and type of mixing (overhead, stirrer, ultrasonic), temperature, and the like.

[0538] 7-Nitroindazole salt formulations: Pharmaceutical compositions containing pharma- ceutically acceptable salts of the compounds of formula I (e.g., salts of 7-nitroindazole) are also contemplated. 7-nitroindazole is a water-insoluble molecule that needs to be taken for extended periods of time. Such compositions can be used in repeated administration, or in continuous extended (sustained) administration over extended periods of time, or in targeted slow and controlled delivery. Formulations can increase the bioavailability of 7-nitroindazole after oral, nasal, short-term and long-term release injectable formulations, as well as transdermal and topical formulations.

[0539] The indazole moiety of 7-nitroindazole tends to form salts by removing a proton from the NH of the azole to form salts with positively charged moieties, such as divalent and trivalent metal ions, ammonium and phosphonium cationic counterparts. In addition, the basic amines that are part of the aromatic ring form salts with proton-containing molecules, including HCl, phosphates, H2SO4, and organic acids. These salts have different properties than unsalted 7-nitroindazole, including solubility, thermal properties and stability, chemical stability, ease of formulation, controlled release, passage through biological membranes, and biodistribution.

[0540] Exemplary salts and compositions containing them are described in the Examples section below.

[0541] Salts according to these embodiments may be included in any of the pharmaceutical compositions or formulations described herein.

[0542] Pharmaceutical compositions for oral or nasal administration: In some embodiments, for oral or nasal dosage forms, the active agent (e.g., 7-nitroindazole or a compound of formula I or a salt thereof) can be dissolved or dispersed as nanoparticles to allow GI absorption. In an exemplary embodiment, an anhydrous pre-concentrated solution of the active agent (e.g., 7-nitroindazole or a compound of formula I or a salt thereof) is formed in a specific mixture of surfactants, lipids and edible solvents, which spontaneously form nanoparticles upon addition to aqueous media, i.e., gastric or intestinal fluids, and are absorbed in high yields to increase the oral bioavailability of the active agent several-fold, as described herein in connection with PNL compositions / formulations. These lipid-based formulations can be delivered in soft gelatin capsules or absorbed into porous silica or another carrier, incorporated into a common tablet composition, and compressed into tablets that are released into GI fluids upon oral ingestion to form nanoparticles that are absorbed into the bloodstream through the GI tract. These lipid-based formulations can be delivered nasally, for example, as a sprayable formulation.

[0543] According to some of these embodiments, pharmaceutical compositions for oral administration as described herein are formulated in a dosage form that allows for at least 25% bioavailability. In exemplary embodiments, the orally administered compositions are used in doses of 10-1000 mg, or 200-1000 mg, or 200-600 mg (including any intermediate values ​​and subranges therebetween), or even up to 2,000 mg per day, which may be in unit dosage forms once per day, twice per day, three times per day, four times per day, or more. At doses as low as 10-100 mg per day, sustained release oral formulations for 1-4 weeks can be designed.

[0544] Lower doses are also contemplated for nasal spray delivery.

[0545] Exemplary Embodiments In an exemplary embodiment, 1 to 10 (e.g., 8) spray doses may be applied per day, each dose containing 1 to 10 mg, to achieve a particular blood level or brain tissue concentration.

[0546] In an exemplary embodiment, each spray is 50-200 microliters which may contain 1-10 mg of active agent. In an exemplary embodiment, daily doses of up to 100 mg are contemplated.

[0547] Injectable pharmaceutical compositions: Injectable polymeric delivery systems are preferably configured to deliver drugs over a period of weeks to months, depending on the daily dose and formulation. An exemplary injectable formulation is based on poly(lactide-glycolide) copolymers, which are used clinically for the long-term delivery of drugs such as risperidone and LHRH analogs.

[0548] Further exemplary formulations for injection that provide sustained release of an active agent include the sustained / controlled release compositions or microparticle compositions / formulations described herein.

[0549] The dosage of sustained release injectable composition is typically a function of the daily dosage multiplied by the number of days that delivery system is intended to deliver active agent.The maintenance dosage of active agent that may be less than the desired daily dosage is also contemplated in the context of these embodiments.This maintenance dosage can be combined with oral dosage or nasal dosage or transdermal patch, for example, at a low daily dosage as described herein.

[0550] Pharmaceutical Compositions for Buccal Administration: According to some embodiments, buccal delivery of an active agent (e.g., 7-nitroindazole or a compound of formula I or a salt thereof) can be carried out using the PNL formulations described herein in any of the respective embodiments absorbed onto a carrier suitable for buccal delivery, e.g., cross-linked polyacrylic acid, Carbopol 940 or Carbopol 970, and / or hydroxypropyl cellulose (HPC) or hydroxypropyl methyl cellulose (HPMC).

[0551] In an exemplary embodiment, the active agent (e.g., 7-nitroindazole or a compound of Formula I or a salt thereof) and PNL absorbing solid are mixed in a w / w ratio of 10:0 to 6:4 carbopol to HPC and compressed into tablets 13 mm in diameter. The more carbopol in the formulation, the stronger the adhesion to the buccal mucosa.

[0552] Other ingredients commonly used in buccal tablets, such as flavorings, different polymer compositions, and colorants, can be added to the powder mixture before compression into a mucoadhesive tablet. In an exemplary formulation, 7-NI (10 mg) is dissolved in 200 microliters of a clear solution of 1:1:0.5:0.2 w / w Tween® 20, Span® 80, sesame oil, and Chremophor 40R. This solution is absorbed into 400 mg of Carbopol 940 and mixed with 100 mg of HPC. The dry powder is compressed into a 13 mm tablet using 2 tons of pressure. The tablet adheres well to the human buccal mucosa, remains in place for 4 hours, and erodes and is removed over time, releasing 7IN to the buccal mucosa.

[0553] Buccal delivery is typically dose limited by penetration through the buccal mucosa, and in some embodiments, administration is low, between 1 and 100 mg per day.

[0554] Pharmaceutical Compositions for Transdermal Administration Transdermal formulations are generally applied to low dose delivery of active agents because skin permeation is low for most molecules, including 7-NI. When a maintenance dose for adults or children from a large surface patch is appropriate, such a patch can be prepared by embedding active agents (e.g., 7-nitroindazole or the compound of formula I or its salt) in a paste-like formulation that is applied onto a common transdermal patch. Enhancers such as Azone, PEG and other agents may be added to promote the permeation of active agents into the skin.

[0555] For transdermal delivery, attention should be paid to the local irritation of the composition. Acceptable doses and formulations are used to achieve the highest possible dose so that the skin surface area applied for delivery of the active agent is adjusted to the desired dose per hour.

[0556] In some embodiments, the transdermal patch may be applied to the skin for one week and may be replaced repeatedly every week if desired. Alternatively, the transdermal patch may be applied to the subject's skin daily, or every 2, 3, or 4 days.

[0557] Delivery of nNOS reducers by a brain-selective delivery system According to some embodiments of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid-based nNOS inhibitor (e.g., a promoter and / or gene and / or other nucleic acid-based material) as described herein in any of the respective embodiments.

[0558] Delivery of such nNOS inhibitors can be by any delivery system suitable for delivery to the CNS, either by direct delivery or systemic delivery.

[0559] Non-limiting delivery systems or vehicles for such nNOS inhibitors to the CNS are nanoparticles, typically having a size of less than 200 nm. These can include lipid-based nanoparticles, polymeric nanoparticles, dendrimers, and inorganic nanoparticles, some of which can be tailored to cross the BBB.

[0560] Another method of administration is through the use of liposomes, which are also able to cross the BBB.

[0561] Preferably, active targeted delivery can be used by using ligands of transporters or receptors to enhance nanoparticle uptake through the BBB.The preferred route for this approach is receptor (or transporter)-mediated transcytosis, whereby cargo (e.g., nanoparticles) are transported between the apical and basolateral surfaces in brain ECs.For example, low-density lipoproteins undergo transcytosis through ECs by receptor-mediated processes, bypassing the lysosomal compartment and being released at the basolateral surface of the brain side.

[0562] Another vehicle for brain delivery includes exosomes, small extracellular vesicles secreted by cells. The main advantage of exosomes over other synthetic nanoparticles is their non-immunogenic nature, resulting in long and stable circulation.

[0563] Because the BBB contains transporters for amino acids, using the BBB's naturally occurring arginine transporter for delivery may be a simple viable approach for delivery to the brain.

[0564] Another approach is to use compounds or electrical stimulation to temporarily open the BBB, allowing high concentrations of systemically administered sequences to reach the brain. Examples of such compounds are Cereport (a bradykinin analogue) or Regadenoson (an adenosine receptor agonist).

[0565] Another way to increase penetration is by ultrasound, which has become an attractive technique in recent years to facilitate drugs to cross the BBB. Microbubble-enhanced diagnostic ultrasound (MEUS), a non-invasive technique, has effectively helped drugs to cross the BBTB. Another approach is transcranial magnetic stimulation (TMS), which stimulates neuronal activity and increases glutamate release, facilitating drug delivery across the BBB. (Reviewed in Xiaowei Dong, Theranostics. 2018;8(6):1481-149, the entire disclosure of which is incorporated herein by reference).

[0566] The route of administration of the desired delivery vehicle may be systemic administration without further radiation-based manipulations (e.g., using particles that inherently enter the BBB); systemic administration using compounds in conjunction with various manipulations to transiently open the BBB (e.g., in conjunction with microbubble-enhanced diagnostic ultrasound (MEUS), transcranial magnetic stimulation (TMS)); or intranasal administration.

[0567] Vehicles for targeted delivery to the CNS (e.g., brain) may also be utilized as carriers in pharmaceutical compositions containing any of the other agents that reduce nNOS activity as described herein in any of the individual embodiments and any combination thereof.

[0568] Delivery of agents that reduce nNOS activity by direct administration to the CNS (e.g., brain) According to one embodiment, administration is by direct injection into the parenchyma or by injection into the cerebrospinal fluid via an intraventricular or intrathecal (cisternal or lumbar) route, as described herein in any of the individual embodiments and any combination thereof.

[0569] A preferred localized administration to the brain can be achieved by administration to the cerebrospinal fluid via the intraventricular route. Another option is delivery to the cisternal injection route, which has recently been adopted as an alternative method for delivery to the cerebrospinal fluid (CSF), resulting in widespread gene delivery throughout the CNS.

[0570] Exemplary uses: According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition as described in any of the respective embodiments herein and any combination thereof, comprising, as an agent for reducing nNOS activity, a compound of formula I as described in any of the respective embodiments herein or a salt thereof.

[0571] According to some embodiments, the pharmaceutical composition is for use in the treatment of any of the diseases or conditions described herein, including, but not limited to, ASD as defined herein, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, schizophrenia, addiction, ALS, epilepsy, bipolar disorder, migraine, as well as all types of neurodevelopmental disorders as well as neurodegenerative disorders as described in Tripathi et al. Redox Biol. 2020:101567.

[0572] According to an aspect of some embodiments of the present invention there is provided a method of treating an autism spectrum disorder as defined herein in any of the respective embodiments and any of the combinations thereof, comprising administering to a subject an effective amount of a composition comprising an active agent that reduces nNOS activity, as described herein in any of the respective embodiments and any of the combinations thereof.

[0573] According to some embodiments, the reduction in nNOS activity is selective or preferential in neuronal cells or neural tissue of the subject (e.g., the CNS or brain) as compared to other tissues, as described herein.

[0574] According to some embodiments, the reduction in nNOS activity is in neural tissue (CNS, e.g., brain) or peripheral organs that contain neural cells that express nNOS, as described herein.

[0575] According to some embodiments, the reduction in nNOS activity is selective relative to other NOS isoforms, as described herein.

[0576] According to some embodiments, the reduction in nNOS activity is selective, such that the amount of physiologically available NO is selectively reduced in neural cells of a subject, as described herein.

[0577] According to some embodiments, the reduction in nNOS activity is selective, such that the amount of physiologically available NO is selectively reduced in the CNS or brain of a subject, as described herein.

[0578] According to an aspect of some embodiments of the present invention there is provided a method of treating an autism spectrum disorder as defined herein in any of each of the embodiments and any combination thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition as described herein in any of each of the embodiments and any combination thereof, including the exemplary compositions described herein.

[0579] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition as described herein, in any of the embodiments, and any combination thereof, including the exemplary compositions described herein, for use for the treatment of an autism spectrum disorder as described herein, in a subject in need thereof.

[0580] According to an aspect of some embodiments of the present invention, there is provided a method of treating a neurally derived cancer as defined herein in any of the respective embodiments and any of the combinations thereof, comprising administering to a subject an effective amount of a composition comprising an active agent that reduces nNOS activity, as described herein in any of the respective embodiments and any of the combinations thereof.

[0581] According to some embodiments, the reduction in nNOS activity is selective or preferential in each neural tissue or each neuronal cell of the subject compared to other tissues, as described herein.

[0582] According to some embodiments, the reduction in nNOS activity is selective relative to other NOS isoforms, as described herein.

[0583] According to some embodiments, the reduction in nNOS activity is selective, such that the amount of physiologically available NO is selectively reduced in each neural tissue or each neuronal cell, as described herein.

[0584] According to an aspect of some embodiments of the present invention, there is provided a method of treating a neurally derived cancer as defined herein in any of the respective embodiments and any combination thereof in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein in any of the respective embodiments and any combination thereof, including the exemplary compositions described herein.

[0585] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition as described herein, in any of the embodiments, and any combination thereof, including the exemplary compositions described herein, for use in treating a neurally derived cancer in a subject in need thereof.

[0586] According to some of any of the embodiments described herein, the neurally derived cancer is neuroblastoma as defined herein.

[0587] According to some of any of these embodiments, the pharmaceutical composition is a sustained release composition as described herein in any of the individual embodiments and any combination thereof.

[0588] According to some of these embodiments, the methods and uses described herein are carried out by repeatedly administering the composition (e.g., by injection as described herein or transdermally) with time intervals between administrations that are at least one day, preferably at least two days, at least three days, at least four days, at least one week, optionally at least two weeks, at least one month, at least two months, and more. According to some of these embodiments, this can be carried out using a controlled sustained release composition as described herein in any of the respective embodiments.

[0589] According to some of any of the embodiments described herein, the subject is a mammal, such as a human.

[0590] According to some of any of the embodiments described herein, the subject is suffering from a disease or condition described herein in any of the respective embodiments, e.g., a chronic disease or condition, such as ASD or neuroblastoma.

[0591] According to some of any of the embodiments described herein, the subject is a newborn or infant, for example, between 0 and 10 years of age.

[0592] According to some of any of the embodiments described herein, the subject is an adolescent, for example, between 10 and 20 years of age.

[0593] According to some of any of the embodiments described herein, the subject is an adult, for example, at least 20 years of age.

[0594] Animal models: The present invention is based on the second finding that administration of S-nitroso-N-acetylpenicillamine (SNAP) is an NO donor that increases NO availability-induced ASD-like phenotypes in a C57BL / 6 mouse model.

[0595] Since the study of ASD is complicated by the lack of good animal models, this finding provides a method to generate ASD models in laboratory animals by administering an effective amount of an NO donor to WT animals.

[0596] Thus, according to one aspect of the present invention there is provided a method of producing an experimental animal model of autism spectrum disorder ASD, the method comprising administering to the experimental animal an effective amount of a composition that increases NO levels in the brain of the animal, thereby resulting in an ASD-like phenotype.

[0597] As used herein, "ASD-like phenotype" refers to a molecular and / or behavioral phenotype. Examples of molecular markers include, but are not limited to, synaptophysin, GAD1, PSD-95, and VGAT.

[0598] Examples of behavioral parameters and methods for testing them include, but are not limited to, motor activity (open field test, which also tests for anxiety-like behavior), social behavior (three-chamber sociability test), anxiety-like behavior (elevated plus maze), exploratory activity, and repetitive and restricted behavior (novel object recognition), and / or restricted behavior (marble burying test).

[0599] According to certain embodiments, the animal model is a mammal.

[0600] According to certain embodiments, the animal is a rodent, such as a mouse, rat or rabbit.

[0601] According to certain embodiments, the mice are young mice (eg, 6 weeks old).

[0602] As used herein, "a composition that increases NO levels in the brain of the animal" refers to any substance or physical condition that increases nNOS activity, prevents its degradation, and prevents depletion of NO from neural tissue above levels that are normal in non-ASD animals.

[0603] According to certain embodiments, the composition comprises a nucleic acid sequence for increasing the expression of heterologous (exogenous) nNOS or a homologue thereof in the brain. Alternatively or additionally, the nucleic acid agent is for increasing the activity of a regulatory region so as to increase the expression of endogenous nNOS.

[0604] Administration of the NO donor may be systemic or may be administered directly to the brain as described herein.

[0605] According to certain embodiments, administration is systemic.

[0606] According to certain embodiments, administration is directly to the brain of the animal.

[0607] According to certain embodiments, the composition comprises an NO donor.

[0608] According to certain embodiments, the NO donor comprises S-nitroso-N-acetylpenicillamine (SNAP).

[0609] Other exemplary NO donors include, but are not limited to, SNAP, nitrates, nitrites, N-nitroso, C-nitroso, S-nitroso, heterocycles, metal / NO complexes, diazeniumdiolates; organic nitrates such as nitroglycerin, isosorbide-5-mononitrate, nicorandil, pentaerythritol tetranitrate, etc.; sodium nitroprusside (SNP), S-nitrosothiols such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione; sydnonimines (e.g., molsidomine, SIN-1); and NONOates (e.g., JS-K, spermine NONOate, and prolithium-NONOate).

[0610] The Examples section below describes the suitability of such animal models. Tissues and cells (as well as cell lines) obtainable from such animals are also contemplated herein.

[0611] Embodiments of the present teachings relate to the use of such animals and cells / tissues derived therefrom in research, such as drug screening and the development of diagnostic assays.

[0612] According to certain embodiments, there is provided an animal model produced according to the methods described herein.

[0613] It is anticipated that many related agents that reduce nNOS activity as described herein will be developed during the life of any patent issued from this application, and the scope of the term "active agent" is intended to include a priori all such new technologies.

[0614] As used herein, the term "about" refers to ±10% or ±5%.

[0615] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0616] The term "consisting of" means "including and limited to."

[0617] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0618] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds (including mixtures thereof).

[0619] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0620] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The phrases "range between" a first indicated number and a second indicated number, and "range from" a first indicated number to a second indicated number, are used interchangeably herein and are meant to include the first and second indicated numbers, and all fractional and integer numbers therebetween.

[0621] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques, and procedures that are known to or readily developed by practitioners in the chemical, pharmaceutical, biological, biochemical, and medical arts.

[0622] When referring to a particular sequence listing, such reference should also be understood to encompass sequences that substantially correspond to their complementary sequences, including minor sequence variations resulting from, for example, sequencing errors, cloning errors, or other changes which result in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0623] It is understood that any sequence number (SEQ ID NO) disclosed in this application, even if the SEQ ID NO is expressed only in that format, can refer to either a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is referenced.

[0624] Any of the active agents described herein (eg, small molecule active agents) may be in the form of a pharma- ceutically acceptable salt, a prodrug thereof, a hydrate or a solvate, as defined herein.

[0625] As used herein, the term "prodrug" refers to a compound that is converted into an active compound in the body. Prodrugs are typically designed to facilitate administration, for example by enhancing absorption. Prodrugs may include active compounds that have been modified, for example, with an ester group, e.g., any one or more of the hydroxyl groups of the compound are replaced with an acyl group, optionally (C 1-4 - modified by an acyl (e.g., acetyl) group to form an ester group, and / or any one or more of the carboxylic acid groups of the compound are modified by an alkoxy or aryloxy group, optionally (C 1-4 -modified by alkoxy (eg methyl, ethyl) groups to form ester groups.

[0626] The term "solvate" refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, etc.) formed by a solute (a heterocyclic compound described herein) and a solvent, where the solvent does not interfere with the biological activity of the solute.

[0627] The term "hydrate" refers to a solvate, as defined above, where the solvent is water.

[0628] The active agents described herein may be available as polymorphs, and the present embodiments further encompass any isomorphic form of the active agent, and any combination thereof.

[0629] The compounds and structures described herein encompass any stereoisomers, including enantiomers and diastereomers, of the compounds described herein, unless a particular stereoisomer is specifically indicated.

[0630] As used herein, the term "enantiomer" refers to a stereoisomer of a compound that can be superimposed on its counterpart only by complete inversion / reflection (mirror image) of each other. Enantiomers are said to have "handedness" because they point to each other like right and left hands. Enantiomers have identical chemical and physical properties except when they are present in an environment that has its own handedness (e.g., all living systems). In the context of this embodiment, a compound may exhibit one or more chiral centers, each of which may exhibit an (R) or (S) configuration and any combination, and compounds according to some embodiments of the present invention may have any of those chiral centers exhibiting an (R) or (S) configuration.

[0631] The term "diastereomers" as used herein refers to stereoisomers that are not mirror images of each other. Diastereomers occur when two or more stereoisomers of a compound have different configurations at one or more, but not all, of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter, they are epimers. Each stereocenter (chiral center) gives rise to two different configurations and thus to two different stereoisomers. In the context of the present invention, embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of configurations, i.e., any diastereomers.

[0632] As used throughout the present specification, the term "alkyl" refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain groups. Preferably, the alkyl group has 1-20 carbon atoms. When a numerical range is described herein, for example, as "1-20," it means that the hydrocarbon group in this case can contain up to 20 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. More preferably, the alkyl is a medium-sized alkyl having 1-10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1-4 carbon atoms. The alkyl group may be substituted or unsubstituted. When substituted, the substituent can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0633] As used herein, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond, including straight-chain and branched-chain groups. Preferably, the alkenyl group has 2-20 carbon atoms. More preferably, the alkenyl is a medium size alkenyl having 2-10 carbon atoms. Most preferably, unless otherwise indicated, the alkenyl is a lower alkenyl having 2-4 carbon atoms. The alkenyl group may be substituted or unsubstituted. A substituted alkenyl can have one or more substituents, where each substituent can independently be, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0634] As used herein, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon triple bond, including straight-chain and branched-chain groups. Preferably, the alkynyl group has 2-20 carbon atoms. More preferably, the alkynyl is a medium size alkynyl having 2-10 carbon atoms. Most preferably, unless otherwise indicated, the alkynyl is a lower alkynyl having 2-4 carbon atoms. The alkynyl group may be substituted or unsubstituted. A substituted alkynyl can have one or more substituents, where each substituent can independently be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0635] A "cycloalkyl" group refers to a saturated or unsaturated all-carbon monocyclic or fused ring (i.e., rings that share adjacent pairs of carbon atoms) group in which one or more rings do not have a completely conjugated pi-electron system. Non-limiting examples of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Cycloalkyl groups can be substituted or unsubstituted. When substituted, the substituent may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.When the cycloalkyl group is unsaturated, it may contain at least one carbon-carbon double bond and / or at least one carbon-carbon triple bond.

[0636] An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic ring having a completely conjugated pi-electron system, i.e., rings that share adjacent pairs of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, and anthracenyl. Aryl groups can be substituted or unsubstituted. When substituted, the substituent can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0637] A "heteroaryl" group refers to a monocyclic or fused ring (i.e., rings that share adjacent pairs of atoms) having one or more atoms in the ring, such as, for example, nitrogen, oxygen, and sulfur, and, in addition, a completely conjugated pi-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituent can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0638] A "heteroalicyclic" group refers to a monocyclic or fused ring group having one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated pi-electron system. Heteroalicyclic groups may be substituted or unsubstituted. When substituted, the substituent can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide and amino, as these terms are defined herein.Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine, etc.

[0639] As used herein, the terms "amine" and "amino" refer to the group -NR'R'' or -N +"R"R"" groups, where R', R" and R"' are each hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (bonded to the amine nitrogen through a ring carbon thereof), aryl, or heteroaryl (bonded to the amine nitrogen through a ring carbon thereof), as defined herein. Optionally, R', R" and R"" are hydrogen or alkyl containing 1 to 4 carbon atoms. Optionally, R' and R" (and R"", if present) are hydrogen. If substituted, the carbon atom of the R', R" or R"' hydrocarbon portion bonded to the nitrogen atom of the amine is not substituted by oxo (unless expressly stated otherwise), and thus R', R" and R"" are not (for example) carbonyl, C-carboxy or amido, as these groups are defined herein.

[0640] An "azido" group is -N=N + =N - Refers to the base.

[0641] An "alkoxy" group refers to any of an -O-alkyl, an -O-alkenyl, an -O-alkynyl, an -O-cycloalkyl, and an -O-heteroalicyclic group, as defined herein.

[0642] An "aryloxy" group refers to both an --O-aryl and an --O-heteroaryl group, as defined herein.

[0643] A "hydroxy" group refers to an --OH group.

[0644] A "thiohydroxy" or "thiol" group refers to a --SH group.

[0645] A "thioalkoxy" group refers to any of the -S-alkyl, -S-alkenyl, -S-alkynyl, -S-cycloalkyl, and -S-heteroalicyclic groups, as defined herein.

[0646] A "thioaryloxy" group refers to both an --S-aryl and an --S-heteroaryl group, as defined herein.

[0647] A "carbonyl" or "acyl" group refers to a -C(=O)-R' group, where R' is as defined hereinabove.

[0648] A "thiocarbonyl" group refers to a -C(=S)-R' group, where R' is as defined herein.

[0649] A "C-carboxy" group refers to a -C(=O)-O-R' group, where R' is as defined herein.

[0650] An "O-carboxy" group refers to an R'C(=O)-O group, where R' is as defined herein.

[0651] A "carboxylic acid" group refers to a -C(=O)OH group.

[0652] An "oxo" group refers to a ═O group.

[0653] An "imine" group refers to a ═N—R′ group, where R′ is as defined herein.

[0654] An "oxime" group refers to a =N-OH group.

[0655] A "hydrazone" group refers to a ═N-NR′R″ group, where R′ and R″ are as defined herein.

[0656] A "halo" group refers to a fluorine, chlorine, bromine or iodine.

[0657] A "sulfinyl" group refers to a -S(=O)-R' group, where R' is as defined herein.

[0658] A "sulfonyl" group refers to a -S(=O)2-R' group, where R' is as defined herein.

[0659] A "sulfonate" group refers to a -S(=O)2-O-R' group, where R' is as defined herein.

[0660] A "sulfate" group refers to an -OS(=O)2-O-R' group, where R' is as defined herein.

[0661] A "sulfonamido" or "sulfonamido" group includes both S- and N-sulfonamido groups as defined herein.

[0662] An "S-sulfonamido" group refers to a -S(=O)2-NR'R'' group, where each of R' and R'' are as defined herein.

[0663] An "N-sulfonamido" group refers to an R'S(=O)2-NR''- group, where each of R' and R'' are as defined herein.

[0664] An "O-carbamyl" group refers to an --OC(.dbd.O)--NR'R'' group, where each of R' and R'' are as defined herein.

[0665] An "N-carbamyl" group refers to an R'OC(=O)-NR''- group, where each of R' and R'' are as defined herein.

[0666] An "O-thiocarbamyl" group refers to an --OC(.dbd.S)--NR'R'' group, where each of R' and R'' are as defined herein.

[0667] An "N-thiocarbamyl" group refers to an R'OC(=S)NR''- group, where R' and R'' are as defined herein.

[0668] An "S-thiocarbamyl" group refers to a -SC(=O)-NR'R'' group, where each of R' and R'' are as defined herein.

[0669] An "amide" or "amido" group includes C-amide and N-amide groups as defined herein.

[0670] A "C-amido" group refers to a -C(=O)-NR'R'' group, where each of R' and R'' are as defined herein.

[0671] An "N-amido" group refers to an R'C(=O)-NR''- group, where R' and R'' are as defined herein.

[0672] A "urea group" refers to a -N(R')-C(=O)-NR''R''' group, where R', R'', and R'' are as defined herein.

[0673] A "thiourea group" refers to a -N(R')-C(=S)-NR''R''' group, where R', R'', and R'' are as defined herein.

[0674] A "nitro" group refers to a -NO2 group.

[0675] A "cyano" group is a -C≡N group.

[0676] The term "phosphonyl" or "phosphonate" refers to a -P(=O)(OR')(OR'') group, with R' and R'' as defined above.

[0677] The term "phosphate" refers to a -OP(=O)(OR')(OR'') group, where each of R' and R'' are defined hereinabove.

[0678] The term "phosphinyl" refers to a -PR'R'' group, where each of R' and R'' are as defined above.

[0679] The term "hydrazine" refers to the group -NR'-NR''R''', where R', R'', and R''' are as defined herein.

[0680] As used herein, the term "hydrazide" describes a -C(=O)-NR'-NR''R''' group, where R', R'', and R''' are as defined herein.

[0681] As used herein, the term "thiohydrazide" describes a -C(=S)-NR'-NR''R'' group, where R', R'' and R''' are as defined herein.

[0682] A "guanidinyl" group refers to a -RaNC(=NRd)-NRbRc group, where each of Ra, Rb, Rc, and Rd can be as defined herein for R' and R''.

[0683] A "guanyl" or "guanine" group refers to a RaRbNC(=NRd)- group, where Ra, Rb, and Rd are as defined herein.

[0684] As used herein, the term "alkylene glycol" refers to -O-[(CR'R'') z -O] y -R''' end group or -O-[(CR'R'') z -O] y Described below are linking groups, where R', R'', and R''' are as defined herein, z is an integer from 1 to 10, preferably 2 to 6, more preferably 2 or 3, and y is an integer greater than or equal to 1. Preferably, R' and R'' are both hydrogen. When z is 2 and y is 1, the group is ethylene glycol. When z is 3 and y is 1, the group is propylene glycol. When y is 2 to 4, the alkylene glycol is referred to herein as an oligo(alkylene glycol).

[0685] When y is greater than 4, the alkylene glycol is referred to herein as a poly(alkylene glycol). In some embodiments of the invention, the poly(alkylene glycol) group or moiety can have 10-200 repeating alkylene glycol units, such that z is 10-200, preferably 10-100, and more preferably 10-50.

[0686] The term "saccharide" as used herein includes monosaccharides, disaccharides, and oligosaccharides. The term "monosaccharide" as used herein and as is well known in the art refers to a simple form of sugar consisting of a single sugar molecule that cannot be further broken down by hydrolysis. The most common examples of monosaccharides include glucose (dextrose), fructose, galactose, and ribose. Monosaccharides can be classified according to the number of carbon atoms as carbohydrates having three carbon atoms, i.e. trioses, such as glyceraldehyde and dihydroxyacetone, tetroses having four carbon atoms, e.g. erythrose, threose and erythrulose, pentoses having five carbon atoms, e.g. arabinose, lyxose, ribose, xylose, ribulose and xylulose, hexoses having six carbon atoms, e.g. allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose and tagatose, heptoses having seven carbon atoms, e.g. mannoheptulose, sedoheptulose, octose having eight carbon atoms, e.g. 2-keto-3-deoxy-manno-octonate, nonoses having nine carbon atoms (e.g. sialose) and decoses having ten carbon atoms. Monosaccharides are the building blocks of disaccharides and oligosaccharides, such as sucrose (the common sugar).

[0687] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0688] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0689] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

[0690] Part I Alleviation of ASD-like phenotypes by nNOS inhibition Materials and Methods Animal models: SHANK3 and CNTNAP2 mutant mice were obtained from Jackson Laboratory and used to study behavioral and synaptic abnormalities associated with autism spectrum disorder. Unless otherwise indicated, WT and mutant mice are 6 weeks old.

[0691] SHANK3 mutations were found to result in increased nitric oxide (NO) levels and altered S-nitroso-proteome in SHANK3 mice. The inventors showed that this mutation leads to S-nitrosylation of key proteins involved in vesicle release and synaptic function, which may result in autistic-like behaviors (Amal et al. Molecular Psychiatry 2018, www(dot)doi(dot)org / 10(dot)1038 / s41380-018-0113-6).

[0692] SNAP Processing Protocol: S-Nitroso-N-acetylpenicillamine (SNAP) was obtained from Tzamal D-Chem. WT or mutant mice were treated intraperitoneally with SNAP. Each mouse was given a dose of 20 mg / kg body weight on each day of the experiment. Behavioral testing began 2 hours after treatment. SNAP was first solubilized in DMSO (solubility is 20 mg / ml) and the additional volume was supplemented with 0.9% saline. Control animals were treated with vehicles (0.9% saline and DMSO). All animals were sacrificed 24 hours after the last treatment [1].

[0693] 7-NI Treatment: 7-Nitro-indazole (7-NI) was obtained from Tzamal D-Chem. WT, SHANK 3-mutant and / or CASPR2-KO mice were intraperitoneally treated with 7-NI. Each mouse was given a dose of 80 mg / kg body weight on each day of the experiment, unless otherwise indicated. Behavioral testing began 2 hours after treatment. 7-NI was first solubilized in DMSO (solubility is 32 mg / ml) and made up to volume with corn oil, unless otherwise indicated. Control animals were treated with the respective vehicles (e.g., corn oil and DMSO). All animals were sacrificed 24 hours after the last treatment.

[0694] Behavior analysis: Open field test: Mice were tested in an open field (45 x 45 cm) virtually divided into a central and peripheral area. Animal activity was recorded by video tracking (Noldus Ethovision). Each mouse was allowed to explore the apparatus for 5 min. The distance traveled, the number of backs and turns, the number of grooming bouts and the cumulative grooming time, the number of head shakes or twitches, the number of central entries, and the time spent in the central and peripheral areas were recorded. Measurements were recorded at 5 min intervals.

[0695] Novel object recognition (see Figure 4A): Novel object testing for object recognition and memory is performed in a white open field arena (45 x 45 cm). The test uses a set of two unique novel objects, each approximately the size of a mouse, composed of two different materials and non-uniform in shape [4]. The test consisted of one 10-min habituation session, a 5-min habituation session, and a 5-min recognition test, each of which was video tracked (Noldus Ethovision). During habituation, animals were allowed to freely explore the empty open field. At the end of the session, they were transferred from the open field to a location in a temporary clean holding cage for approximately 2 min. Two identical objects were placed on the midline 10 cm from each wall, and animals were returned to the open field and allowed to explore the objects for 5 min before being returned to their home cage. The next day, one familiar object and one novel object were placed in the open field relative to the location where the same objects were placed during the habituation session, and mice were allowed to explore them for a 5-min recognition test. The side of the location of the novel object was randomly assigned such that half of the animals were exposed to the novel object placed on the right side of the open field and half of the animals were exposed to the novel object placed on the left side of the open field. Between each session, the open field and objects were carefully washed with 70% ethanol and dried. The familiarization and recognition sessions were scored for the total time spent exploring each object, the number of object interactions, and the latency of the first object interaction. The time spent on each side during habituation and habituation, as well as the time spent sniffing two identical objects during the habituation phase, were used to examine innate side bias. The total time spent sniffing both objects was used as a measure of general exploration.

[0696] Three-chamber sociability test (see FIG. 4B): Sociability and preference for social novelty and social recognition were tested in a three-chamber apparatus. The test mice were first placed in the central neutral chamber and allowed to explore for 10 min with all doors closed. The doors were then opened and the mice were allowed to freely explore the other two empty chambers (1 and 2) for another 10 min. During this habituation, the lack of sub-preference was confirmed.

[0697] Two cylindrical wire cages were placed, one in chamber 1 and the other in chamber 2. For the sociability test, the test animal was introduced into the center chamber and allowed to adjust for 5 min. An unfamiliar mouse was then introduced into the wire cage in one side chamber, while the other side chamber was left empty. The time the test mouse spent exploring the wire cage with the unfamiliar mouse inside was recorded for 5 min.

[0698] Next, to examine social memory, a novel mouse is introduced into the empty cage and it is examined whether the tested mouse is more interested in the novel mouse compared to the familiar mouse.

[0699] Elevated plus maze test (see FIG. 4C): The elevated plus maze consisted of four arms (30×5 cm), two open and two closed. The platform was made of white plexiglass. The apparatus was elevated 45 cm above the floor. The test was started by placing the mouse on the central platform of the maze, facing one of the open arms and allowing it to move freely. Each session lasted 5 min. The time spent in the closed and open arms was recorded.

[0700] Marble burying test (see FIG. 4D): The marble burying assay is a tool to evaluate either anxiety-like and / or repetitive-like behavior in mice. The subject was tested in a normal clean cage (28 cm long x 18 cm wide x 12 cm high) with 3 cm of fresh bedding. The subject was first placed in an empty cage and allowed to habituate for 5 min. It was then briefly placed in an empty clean cage, and 20 dark blue marbles (15 mm diameter) were placed equidistantly on the bedding in a 4×5 arrangement to cover the entire cage surface. The subject was then returned to the test cage and allowed to explore and bury the marbles during a 15-minute session, which was videotaped. At the end of the session, the subject was removed and the number of buried marbles (50% of the marbles were covered with bedding material) was recorded.

[0701] Example 1 Increased 3-nitrotyrosine (Ntyr) in ASD models 3-Nitrotyrosine (3-Ntyr) is a post-translational modification in proteins that occurs through the action of nitrating agents resulting in the addition of a NO2 group (ortho to the phenolic hydroxyl group), resulting in protein tyrosine nitration (PTN). The essential feature of PTN is that it is a stable post-translational modification and does not occur randomly. 3-Ntyr represents a reliable marker of nitrosative stress.

[0702] Ten participants with ASD (ages: 2–6 years, male) and five unrelated, age- and sex-matched control participants (ages: 2–6 years, male) with normal education and no neuropsychiatric diagnosis were recruited from the outpatient neuropediatric clinic at Shaare Zedek Medical Center as part of an ongoing clinical trial (0501-20-SZMC) evaluating biomarkers in children with ASD. The study was approved by the Institutional Review Board at Shaare Zedek Medical Center, and participants' parents provided written informed consent prior to participant enrollment.

[0703] Western blots were performed to determine NTyr leve...

Claims

1. 1. A pharmaceutical composition comprising an active agent that reduces or inhibits nNOS enzyme activity for use in the treatment of a disease or condition in which a beneficial clinical effect is achieved by reducing or inhibiting nNOS activity, said disease or condition being autism spectrum disorder (ASD).

2. The composition for use according to claim 1, wherein the active agent is selected from L-N G -methyl-L-arginine (L-NMMA), L-N G -propyl-L-arginine (N-PLA), L-N G -nitroarginine (L-NNA), L-N G -nitroarginine methyl ester (L-NAME), L-thiocitrulline, S-methyl-L-thiocitrulline, ethyl-L-NIO, vinyl-L-NIO and methylene blue.

3. The composition for use according to claim 1 or 2, wherein the agent is L-N G -propyl-L-arginine (N-PLA).

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