Neurodevelopmental disorder treatment

Abstract

The present disclosure relates to the treatment and / or prevention of neurodevelopmental disorders, especially Autism Spectrum Disorders (ASD), using P2Y2 agonists such as: Diquafosol, Denufosol, MRS2698, MRS2768, PSB1114, ATP, ATP derivatives, UTP, UTP derivatives, 2-((ethyl(4-fluorobenzyl)amino)methyl)-7,8-dimethylquinolin-4(1H)-one, or a pharmaceutically acceptable salt or solvate thereof. Also disclosed herein are combination therapies, compositions, and kits for the treatment and / or prevention of neurodevelopmental disorders using said P2Y2 agonists.

Description

NEURODEVELOPMENTAL DISORDER TREATMENTRELATED APPLICATION

[0001] This application claims benefit of United States Provisional Patent Application serial no. 63 / 665,397 filed June 28, 2024, incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to methods, uses and kits for treating or preventing a neurodevelopmental disorder. In particular, the present disclosure pertains to the use of a P2Y2 agonist for the treatment or prevention of Autism Spectrum Disorders.BACKGROUND

[0003] Autism spectrum disorders (ASD) are neurodevelopmental disorders associated with social interaction deficits, communication impairments and repetitive behaviors. These disorders vary in symptom severity with children ranging from high functioning to severely impaired individuals requiring constant assistance. Increased awareness and advancements in diagnostic / medical tools have led to a growing number of children being diagnosed with ASD. These pervasive neurodevelopmental disorders are characterized by early-onset social interaction deficits, communication impairments, repetitive behaviors and restricted interests.

[0004] Currently, there are no treatments to treat ASD root causes, nor slow down their progression. However, there are methods to reduce debilitating symptoms such as aggression, self-injurious behaviors, agitation and hyperactivity. For instance, pharmacological treatments including antipsychotics, anticonvulsants, antidepressants and mood stabilizers can help moderate some of these symptoms. Furthermore, the high prevalence of comorbidities will determine the medication that can be given to individuals with ASD, thus adding to the difficulty of treating and managing the symptoms of these disorders.

[0005] The causes of ASD are unknown. In the past decade, research has identified some neurobiological and genetic underpinnings associated withASD. Consequently, ASD have been associated with both environmental and genetic origins. In individuals with ASD, a large number of genes have been associated with ASD, some of which interfere with neurodevelopment in utero through childhood as well as in synaptogenesis and axon motility. For example, mutations in SHANK1, SHANK3, SYNGAP1, SYNAPSIN, NCAM MY016, ASTN2, PTEN, CHD2, CHD4, CHD8, NLGN3, NLGN4X, CNTN4, CNTNAP2, or NRXN genes which are involved in synaptic function and axon motility can lead to an ASD diagnosis.

[0006] The only current treatments relate to symptom management such as cognitive and behavioural therapies, skills training, medications for symptom management. Accordingly, treatments to address the causes of ASD or to slow the progression of ASD are needed.SUMMARY

[0007] According to the present disclosure, a link between brain endothelial cell energy metabolism and ASD has been identified. Further, the P2Y2 purinergic receptor on endothelial cells has also been identified as a target for rescuing endothelial defects leading to ASD symptoms, thereby a target for treating ASD causes at an early stage, rather than mere symptom management.

[0008] Accordingly, an aspect of the disclosure is a method of treating or preventing a neurodevelopmental disorder comprising administration of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof to a subject in need thereof. Another aspect of the disclosure is a use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof for treating or preventing a neurodevelopmental disorder in a subject in need thereof. In yet another aspect of the disclosure is a use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a neurodevelopmental disorder in a subject in need thereof. A further aspect of the disclosure is a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof foruse for treating or preventing a neurodevelopmental disorder in a subject in need thereof. In yet a further aspect of the disclosure is a composition for use in treating or preventing a neurodevelopmental disorder in a subject in need thereof, the composition comprising a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

[0009] In some embodiments, the neurodevelopmental disorder is Autism Spectrum Disorders (ASD).

[0010] In some embodiments, the ASD comprise a mutation in a gene comprising SHANK1, SHANK3, SYNGAP1, SYNAPSIN, NCAM, MY016, MECP2, ADNP, FMR1, KCTD13, ASTN2, PTEN, CHD2, CHD4, CHD8, NLGN3, NLGN4X, CNTN4, CNTNAP2, and / or NRXN.

[0011] In some embodiments, the ASD comprise a mutation in a gene selected from: SHANK3, SYNGAP1, SYNAPSIN, NCAM, MY016, MECP2, ADNP, FMR1, and KCTD13.

[0012] In some embodiments, the ASD comprise a genomic copy number variation (CNV) in 16p11 .2.

[0013] In some embodiments, the P2Y2 agonist is Diquafosol, Denufosol, MRS2698, MRS2768, PSB1114, ATP, ATP derivatives, UTP, UTP derivatives, 2-((ethyl(4-fluorobenzyl)amino)methyl)-7,8-dimethylquinolin-4(1 / 7)- one , or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0014] In some embodiments, the P2Ys agonist is Diquafosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0015] In some embodiments, the P2Y2 agonist is Denufosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0016] In some embodiments, the P2Y2 agonist is MRS2698 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0017] In some embodiments, the P2Y2 agonist is MRS2768 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0018] In some embodiments, the P2Y2 agonist is adenosine triphosphate (ATP), a derivative thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0019] In some embodiments, the P2Y2 agonist is uridine triphosphate (UTP), a derivative thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0020] In some embodiments, the P2Y2 agonist is 2-((ethyl(4- fluorobenzyl)amino)methyl)-7,8-dimethylquinolin-4(1 / 7)-one, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0021] In some embodiments, the P2Y2 agonist is administered or for use parenterally or orally.

[0022] In some embodiments, the P2Y2 agonist is administered or for use daily, optionally the P2Y2 agonist is administered daily for one week.

[0023] In some embodiments, the P2Y2 agonist is permeable to the blood-brain barrier.

[0024] In some embodiments, the P2Y2 agonist is impermeable to the blood-brain barrier.

[0025] In some embodiments, the P2Y2 agonist is administered or for use as a pharmaceutical composition comprising the P2Y2 agonist and at least one pharmaceutically acceptable carrier, excipient or diluent.

[0026] In some embodiments, the pharmaceutically acceptable carrier, excipient or diluent is a nanoparticle.

[0027] In some embodiments, the P2Y2 agonist is administered or for use as a combination therapy.

[0028] In some embodiments, combination therapy comprises a behavioral therapy, a physical exercise, a speech and language pathology therapy, an ergotherapy, a pediatric occupational therapy, or a psychotherapy

[0029] In some embodiments, the subject is an infant or a child, wherein the infant or child has not been diagnosed with the neurodevelopmental disorder.

[0030] In some embodiments, the subject is an infant, a child, teenager, or an adult, wherein the infant, child, teenager or adult has been diagnosed with the neurodevelopmental disorder.

[0031] These and other features and advantages of the present disclosure will become apparent from the following detailed description taken together with the accompanying drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred implementations of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0032] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0033] FIGs. 1A, 1 B show that extracellular ATP rescues 16p11.2- deficient angiogenic activity, and that this is mediated by P2-class purinergic receptors. FIG. 1A (top panel) and FIG. 1 B (top 2 panels) show representative images at the 8h post-seeding time point of WT and 16p 1 1 ,2-deficient untreated ECs and 16p11 ,2-deficient ECs treated with 100pM of extracellular ATP and with 50 pM PPADs. FIG. 1A and FIG. 1 B (bottom panels) show quantifications of network densities (total endothelial tube length) and network nodes (total number of branching hubs) with representative percent of WT panels. All data shown are from ECs isolated from male mice. Data are shown as the mean + s.e.m. * / # / $P<0.05, ** / ## / $$p<0.01 , *** / ### / $$$p<0.001 (two-way ANOVA and Sidak’s multicomparison post hoc test).

[0034] FIG. 2 shows that adenosine does not rescue 16p11 ,2-deficient angiogenic activity. The top panel shows representative images at the 8h postseeding time point of WT and 16p11 ,2-deficient untreated ECs and 16p11 ,2- deficient ECs treated with 100pM of adenosine. The bottom panel shows quantifications of network densities (total endothelial tube length) and network nodes (total number of branching hubs). All data shown are from ECs isolated from male mice. Data are shown as the mean + s.e.m. * / #P<0.05, ** / ##P<0.01 , *** / ###P<0.001 (two-way ANOVA and Sidak’s multicomparison post hoc test).

[0035] FIG. 3 shows five graphs depicting that messenger RNAs (mRNAs) encoding P2-class receptors P2rx1 , P2rx4, P2rx7 and P2ry14 were found at high expression levels in both genotypes. The mRNA encoding P2Y2 (P2ry2), was found at relatively low expression levels in WT mice, however its expression nearly doubled in 16p11 ,2-deficient brain ECs.

[0036] FIG. 4 shows that both WT and 16p11 .2 mice endothelial cells express the P2Y2 receptor to comparable levels. Therefore, any functional rescue from this mouse model is not due to a change in protein expression level or locus.

[0037] FIG. 5 shows that DQS rescued vascular network growth defects in 16p11 .2 deficient mice.

[0038] FIG. 6 shows ATP supplementation significantly increased intracellular steady-state Ca2+levels in WT ECs as compared to WT ECs without ATP, no difference was observed between 16p11.2df / +ECs and 16p11.2df / +with ATP supplementation.

[0039] FIG. 7 shows that supplementation with ATP decreased the frequency of Ca2+transients in 16p11.2df / +ECs.

[0040] FIG. 8 shows that the supplementation of ATP in 16p 11 ,2d / z+mice decreased marble burying tendencies.

[0041] FIG. 9 shows an exemplary electron microscopy with immunogold labeling imaging demonstrating that P2Y2 receptors were localized at the luminal membrane.

[0042] FIG. 10 shows a schematic representation of DQS injections and behavioral timeline.

[0043] FIGs. 11 A, 11 B and 11C show behavioral assessments (Marble Burying and Novel Object Recognition) of P50 adult 16p11.2df / +males. FIG. 11A shows that 16p11.2df / +males demonstrated reduced marble burying (MB) following a one-week DQS treatment compared with PBS treated 16p11.2df / +males. FIG. 11 B shows 16p11.2df / +males treated with DQS demonstrated improved recognition memory following a novel-object recognition task (NOR) compared to PBS treated 16p11.2df / +males. FIG. 11 C shows that 16p11.2df / +males demonstrated reduced MB following a two-week DQS treatment compared with PBS treated 16p11.2df / +males. *Data shown are mean + s.e.m (n=7-13 animals per group). ***P<0.001 (one-way ANOVA and Tukey multicomparison post hoc). Circles represent WT mice treated with PBS, squares represent 16p11.2df / +males treated with PBS and diamonds represent 16p11.2df / +males treated with DQS.

[0044] FIG. 12 shows DQS improved evoked cerebral blood flow responses in ASD mice compared to PBS (vehicle) treated ASD mice.

[0045] FIG. 13A demonstrates no change in vascular permeability in both untreated ASD mice and DQS-treated ASD mice. FIG. 13B shows DQS does not impact brain blood supply following assessment of pial artery perfusion in ASD mice. FIG. 13C demonstrates 100% survival of WT, untreated ASD mice and DQS-treated ASD mice. FIG. 13D demonstrates no significant changes in body weight between WT, untreated ASD mice and DQS-treated ASD mice.

[0046] FIG. 14 shows DQS rescues vascular reactivity of cortical parenchymal arterioles (PAs) ex vivo in ASD mice. Changes in arteriole diameter were measured in response to adenosine. Representative image of PA preparations (left) and traces of arteriolar diameter of WT (top middle) and ASD (bottom middle) PA during DQS treatment are shown. The graph (right) shows quantification of ex vivo vascular reactivity following DQS treatment demonstrating a rescue in vasodilatory responses of ASD arterioles.

[0047] FIG. 15 shows brain endothelial cells isolated from ASD mice displayed an impaired ability to form networks in vitro compared to brain endothelial cells isolated from wild-type mice. Administration of 10pM DNS following brain endothelial cell isolation from ASD mice and throughout endothelial cell culturing improved tube formation (branching) ability of ASD mice brain endothelial cells compared to untreated ASD mice brain endothelial cells.DESCRIPTION OF VARIOUS EMBODIMENTS

[0048] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0049] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. Definitions

[0050] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, thepresent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0051] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0052] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0053] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0054] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0055] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0056] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1 .5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0057] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0058] The term “neurodevelopmental disorder” as used herein refers to disorders which typically emerge in childhood, and which can persist into adulthood. Without wishing to be bound by theory, neurodevelopmental disorders may impact multiple aspects of life, including without limitation, personal, social, academic and occupational functioning. Neurodevelopmental disorders include, without limitation, intellectual developmental delays, communication disorders (speech and language), autism spectrum disorders, attention deficit / hyperactivity disorders, motor impairments, repetitive movements, focused interests and specific learning disabilities.

[0059] The term “autism spectrum disorders” or “ASD” as used herein refers to a neurodevelopmental disorder characterized by impairments in social interaction, stereotyped behavioural patterns, and narrow interests. ASD include subtype disorders, including without limitation autistic disorder, Asperger’s disorder, childhood disintegrative disorder and pervasive developmental disorder (not otherwise specified). The etiology of ASD remains unknown, however may be influenced by genetic and / or environmental factors. For example, mutations can be in any ASD risk gene including, but not limited to, SHANK1 , SHANK3, SYNGAP1 , SYNAPSIN, NCAM, MY016, MECP2, ADNP, KCTD13, CNTNAP2, ASTN2, PTEN, CHD2, CHD4, CHD8, NLGN3,NLGN4X, CNTN4, CNTNAP2, NRXN genes, which are involved in neuronal morphogenesis, synaptic function, plasticity and axon motility, are considered ASD risk genes and can lead to an ASD diagnosis.

[0060] The term “purinergic receptor” as used herein refers to a family of receptors which bind to adenosine triphosphate (ATP), and other nucleotide analogs, as well as the byproduct adenosine. Purinergic receptors include P1 and P2 receptors. P1 receptors are adenosine receptors, while P2 receptors are ATP / adenosine diphosphate (ADP) and uridine triphosphate (UTP) / uridine diphosphate (UDP). P2 receptors include P2X and P2Y receptor types which mediate glial cell hyperactivation.

[0061] The term “P2Y2 agonist” as used herein refers to an agent, such as a molecule, drug, antibody or ligand, which stimulates the purinergic receptor P2Y2. P2Y2 agonists include, both selective and non-selective agonists and positive allosteric modulators of P2Y2 activity.

[0062] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Optionally, the term “subject” includes mammals that have been diagnosed with a neurodevelopmental disorder. In one embodiment, the term “subject” refers to a human having, or suspected of having, a neurodevelopmental disorder. The subject may be an infant (0-24 months), a child (2-8 years), a teenager (9-17 years) or an adult (18+ years).

[0063] The term "subject in need thereof" refers to a subject that could benefit from the method(s) or treatment(s) described herein, and optionally refers to a subject with a neurodevelopmental disorder, or optionally a subject with increased risk of a neurodevelopmental disorder, ora subject with a strong genetic disposition.

[0064] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.

[0065] “Palliating” a disease, disorder or condition means that the extent and / or undesirable clinical manifestations of a disease, disorder or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

[0066] The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition.

[0067] The term “administered” or “administering” as used herein means administration of a therapeutically effective amount of a compound or composition of the disclosure to a cell either in cell culture or in a subject.

[0068] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context of treating ASD, an effective amount is an amount that for example rescues endothelial cell defects, reduces disease symptoms, and / or reduces disease severity compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the animal. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0069] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts ofthe method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0070] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.II. Methods and Uses

[0071] As shown herein, P2Y2 agonists are efficacious in treating and / or preventing neurodevelopmental disorders. The present disclosure shows a link between brain endothelial cell energy metabolism and neurodevelopmental disorders, such as Autism Spectrum Disorders (ASD). These endothelial cell defects can be rescued by treatment with P2Y2 agonists.

[0072] Accordingly, an aspect of the present disclosure is a method of treating or preventing a neurodevelopmental disorder comprising administration of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof to a subject in need thereof. Another aspect of the disclosure is use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof for treating or preventing a neurodevelopmental disorder. In another aspect of the disclosure is use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a neurodevelopmental disorder. In yet another aspect of the disclosure is a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof for treating or preventing a neurodevelopmental disorder. In yet another aspect of the disclosure is a composition for treating or preventing a neurodevelopmental disorder comprising a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

[0073] In an embodiment, the neurodevelopmental disorder is an Autism spectrum disorders (ASD).

[0074] In an embodiment, ASD comprise genetic variations including several mutation(s) in one or more ASD risk gene(s). It is understood that there are many ASD risk genes, and ASD relevant mutations, including, without limitation, sequence number variants (SNVs) copy number variations (CNVs), truncation mutations, loss-of-fu notion mutations, gain-of-function mutations, missense variants and single nucleotide polymorphisms (SNPs). ASD risk genes are described in Viggiano, M., Ceroni, F., Visconti, P. et al. Genomic analysis of 116 autism families strengthens known risk genes and highlights promising candidates, npj Genom. Med. 9, 21 (2024). https: / / doi.org / 10.1038 / s41525-024-00411-1 ; Forrest MP, Penzes P. Autism Genetics: Over 100 Risk Genes and Counting. Pediatr Neurol Briefs. 2020 Dec 4;34:13. doi: 10.15844 / pedneurbriefs-34-13. PMID: 33304087; PMCID: PMC7718098; Liao C, Moyses-Oliveira M, De Esch CEF, Bhavsar R, Nuttie X, Li A, Yu A, Burt ND, Erdin S, Fu JM, Wang M, Morley T, Han L; CommonMind Consortium; Dion PA, Rouleau GA, Zhang B, Brennand KJ, Talkowski ME, Ruderfer DM. Convergent coexpression of autism-associated genes suggests some novel risk genes may not be detectable in large-scale genetic studies. Cell Genom. 2023 Mar9;3(4):100277. doi: 10.1016 / j.xgen.2O23.100277. PMID: 37082147; PMCID: PMC10112287; Yuen, R., Thiruvahindrapuram, B., Merico, D. et al. Whole-genome sequencing of quartet families with autism spectrum disorder. Nat Med 21 , 185-191 (2015). https: / / doi.org / 10.1038 / nm.3792; and Nakanishi M, Anderson MP, Takumi T. Recent genetic and functional insights in autism spectrum disorder. Curr Opin Neurol. 2019 Aug;32(4):627-634. doi: 10.1097 / WCO.0000000000000718. PMID: 31135459; PMCID: PMC6959126; each of which are hereby incorporated by reference.

[0075] In some embodiments, the ASD comprise a mutation in a gene comprising SHANK1, SHANK3, SYNGAP1, SYNAPSIN, NCAM, MY016, MECP2, ADNP, FMR1, KCTD13, ASTN2, PTEN, CHD2, CHD4, CHD8, NLGN3, NLGN4X, CNTN4, CNTNAP2, and / or NRXN.

[0076] In an embodiment, the ASD comprise a mutation in a gene selected from: SHANK3, SYNGAP1, SYNAPSIN, NCAM, MYO16, ADNP, MECP2, FMR1, and KCTD13.

[0077] SH3 and multiple ankyrin repeat domains 1 (SHANK1) is an adapter protein in the postsynaptic density of excitatory synapses that interconnects receptors of the postsynaptic membrane (NMDA-type) and metabotropic glutamate receptors. It plays a role in the structural and functional organization of the dendritic spine and synaptic junction. Mutations in the SHANK1 gene have been associated with ASD. For example, the nucleotide and amino acid sequences of human SHANK1 , including variants and isoforms thereof can be found at GenelD: 50944, NCBI Reference Sequence: NM_016148.5, and UniProt ID: Q9Y566.

[0078] SH3 and multiple ankyrin repeat domain protein 3 (SHANK3) is a synaptic scaffolding protein found in the postsynaptic density of excitatory synapse, and plays a role in the formation, maturation and maintenance of synapses. Mutations in the SHANK3 gene have been associated with ASD. For example, the nucleotide and amino acid sequences of human SHANK3, including variants and isoforms thereof can be found at GenelD: 85358, NCBI Reference Sequence: NG_070230.1 and UniProt ID: Q9BYB0.

[0079] Synaptic Ras GTPase activating protein 1 (SYNGAP1) negatively regulates Ras, Rap and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking to the postsynaptic membrane to regulate synaptic plasticity and neuronal homeostasis. Allelic variants of this gene are associated with intellectual disability and autism spectrum disorders. For example, the nucleotide and amino acid sequences of human SYNGAP1 , including variants and isoforms thereof can be found at GenelD: 8831 , NCBI Reference Sequence: NG_016137.2 and UniProt ID: Q96PV0.

[0080] Synapsin (SYN1) is a neuronal phosphoprotein associated with the cytoplasmic surface of synaptic vesicles. Synapsin is implicated involved in synaptogenesis and the modulation of neurotransmitter release, thus suggestive of a role in several neuropsychiatric diseases. For example, the nucleotide and amino acid sequences of human synapsin, including variantsand isoforms thereof can be found at GenelD: 6853, NCBI Reference Sequence: NG_008437.1 and UniProt ID: P17600.

[0081] Neural cell adhesion molecule 1 (NCAM1) is a cell adhesion protein of the immunoglobulin superfamily. The NCAM1 protein plays a role in the development of the nervous system by regulating neurogenesis, neurite outgrowth, and cell migration. For example, the nucleotide and amino acid sequences of human NCAM1 , including variants and isoforms thereof can be found at GenelD: 4684, NCBI Reference Sequence: NG_032036.2 and UniProt ID: P13591.

[0082] Myosin XVI (MY016) is an unconventional myosin protein suspected to act as a serine-threonine phosphatase-1 targeting or regulatory subunit. For example, the nucleotide and amino acid sequences of human MYO16, including variants and isoforms thereof can be found at GenelD: 23026, NCBI Reference Sequence: NW_013171811 .1 and UniProt ID: Q9Y6X6.

[0083] Activity dependent neuroprotection homeobox (ADNP) contains one homeobox and nine zinc finger domains, suggested to function as a transcription factor. For example, the nucleotide and amino acid sequences of human ANDP, including variants and isoforms thereof can be found at GenelD:23394, NCBI Reference Sequence: NC_000020.11 and UniProt ID: Q9H2P0.

[0084] Methyl-CpG binding protein 2 (MECP2) is part of a family of nuclear proteins which comprise a methyl-CpG binding domain (MBD). These proteins specifically bind methylated DNA. MECP2 can also repress transcription from methylated promoters. MECP2 is an X-linked gene and subject to X-inactivation. For example, the nucleotide and amino acid sequences of human MECP2, including variants and isoforms thereof can be found at GenelD:4204, NCBI Reference Sequence: NG_007107.3 and UniProt ID: P51608.

[0085] Fragile X messenger ribonucleoprotein 1 (FMR1) is an RNA binding protein associated with polysomes and may be involved in mRNAtrafficking from the nucleus to the cytoplasm. For example, the nucleotide and amino acid sequences of human MECP2, including variants and isoforms thereof can be found at GenelD:2322, NCBI Reference Sequence: NG_007529.2 and UniProt ID: Q06787.

[0086] Potassium channel tetramerization domain containing 13 (KCTD13) enables identical protein binding activity and small GTPase binding activity. KCTD13 is involved in several processes, including cellular protein metabolic process; negative regulation of Rho protein signal transduction; and stress fiber assembly. For example, the nucleotide and amino acid sequences of human MECP2, including variants and isoforms thereof can be found at GenelD:253980 and UniProt ID: Q8WZ19.

[0087] Activity dependent neuroprotector homeobox (ADNP) is a transcription factor that modulates WNT-beta-Catenin / CTNN1 B signaling. It is required for neural induction and neuronal differentiation. Mutations in ADNP are associated with ASD, intellectual disability and hypotonia. For example, the nucleotide and amino acid sequences of human ADNP, including variants and isoforms thereof can be found at GenelD: 23394, NCBI Reference Sequence: NG_034200.2, and UniProt ID: Q9H2P0.

[0088] Astrotactin 2 (ASTN2) mediates neuronal cell adhesion molecule ASTN1 of the cell membrane in migrating neurons while promoting ASTN1 internalization and intracellular transport of endocytosed ASTN1. The protein expressed in the brain is involved in neuronal migration. Mutations in ASTN2 are associated with ASD and attention-deficit / hyperactivity disorder (ADHD). For example, the nucleotide and amino acid sequences of human ASTN2, including variants and isoforms thereof can be found at GenelD:23245, NCBI Reference Sequence: NG_021409.2, and UniProt ID: 075129.

[0089] Phosphatase and tensin homolog (PTEN) is a tumor suppressor that mediates cell cycle progression and cell survival. PTEN modulates correct neuron position, dendritic development and synapse formation. Mutations in PTEN are associated with ASD, particularly in individuals with macrocephaly. For example, the nucleotide and amino acid sequences of human PTEN,including variants and isoforms thereof can be found at GenelD:5728, NCBI Reference Sequence: NG_007466.2, and UniProt ID: P60484.

[0090] Chromodomain helicase DNA binding protein 2 (CHD2) is a DNA- binding helicase that binds to the promoter of target genes for chromatin remodeling and transcription regulation. Mutations in CHD2 are associated with ASD, intellectual disability and epilepsy. For example, the nucleotide and amino acid sequences of human CHD2, including variants and isoforms thereof can be found at GenelD:1106, NCBI Reference Sequence: NG_012826.2, and UniProt ID: 014647.

[0091] Chromodomain helicase DNA binding protein 4 (CHD4) is a ATP- dependent helicase that binds and distorts nucleosomal DNA. It participates in the remodeling of chromatin through the histone deacetylase NuRD complex and plays a role in epigenetic transcriptional repression. Mutations in CHD4 are associated with neurodevelopmental disorders including ASD and intellectual disability. For example, the nucleotide and amino acid sequences of human CHD4, including variants and isoforms thereof can be found at GenelD:1108, NCBI Reference Sequence: NG_052823.1 , and UniProt ID:Q14839.

[0092] Chromodomain helicase DNA binding protein 8 (CHD8) acts as a chromatin remodeling factor and regulates transcription. It is known to have functions involved in transcriptional regulation, epigenetic remodeling, promotion of cell proliferation, and regulation of RNA synthesis. Mutations in CHD8 are associated with ASD and macrocephaly. For example, the nucleotide and amino acid sequences of human CHD8, including variants and isoforms thereof can be found at GenelD:57680, NCBI Reference Sequence: NG-021249.2, and UniProt ID:Q9HCK8.

[0093] Neuroligin 3 (NLGN3) is a cell surface protein involved in cell-cell- interactions. It is involved in synapse function, synaptic signal transmission and promotes synapse formation. Mutations in NLGN3 are associated with ASD and intellectual disability. For example, the nucleotide and amino acid sequences of human NLGN3, including variants and isoforms thereof can be found at GenelD:54413, NCBI Reference Sequence: NG_015874.2, and UniProt ID: Q9NZ94.

[0094] Neuroligin 4 X-linked (NLGN4X) is a cell surface protein involved in cell-cell-interactions. It plays a role in the formation and remodeling of synapses. Mutations in NLGN4X are associated with ASD and intellectual disability. For example, the nucleotide and amino acid sequences of human NLGN4X, including variants and isoforms thereof can be found at GenelD:57502, NCBI Reference Sequence: NG_008881.2, and UniProt ID: Q8N0W4.

[0095] Contactin 4 (CNTN4) is an axon -associated cell adhesion molecule that plays a role in neuronal network formation and plasticity. Mutations in CNTN4 are associated with ASD. For example, the nucleotide and amino acid sequences of human CNTN4, including variants and isoforms thereof can be found at GenelD:152330, NCBI Reference Sequence: NG_012827.2, and UniProt ID: Q8IWV2.

[0096] Contactin associated protein 2 (CNTNAP2) mediates interactions between neurons and glia during nervous system development. It plays a role in gap junction formation and with CNTNAP1 is required for radial and longitudinal organization of myelinated axons. CNTNAP2 is involved in the formation of functional distinct domains critical for saltatory conduction of nerve impulses in myelinated nerve fibers. Mutations in CNTNAP2 are associated with ASD, schizophrenia, ADHD, and intellectual disability. For example, the nucleotide and amino acid sequences of human CNTNAP2, including variants and isoforms thereof can be found at GenelD:26047, NCBI Reference Sequence: NG_007092.3, and UniProt ID: Q9UHC6.

[0097] The neurexin (NRXN) gene family are cell surface receptors that bind to neuroligins to form Ca(2+)- dependent neurexin / neuroligin complexes at synapses. They are involved in cell recognition and cell adhesion. Mutations in NRXN genes are associated with ASD.

[0098] In an embodiment, the ASD comprise a copy number variation in 16p11.2, 16p13.11 , 15q11-q13, 1q21.1 , 22q13, and / or 22q11.2. In some embodiments, the ASD comprise a genomic copy number variation (CNV) in the 16p11.2 locus, known as 16p11.2 deletion. The copy number variations(CNV) refer to a structural variation of a chromosome, such as deletion on a specific location of a chromosome.

[0099] In an embodiment, the P2Y2 agonist is Diquafosol, Denufosol, MRS2698, MRS2768, ATP, ATP derivatives, UTP, UTP derivatives, compound 89, reported by Sakuma, K., et al. Effects of 4(1 / 7)-quinolinone derivative, a novel non-nucleotide allosteric purinergic P2Y 2 agonist, on cardiomyocytes in neonatal rats. Sei Rep 7, 6050 (2017). https: / / doi.org / 10.1038 / s41598-017- 06481 -9, incorporated herein by reference insofar as it relates to compound 89, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0100] In an embodiment, the P2Y2 agonist is Diquafosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0101] In an embodiment, the P2Y2 agonist is Denufosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0102] In an embodiment, the P2Y2 agonist is MRS2698 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0103] In an embodiment, the P2Y2 agonist is MRS2768 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0104] In an embodiment, the P2Y2 agonist is ATP, or derivatives thereof or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0105] In an embodiment, the P2Y2 agonist is UTP, or derivatives thereof or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0106] In an embodiment, the P2Y2 agonist is compound 89, or derivatives thereof or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0107] The structure of P2Y2 receptor agonists, including Diquafosol, are described in WO 2012 / 033189A1 , incorporated herein by reference insofar as it relates to P2Y2 agonists, the P2Y2 agonists having the following general structure:where X1 and X3 are uridine and X2 is a phosphate group.

[0108] Diquafosol, also known as Diquas® is commonly used as an ophthalmic treatment for dry eye. Diquafosol is described in US Patent No. 10,632,139, incorporated herein by reference insofar as it relates to the compound Diquafosol. The structure of Diquafosol is shown below:

[0109] In one embodiment, Diquafosol is Diquafosol sodium, shown below:

[0110] Denufosol, also known by the chemical name P1-(uridine 5'-)-P4- (2'-deoxycytidine 5'-) tetraphosphate; its chemical registry number is 211448- 85-0. Denufosol is a P2Y2 receptor agonist which was previously used for cystic fibrosis treatment. Denufusol is described in United States Patent ApplicationNo. 12 / 603,506, incorporated herein insofar as it relates to the compound Denufosol.

[0111] MRS2698 is a selective P2Y2 agonist, with the chemical name(((2S,3S,4R,5R)-4-amino-3-hydroxy-5-(4-oxo-2-thioxo-3,4-dihydropyrimidin- 1 (2H)-yl)tetrahydrofuran-2-yl)methyl)phosphonic diphosphoric anhydride. The structure of MRS2698 is shown below:

[0112] MRS2768 is a tetrasodium salt and is a selective P2Y2 agonist, with the chemical name Uridine-5'-tetraphosphate b-phenyl ester tetrasodium salt. The structure of MRS2768 is shown below:

[0113] Compound 89 is a selective non-nucleotide P2Y2 allosteric agonist with the chemical name 2-((ethyl(4-fluorobenzyl)amino)methyl)-7,8- dimethylquinolin-4(1 / 7)-one. Compound 89 is described in Sakuma, K., et al. The structure of compound 89 is shown below:

[0114] In some embodiments, the salt of a compound of the application is an acid addition salt or a base addition salt. In some embodiments, for pharmaceutical methods and uses on human or animal subjects, the salt is a pharmaceutically acceptable salt. The selection of a suitable salt may be made by a person skilled in the art. Suitable salts include acid addition salts that may, for example, be formed by mixing a solution of a compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. Additionally, acids that are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) and Handbook of Pharmaceutical Salts. Properties,Selection and Use. (2002) Zurich: Wiley VCH; S. Berge et al, Journal of Pharmaceutical Sciences 1977 66(1 ) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201 -217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website).

[0115] An acid addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids, as well as acidic metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such organic acids are, for example, acetic, trifluoroacetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, mandelic, salicylic, 2-phenoxybenzoic, p-toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2- hydroxyethanesulfonic acid. In some embodiments, exemplary acid addition salts also include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates (“mesylates”), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates) and the like. In some embodiments, the mono- or di-acid salts are formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents and generally demonstrate higher melting points in comparison to their free base forms. The selection criteria for the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts such as but not limited to oxalates may be used, for example in the isolation of compounds of theapplication for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0116] A base addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compounds that form a basic addition salt include, for example, compounds comprising a carboxylic acid group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide as well as ammonia. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. The selection of the appropriate salt may be useful, for example, so that an ester functionality, if any, elsewhere in a compound is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art. In some embodiments, exemplary basic salts also include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamine, Abutyl amine, choline and salts with amino acids such as arginine, lysine and the like. Basic nitrogen containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl and dibutyl sulfates), long chain halides (e.g., decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides) and others. Compounds carrying an acidic moiety can be mixed with suitable pharmaceutically acceptable salts to provide, for example, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium ormagnesium salts) and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (-COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.

[0117] All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the application and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the application. In addition, when a compound of the application contains both a basic moiety, such as, but not limited to an aliphatic primary, secondary, tertiary or cyclic amine, an aromatic or heteroaryl amine, pyridine or imidazole and an acidic moiety, such as, but not limited to tetrazole or carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the terms “salt(s)” as used herein. It is understood that certain compounds of the application may exist in zwitterionic form, having both anionic and cationic centers within the same compound and a net neutral charge. Such zwitterions are included within the application.

[0118] Solvates of compounds of the application include, for example, those made with solvents that are pharmaceutically acceptable. Examples of such solvents include water (resulting solvate is called a hydrate) and ethanol and the like. Suitable solvents are physiologically tolerable at the dosage administered.

[0119] Prodrugs of the compounds of the present application may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0120] In some embodiments, the compounds of the present application are suitably formulated in a conventional manner into compositions using one or more carriers, optionally in combination with one or more viruses. Accordingly, the present application also includes a composition comprising one or more compounds of the application and a carrier. The present application also includes a composition comprising one or more compounds ofthe application, one or more viruses and a carrier. The compounds of the application are suitably formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present application further includes a pharmaceutical composition comprising one or more compounds of the application and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising one or more compounds of the application, one or more viruses and a pharmaceutically acceptable carrier. In embodiments of the application the pharmaceutical compositions are used in the treatment of any of the diseases, disorders or conditions described herein.

[0121] The compositions of the application are administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. For example, a composition of the application is formulated for administration by oral, ocular, inhalation, parenteral (including subcutaneous, intramuscular, and intravenous), intracerebroventricular, buccal, sublingual, insufflation, epidurally, intraosseous, intranasal, rectal, vaginal, patch, pump, minipump, topical or transdermal administration and the pharmaceutical compositions formulated accordingly. In some embodiments, administration is by means of a pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0122] Parenteral administration includes systemic delivery routes other than the gastrointestinal (Gl) tract and includes, for example intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (for example, by use of an aerosol), intrathecal, rectal and topical (including the use of a patch or other transdermal delivery device) modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0123] Intracerebroventricular administration includes direct delivery, such as injection or infusion, into the brain.

[0124] In some embodiments, a composition of the application is orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it is enclosed in hard or soft shell gelatin capsules, or it is compressed into tablets, or it is incorporated directly with the food of the diet. In some embodiments, the compound is incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions and suspensions and the like. In the case of tablets, carriers that are used include lactose, com starch, sodium citrate and salts of phosphoric acid. Pharmaceutically acceptable excipients include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate), or solvents (e.g. medium chain triglycerides, ethanol, water). In embodiments, the tablets are coated by methods well known in the art. In the case of tablets, capsules, caplets, pellets or granules for oral administration, pH sensitive enteric coatings, such as Eudragits™ designed to control the release of active ingredients are optionally used. Oral dosage forms also include modified release, for example immediate release and timed-release, formulations. Examples of modified-release formulations include, for example, sustained-release (SR), extended-release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet. Timed-release compositions are formulated, for example as liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc.Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. In some embodiments, liposomes are formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. For oral administration in a capsule form, useful carriers, solvents or diluents include lactose, medium chain triglycerides, ethanol and dried com starch.

[0125] In some embodiments, liquid preparations for oral administration take the form of, for example, solutions, syrups or suspensions, or they are suitably presented as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and / or emulsions are administered orally, the compound of the application is suitably suspended or dissolved in an oily phase that is combined with emulsifying and / or suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents are added. Such liquid preparations for oral administration are prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., medium chain triglycerides, almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0126] It is also possible to freeze-dry the compositions of the application and use the lyophilizates obtained, for example, for the preparation of products for injection.

[0127] In some embodiments, a composition of the application is administered parenterally. For example, solutions are prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. For parenteral administration, sterile solutions are usually prepared and the pH's of the solutions are suitablyadjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids are delivered, for example, by ocular delivery systems known to the art such as applicators or eye droppers. In some embodiments, such compositions include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzyl chromium chloride and the usual quantities of diluents or carriers. For pulmonary administration, diluents or carriers will be selected to be appropriate to allow the formation of an aerosol.

[0128] In some embodiments, a composition of the application is formulated for parenteral or intracerebroventricular administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection are, for example, presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In some embodiments, the compositions take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles and contain formulating agents such as suspending, stabilizing and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, the compositions of the application are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0129] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, drops, gels and powders. For intranasal administration or administration by inhalation, the compositions of the application are conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which, for example, take the formof a cartridge or refill for use with an atomising device. Alternatively, the sealed container is a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which is, for example, a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. Suitable propellants include but are not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or another suitable gas. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. In some embodiments, the pressurized container or nebulizer contains a solution or suspension of the active components. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator are, for example, formulated containing a powder mix of a compound of the application and a suitable powder base such as lactose or starch. The aerosol dosage forms can also take the form of a pump-atomizer.

[0130] Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein a composition of the application is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0131] Suppository forms of the compositions of the application are useful for vaginal, urethral and rectal administrations. Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature. The substances commonly used to create such vehicles include but are not limited to theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. See, for example: Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533 for further discussion of suppository dosage forms.

[0132] In some embodiments a compound of the application is coupled with soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide- phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, in some embodiments, a compound of the application is coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0133] The compositions of the application are particularly amenable to administration with the aid of nano-carrier systems, such as liposomes, micelles, nanoparticles, nano-emulsions, lipidic nano-systems and the like (see for example, Bhat, M. et al. Chem. and Phys, of Lipids, 2021 , 236, 105053). Accordingly, the present application includes a composition comprising one or more compounds of the application, optionally one or more viruses and one or more components of a nano-carrier system.

[0134] Depending on the mode of administration, a pharmaceutical composition will comprise from about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt%, of the active ingredient (compound(s) of the application and optionally one or more viruses) and from about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of a pharmaceutically acceptable carrier, all percentages by weight being based on the total composition.

[0135] In some embodiments, the P2Y2 agonist is administered or for use daily. In some embodiments, the P2Y2 agonist is administered or for use as an intraperitoneal injection. In some embodiments, the P2Y2 agonist is administered or for use as a daily intraperitoneal injection. In some embodiments, the P2Y2 agonist is administered or for use daily for one-week. In some embodiments, the P2Y2 agonist is administered or for use daily for two weeks.

[0136] In some embodiments the P2Y2 agonist is administered or for use at a dosage of about 10 mg / mL to about 30 mg / mL. Suitable dosages in humans can be estimated for example by calculating the human equivalent dose (HED) using for example the methods of Nair & Jacob (2016)

[0068] ,

[0137] Suitable use or administration schedules may include, without limitation, at least once a week, from about once in lifetime, one time per two weeks, three weeks or one month, about one time per week to about once daily. The length of the treatment period may depend on a variety of factors, such as the severity of the disease, disorder or condition, the age of the subject, the concentration and / or the activity of the P2Y2 agonist or composition described herein. It will also be appreciated that the effective dosage of the P2Y2 agonist or composition described herein used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration or use is required. For example, the P2Y2 agonist or composition described herein are administered to or for use in the subject in an amount and for duration sufficient to treat the subject.

[0138] In some embodiments, the compositions of the application comprise an additional therapeutic agent. Therefore, the present application also includes a pharmaceutical composition comprising one of more compounds of the application, and an additional therapeutic agent, and optionally one or more pharmaceutically acceptable excipients.

[0139] In some embodiments, the compounds or compositions of the application are administered as a combination therapy. In some embodiments, the combination therapy is a behavioral therapy, physical exercise, speech and language pathology therapy, ergotherapy (such as pediatric occupational therapy) or a psychotherapy.

[0140] The term co-administration means that at least two compounds or compositions are administered to the subject concurrently, such that effective amounts or concentrations of each of the two or more compounds may be found in the subject at a given time point. Although compounds according to the present disclosure may be co-administered to a subject at the same time, theterm embraces both administration of two or more agents at the same time or different times, provided that effective concentrations of all co-administered compounds or compositions are found in the subject at a given time. Coadministration also embraces the use or administration of two or more agents in a single administration or a series of administrations, and / or in the same dosage form or separate dosage forms.

[0141] In some embodiments, the subject is an infant or a child, wherein the infant or child has not been diagnosed with the neurodevelopmental disorder.

[0142] In some embodiments, the subject is an infant, a child or an adult, wherein the infant, child or adult has been diagnosed with the neurodevelopmental disorder.

[0143] Also provided are kits comprising a P2Y2 agonist or pharmaceutical composition as described herein, along with suitable container or packaging and / or instructions for the use thereof, such as for the treatment or prevention of a neurodevelopmental disorder in a subject.

[0144] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.Examples

[0145] The following non-limiting examples are illustrative of the present disclosure:Example 1 : Study Rationale16p11 .2 deletion syndrome

[0146] Both single gene and chromosomal mutations have been linked to ASD diagnosis, including deletions and duplications at chromosome 1 q21 .1 , 15q11-13, 22q11-13 and 16p11 .2, which is one of the most common genetic causes of ASD. 16p11.2 microduplications are also risk factors for schizophrenia, and children with intellectual disabilities. Approximately 2% of all patients with ASD possess a 16p11.2 microdeletion, which can be inherited or 75% of cases are de novo. Among the highly-conserved genes found in the human 16p11 .2 locus, several genes control critical features of brain development and neuronal function. For instance, Mvp or Yelp3 control cell cycle progression, cellular motility, proliferation, differentiation or senescence. In addition, Taok2 or Kctd13 regulate neuronal differentiation and maturation, while Doc2A and Prrt2 control synaptic transmission and neuronal excitability.

[0147] It is possible to study the 16p11.2 deletion syndrome at the preclinical level in animal models. In particular, the 16p11.2 locus is highly- conserved in the syntenic 7qF3 region in mice, thus making it possible to accurately replicate the deletion in a rodent model. Although no rodent model can perfectly encompass all aspects of ASD, the 16p11 .2 deletion mouse model is representative of a significant population of individuals with ASD. Furthermore, it displays several ASD-related characteristics which are fundamental for a diagnostic.Energy metabolism and ASD

[0148] The brain requires a healthy vasculature to properly develop and regulate its metabolism. While the neuronal underpinnings of autism spectrum disorders (ASD) have been studied extensively, recent findings have identified neurovascular alterations in ASD, including brain endothelial dysfunction. Described herein is a characterization of brain endothelial cells (ECs) in a mouse model of 16p11 .2 deletion ASD syndrome.

[0149] The brain is highly reliant on metabolic health, including uninterrupted delivery of nutrients and oxygen from its vasculature. While neurons are responsible for massive energy consumption, the brain’s highly specialized endothelium takes part in modulating fundamental functions suchas vascular permeability and tone, maintenance of brain homeostasis, and promotion of neurogenesis. Although the metabolic characteristics of brain endothelial cells (ECs) are not fully elucidated, ECs are known to meet their own metabolic demands predominantly through glycolysis and in part via oxidative phosphorylation to generate ATP. While these cells exist in oxygenrich environment, glycolytic metabolism enables fast ATP production for rapid proliferation and migration to efficiently vascularize and / or revascularize tissues and maintain brain energy balance.

[0150] Energy requirements during critical periods of brain development further increase the susceptibility of the brain to metabolic abnormalities. During development, brain ECs help guide and coordinate neurogenesis, while neuronal cues regulate vascularization of the central nervous system. As such, brain endothelial dysfunction can compromise metabolic support to neuronal cells, leading to the progression and / or onset of neurological conditions.

[0151] There is an association between brain endothelial dysfunction and ASD onset in a mouse model of the 16p11 .2 deletion syndrome, which mimics a mutation commonly found in human ASD. While 16p11 ,2-deficient mouse brain ECs were healthy at baseline, they were unable to respond to functional challenges both in vitro (i.e. , lack of angiogenic capacity in a Matrigel- based tube formation assay) and in vivo (e.g., lack of endothelial-dependent vasodilation). These results were consistent with delayed endothelial network growth at postnatal (P) day 14 and reduced neurovascular coupling at P50 in vivo, underscoring that 16p11 ,2-deficient ECs fail to execute intrinsic functions. As a result, endothelium-specific 16p11 .2 haploinsufficiency was sufficient to drive a subset of ASD-associated behaviors in mice.

[0152] Accordingly, it was demonstrated that the 16p11.2 deletion is associated with male-specific endothelial dysfunction characterized by defective angiogenesis and lack of endothelium-dependent vasodilation in vivo, coinciding with the emergence of ASD-related phenotypes (14).

[0153] It was confirmed that 16 p 11 ,2-deficient ECs isolated from 14-day old mice, a landmark of cerebrovascular maturation (28), do not establish a vascular network in vitro. This was evidenced by absence of endothelial tubesfollowing seeding of 16p11 ,2-deficent ECs in an extracellular matrix (data not shown).

[0154] As these cells are fundamental for regulating energy allocation to the brain, brain metabolism was assessed in adult 16p 11 ,2-deficient male mice. Reduced mitochondrial biogenesis was identified as a possible player in 16p11.2 deletion-induced endothelial dysfunctions and it was found that endothelium-specific 16p11 .2 haploinsufficiency led to compensatory shifts in adult brain metabolism (data not shown).

[0155] Accordingly, ATP can be used to show energy metabolism deficiency rescue.Example 2: Extracellular exposure to ATP rescues 16p11.2 deletion- associated endothelial dysfunction in vitro via P2-class purinergic receptor-mediated signaling.

[0156] In addition to being the main energy source for the cell, ATP also acts as a signaling molecule on brain ECs (37). Brain ECs are sensitive to ATP via expression of P2-class purinergic receptors (38). Recent work by Thakore et al. also demonstrated that intracellular ATP can exit brain ECs through pannexin-1 (Panxl ) channels to subsequently activate membrane P2 purinergic receptors on adjacent ECs to regulate vascular tone (39). It was thus tested whether extracellular ATP can also rescue the angiogenic capacity of 16p11 ,2-deficient ECs in vitro. ECs were treated with exogenous ATP at 1 pM, 10 pM and 100 pM from cell isolation for 7 days (6 days in culture and 24-hour tube formation assay). It was found that extracellular ATP supplementation was able to rescue capillary network formation, in a dose-dependent manner (100pM shown in FIG. 1A). These findings indicate that dysfunction of 16p11 ,2- deficient ECs might result from lack of ATP signaling, rather than lack of ATP itself. Of note, adenosine, a by-product of ATP hydrolysis, failed to influence endothelial network growth (FIG. 2).

[0157] Brain ECs are known to express various purinergic receptors that are activated by ATP, namely most ionotropic P2X isoforms and several metabotropic P2Y isoforms, all of which take part in maintaining vascularfunction (39, 40). Considering this, it was tested whether rescue of endothelial function by extracellular ATP is receptor-mediated. To test this, ECs were treated with a non-selective P2 purinergic antagonist (pyridoxalphosphate-6- azophenyl-2’,4’-disulfonic acid, PPADS) for 10 minutes prior to each ATP administration ( / .e., each culture medium change). Administration of 50pM of PPADs abolished the extracellular ATP-mediated rescue in 16p11.2df / +ECs (FIG. 1 B). At a concentration of 10pM or 100pM, PPADs was less efficient at preventing ATP-mediated rescue (data not shown). These results demonstrate that extracellular ATP acts on purinergic P2 receptors to rescue the angiogenic ability of 16p11.2-deficient brain ECs.Example 3: Activation of purinergic P2Y2 receptors restores the angiogenic function of 16p11.2-deficient endothelial cells.

[0158] Consulting the previously published RNAseq database (14), the expression of several purinergic receptors expressed in both WT and 16p11.2df / +brain ECs at P14 was detected. Messenger RNAs (mRNAs) encoding P2-class receptors were found at high expression levels in both genotypes, including P2rx1, P2rx4, P2rx7 and P2ry14 (FIG. 3). The mRNA encoding P2Y2 (P2ry2), a metabotropic receptor important for angiogenesis (40), was found at relatively low expression levels in WT mice, however its expression nearly doubled in 16p11 ,2-deficient brain ECs (FIG. 3). A slight, albeit non-significant, increase in P2Y2 protein levels was seen in 16p11.2- deficient ECs (FIG. 4). Considering this, P2Y2 was tested for its ability to modulate endothelial function in 16p11.2-deficient ECs. 16p11.2-deficient ECs were treated with a selective P2Y2 agonist, Diquafosol tetrasodium (DQS), a compound with efficacy in treating Dry Eye Syndrome post-cataract surgery (41 , 42) and clinically-approved in Japan and South Korea (Diquas®). Activation of P2Y2 receptors with 100pM of DQS rescued capillary network formation in 16p11.2df / +ECs (FIG. 5). These results suggest that P2Y2 receptor activation may represent a strategy to maintain brain EC function in the 16p11 .2 deletion ASD syndrome via metabolic rescue.Example 4: Ca2+-mediated responses to ATP are altered in 16p11.2- deficient brain ECs.

[0159] Purinergic receptor signaling relies on G protein-coupled receptors resulting in IP3-mediated Ca2+release from internal stores (P2Y class) (43) as well as Ca2+permeable ligand-gated ion channels (P2X class) (44) to establish its function. To gain further insight into ATP-mediated cellular responses, both steady-state intracellular Ca2+levels and Ca2+transients were measured. To assess the steady-state Ca2+level, Twitch-2B, a FRET-based ratiometric Ca2+indicator, was used. This analysis revealed similar steady-state intracellular Ca2+levels under baseline conditions, in non-treated WT and 16p11.2df / +ECs (FIG. 6). While ATP supplementation significantly increased intracellular steady-state Ca2+level in WT ECs as compared to WT ECs without ATP, no difference was observed between 16p11.2df / +ECs and 16p 11.2df / +with ATP supplementation (FIG. 6).

[0160] Next ECs derived from WT and 16p11.2df / +mice were loaded with Cal590-AM organic Ca2+indicator to record Ca2+transients. No difference in the frequency and amplitude of Ca2+transients were observed under baseline conditions, in non-treated WT and 16p11.2df / +ECs (FIG. 7). However, while supplementation with ATP decreased the frequency in both WT and 16p11.2df / +ECs, the effect was larger in 16p11.2df / +ECs (FIG. 7), revealing statistically significant differences in WT + ATP vs. 16p11.2df / ++ ATP conditions (FIG. 7). Altogether, this data suggests distinct Ca2+signatures in WT and 16p11.2df / +ECs in response to extracellular ATP supplementation.Example 5: ATP injection restored Marble Burying behavioral trait in 16p11.2df / +mice

[0161] Autism is a neurodevelopment disorder primarily characterized by repetitive / stereotypic behaviors. A marble burying test was performed as a proxy for repetitive behavior assessment using 16p11.2df / +mice (ASD mice). ASD mice displayed an increased number of buried marbles compared to non- ASD mice (FIG. 8), suggesting that these mice have increased repetitive behaviors as found in the human condition. To test whether ATP rescues repetitive behaviors in ASD mice, the mice were treated with 125mg / kg of ATP systemically via intraperitoneal injection 30 minutes prior behavioral testing. Dose and treatment regimen were based on previous reports in mice showingefficacy at increasing ATP in the brain and ameliorating behavior impairments [69, 70], The marble burying behavioral assay consisted of a 30-min trial per mouse. For each trial, a standard polycarbonate cage (26 x 48 x 20 cm) was filled with 5-cm-thick of bedding. A total of 20 marbles were evenly distributed (5 rows of 4 marbles) in each cage. Each mouse was placed at the bottom-left corner of the cage and left to explore. During the test, the cage was covered by transparent Plexiglas. Once the trial was completed, the number of marbles that were either fully buried or at least two-thirds covered by bedding were counted as buried. Following ATP injection, ASD mice buried less marbles compared to saline treated ASD mice suggesting that ATP injection improves autism-related repetitive behaviors.Example 6: Discussion

[0162] As ATP is required for EC function, it was hypothesized to rescue 16p11 ,2-deficient EC associated phenotypes by increasing ATP bioavailability intra- or extra-cellularly. Strikingly, both intra- and extra-cellular delivery of ATP rescues 16p11 ,2-deficient EC function, suggesting that a lack of ATP might be causing deficits. However, extracellular ATP has emerged as a signaling regulator in several cellular processes including proliferation, angiogenesis, and vessel homeostasis. Brain ECs release ATP in response to various stimuli, triggering signaling events via ligand gated (P2X) and / or G-protein-coupled (P2Y) purinergic receptors (52, 53). In line with ATP regulating key vascular features via activation of purinergic receptors, provided herein is novel evidence that ATP-mediated rescue of 16p11.2-related dysfunction relies on P2-class receptors expressed by ECs. Indeed, non-selective pharmacological blockade of P2 receptors abolished the extracellular ATP-mediated rescue. The fact that 16p11 ,2-deficient EC function was also reinstated following intracellular ATP delivery brings into question whether this effect was mediated in similar manner as extracellular ATP. It has recently been established that ATP can exit brain ECs through pannexin-1 channels to activate P2 receptors on adjacent ECs (39). Inhibition of P2-class receptors with PPADs prior to intracellular delivery of ATP prevented ATP-mediated rescue of 16p11 ,2-deficient EC function (data not shown). These findings demonstrate that ATP acts on membrane-bound P2receptors in both treatment conditions (extra- and intra-cellular ATP), and pannexin-1 channel activity may be involved in this response.

[0163] Disclosed herein is the involvement of P2Y2 receptor activation as a mechanism to rescue 16p11.2-related endothelial dysfunction. P2Y2 receptors have been associated with vascular function (40). Mice with endothelial P2Y2-knockout displayed dysregulated vasodilatory responses during increased blood flow (54). Furthermore, P2Y2 receptors have a prominent role in angiogenesis, as their activation promotes endothelial sprouting and vascular tube formation. Inhibition of P2Y2 with a selective antagonist reduced tube length in human umbilical vein endothelial cells (HUVECs) (40). These findings emphasize that lack of P2Y2 activation in 16p 11 ,2-deficient ECs due to decreased ATP availability might be sufficient to drive functional changes.

[0164] The purinergic system has been linked to ASD; however, anti- purinergic therapies improved autism-like behavior (47, 55). In certain instances, elevated extracellular ATP in the blood of ASD patients have been associated with metabolic changes and suggested to activate the cellular danger response (CDR), leading to detrimental stress responses ( / .e. changes in mitochondrial function, oxidative stress and innate immune activation) (47). As such, higher extracellular ATP may negatively impact EC function. These findings show that an ATP delivery threshold should be taken into consideration when designing purinergic signaling therapies. Furthermore, that functional rescue was specifically conferred by ATP and not by adenosine, a by-product of ATP hydrolysis, acting on P1 -class receptors was confirmed herein. Indeed, administration of adenosine to 16 p 11 ,2-deficient cultured ECs failed to promote angiogenic ability of these cells. Of note, adenosine was recently associated with improved social novelty behaviors in adult female 16p11.2df / +mice (56). It thus possible that the beneficial effects of adenosine in female mice behavior are independent of vascular features.

[0165] P2-class purinergic receptor activation via ATP can modulate steady-state intracellular Ca2+levels as well as Ca2+transients. Marked differences in Ca2+responses in 16p11 ,2-deficient ECs, as compared to WTECs, in response to extracellular ATP was observed. In contrast to WT ECs, supplementation with ATP did not affect the steady-state intracellular Ca2+level in 16p11 ,2-deficient ECs. Furthermore, the decrease in frequency of Ca2+transients was more pronounced in 16p11 ,2-deficient ECs, as compared to WT ECs. While it is yet unclear how these distinct Ca2+responses to ATP may underlie the rescue effects observed in 16p11 ,2-deficient ECs, this data hints on Ca2+as a possible signaling molecule mechanistically linking purinergic receptor activation to intracellular pathways leading to restoration of angiogenic ability of 16p 11 ,2-deficient ECs. Notably, Pannexin-1 channels are activated by increases in intracellular calcium leading to the release of ATP from the cell and so ATP acting on P2 receptors (39). These findings emphasize the possible role of Ca2+signaling and pannexin-1 in mediating the intracellular ATP rescue response in 16p11 ,2-deficient ECs.Example 7: Materials and MethodsAnimals.

[0166] Male 16p11.2df / +(Jackson laboratory, stock #013128; mixed B6 / 129 background) were crossed with WT females of the same background (Jackson laboratory, stock #101043) to obtain hemizygous 16p 11.2df / +offspring as well as WT littermates. To improve 16p11.2^ pup survival, breeding cages were supplemented with breeding chow (#2019, Envigo Teklad) and DietGel™ (#76A, ClearH2O) up to weaning age. All experiments were conducted using post-natal 14-day old (P14) mice.Primary mouse brain EC isolation.

[0167] All mice were euthanized by cervical dislocation. The cerebral cortex was dissected in cold HBSS without calcium and magnesium using autoclaved tools submerged in 100% ethanol for 30 minutes prior dissections. The cortex was minced in 2-3 mm pieces and dissociated in Neural Tissue Dissociation Kit P compounds (Miltenyi Biotec, 130-092-628) to obtain a cell suspension. Cell isolation procedures were completed according to the manufacturer’s instructions. The cell suspension was incubated with CD31 - coated magnetic microbeads (Miltenyi Biotec, 130-097-418) and placed on amagnetic MACs separator to isolate endothelial cells (ECs). ECs isolated from one mouse were seeded in a plate coated with attachment factor protein 1X (ThermoFisher Scientific, S006100). For a pure EC population, ECs were cultured in an EC specific medium (Lonza, CC-3202) which was replaced 48 hours post-seeding and every 48 hours until appropriate level of confluency was reached for subsequent processing (40-60 % for immunocytochemistry; 90-100% for tube formation).Immunocytochemistry.

[0168] Primary ECs were cultured until 40-60% confluence (3 days postisolation) on glass cover-slip cover glass for microscopy analysis. Before staining, ECs were washed twice in pre-warmed PBS and fixed for 10 min in cold 4% PFA. Fixed cells were permeabilized in a blocking solution containing 10% donkey serum, 0.1 % PBT and 0.5% cold water fish skin gelatin. The primary antibodies anti-P2Y2 (1 :500, ab272891 Abeam) and anti-CD31 (1 :200, BD Pharmingen) were diluted in blocking solution and incubated for 2 hours at room temperature. Cells were then rinsed with 0.2% PBST. Alexa Fluor™ species-specific secondary antibodies were diluted in blocking solution at 1 :300 and incubated with cells for 60 minutes. Cells were washed with 0.2% PBST then 0.1 M PB. Glass cover-slips, with stained ECs, were mounted on slides using Fluoromount G and imaged (40x objective) with a Zeiss Axio Imager M2 microscope equipped with a digital camera and ApoTome™.2 module. Images consisted of 4uM thick z-stacks (0.250uM / slice).Western blots.

[0169] Protein extraction: For endothelial proteins, primary brain endothelial cells were isolated and cultured as previously described. To lyse cells, 50uL of RIPA buffer (NaCI 150mM, Sodium Deoxycholate 12mM, SDS 3.5mM, Tris 50mM, Triton™ X-100 1 %v / v, pH 8.0) with protease and phosphatase inhibitors was added to each well and left on ice for 30 min. The supernatant was transferred to a 1 ,5m L tube and then centrifuged at 13,000rpm for 10 min. The protein concentration of the collected supernatant was quantified using Pierce BCA Protein assay (kit #23227, ThermoScientific, Illinois, USA).

[0170] Immunoblotting: Twenty five (25) pg of protein were loaded into the wells of SDS-acrylamide gels (GX Stain-free FastCast 12% Bio-rad™ gels, Bio-Rad, ON, CAN, #1610184) and separated by a constant current of 100 V for 120 min in running buffer (SDS 35mM, Tris 250mM, Glycine 1865mM). After UV activation of the gels, the proteins were then transferred to a nitrocellulose membrane in ice-cold transfer buffer (Tris 48m M, Glycine 38mM, methanol 20% v / v) for 30 min at 100 V. After the transfer, the nitrocellulose membranes and gels were imaged to quantify the total protein transferred. The membranes were then blocked with 5% skim milk in 1 M PBS for 1 h at room temperature and incubated with primary antibodies raised against P2Y2 in TBST (Tris 50mM, NaCI 150mM, Tween™ 20 1 % v / v) overnight at 4°C. The membranes were then washed with TBST (3 x 10 min) and incubated at room temperature for 1 h with the secondary antibody (1 / 10,000, Fisher Scientific, #PR-W4011 ), also diluted in TBST. After a final wash (TBST, 3 x 10 min), the proteins were detected by enhanced chemiluminescence (ECL; 1 / 4, Fisher Scientific, #34579) and imaged with the Odyssey Imaging system.

[0171] Extracellular ATP supplementation: Primary ECs were treated with 1 pM, 1 OpM or 1 OOpM of ATP (Sigma Aldrich, A7699) following cell isolation and once cells reached 90-100% confluency, underwent an in vitro networkformation assay. ATP was directly added to EC specific culture media (Lonza, CC-3202) and was replaced every 48hrs until required confluency was reached.

[0172] Adenosine supplementation: Primary ECs were treated with 100pM of adenosine (Sigma Aldrich, A4036) or NF OH (vehicle, Sigma Aldrich, 09859) following cell isolation, once cells reached 90-100% confluency, underwent an in vitro network-formation assay. Adenosine was prepared according to the manufacturers. Briefly, adenosine was dissolved in 1 M NH4OH using heat (70°C) for 5 mins and maintained at room temperature protected from light. Adenosine was directly added to EC specific culture media (Lonza, CC-3202) and was replaced every 48hrs until required confluency was reached.

[0173] Non-selective P2 antagonist treatment: Primary ECs were treated with 10pM, 50pM or 100pM of pyridoxalphosphate-6-azophenyl-2',4'- disulfonic acid (PPADs) tetrasodium salt (Tocris, 0625) for 10 minutes prior toaddition of 100pM of ATP and replaced every 48hours until cells reached 90- 100% confluency. Once confluency was reached, ECs underwent an in vitro network-formation assay.

[0174] Non-selective P2 antagonist (PPADs) and ATP-LNP treatment: Primary ECs were treated with 50pM of PPADs for 10 minutes prior administration of 1 mM of ATP-LNPs. Media was replaced every 48 hours until cells reached 90-100% confluency. Once confluency was reached, ECs underwent an in vitro network-formation assay.

[0175] P2Y2 agonist: Primary endothelial cells were treated with 100pM of Diquafosol tetrasodium (Sigma Aldrich, SML3058) following cell isolation, once cells reached 90-100% confluency, cells underwent an in vitro networkformation assay. Diquafosol tetrasodium was directly added to EC specific culture media (Lonza, CC-3202) and was replaced every 48hrs until required confluency was reached.In vitro network-formation assay.

[0176] EC network-formation assays were performed using growth- factor-reduced Matrigel (BD Bioscience, cat. no. C354230). Growth-factor- reduced condition included: EGF < 0.5 ng ml— 1 , PDGF < 5 pg ml— 1 , IGF1 = 5 ng ml-1 and TGF-p = 1.7 ng ml-1. In brief, each well of a 96-well plate was coated with 50 pl of Matrigel and incubated at 37 °C for 30 min to promote polymerization. ECs were collected with TryplE (Gibco, 12604013) and counted. A total of 2 x 104cells were seeded in each well with 150 pl EC specific media (Lonza, CC-3202) or EC media supplemented with ATP. TIFF images of capillary-like networks were captured using a Zeiss Axio Image M2 microscope equipped with a digital camera at 4, 8, and 24h post-seeding (mouse ECs). Images were processed using the Angiogenesis function of Imaged (63).Steady-state cytosolic Ca2+level.

[0177] To assess the cytosolic levels of intracellular Ca2+in endothelial cells we used a ratiometric FRET-based Ca2+indicator Twitch-2B, driven by the CMV promoter. Primary brain ECs were isolated and seeded in a 12-well plate on 12mm glass coverslips and maintained in an EC specific medium (Lonza,CC-3202) with or without 100uM of ATP at 37 °C and 5% CO2 atmosphere, replacing the growth medium every 48 hours. Transfection with CMV-Twitch- 2B was performed when cell cultures were at -80% confluency. On the day of transfection, Lipofectamine / DNA complexes were prepared in 10Oul of opti- MEM (Invitrogen) for each well of 12-well plates by mixing 1.5 pg of plasmid with 3 pl of Lipofectamine LTX and 1 ul of Plus transfection reagent according to manufacturer’s instructions (Invitrogen) and added dropwise to each well (100 pl / well). Cells were incubated at 37 °C in a CO2 incubatorfor24 h, followed by fixation in 4% PFA. The images were acquired using the CBIA Core Facility confocal microscope (LSM880 AxioObserver Z1 ; Zeiss) at the University of Ottawa with a 20x air objective (Plan-Apochromat 20x / NA 0.8; Zeiss). Images were acquired using a 458 nm laser to excite CFP, and the fluorescence signals were collected at the CFP and YFP emission wavelengths (461 -519 and 522- 578 nm bandwidth, respectively). The fluorescence signals from the regions of interest (ROIs) were calculated as a ratio of YFP to CFP emissions.Analysis of Ca2+transients.

[0178] To assess spontaneous Ca2+activity in primary mouse cerebral cortex endothelial cells we used Cal-590™ AM, a membrane-permeable organic dye that allows detection of intracellular Ca2+fluctuation (64). ECs were cultured in an EC specific medium (Lonza, CC-3202) with or without 100uM of ATP at 37 °C and 5% CO2. EC medium was replaced every second day up until 80% confluence was reached (~6 days). On the day of experiment once ECs were 80% confluent, cells were treated with 2 pM of Cal-590™ AM (AAT Bioquest, CA, USA) and were further incubated for about 1 hour in a humidified incubator at 37 °C and 5% CO2. After incubation, the culture dish was transferred on the stage of fluorescent microscope (DeltaVision Elite Olympus XI-71) of the CBIA Core Facility at the University of Ottawa. The microscope was equipped with an incubation chamber allowing to maintain the temperature at 37°C and CO2 at 5%. Images were acquired every 10 s for 5 min using a 40x oil objective, (UPLFLN 40x / NA 1.3; Olympus) and the recorded videos were analyzed using a custom-written script (65) in MATLAB (MathWorks). Briefly, regions of interests (ROI)s were traced around the entire cell and thefluorescence intensity in each ROI and for each time point was extracted. Ca2+activity was calculated as relative changes in the percentage of AF / F = (F-Fback) / Fback, where F is the Cal-590™ AM intensity in the ROI and Fback is the background signal taken in a “cell-free” region of the imaging field. Analysis of spontaneous Ca2+activity was performed using a multiple threshold algorithm(66, 67). Briefly, mean standard deviation (SD) of Ca2+trace for each cell was first calculated, and all peaks with an amplitude greater than 1 .5 times the SD were measured. These peaks were then removed from the Ca2+traces, and the mean SD of Ca2+trace was re-calculated to depict events greater than 1 .5 times the new SD. This procedure allowed us to depict all Ca2+events regardless of their amplitudes. Ca2+frequency was calculated as the number of peaks over video duration. The statistical analysis was performed in a total number of n videos collected from n wells for n cells in total in Cal-590 treated cells for 4 distinct conditions.Statistical analyses

[0179] No statistical methods were used to predetermine sample sizes. Sample sizes were similar to those reported in previous publications (14, 15). All sample numbers in this study are in line with well-accepted standards from the literature for each method. All data presented in this work were obtained from experimental replicates (e.g. multiple animal cohorts from different litters, at least three experimental repeats for each assay). All attempts of replication were successful. All data analyses were conducted blinded to the genotype and experimental condition. Groups were reassembled following completion of the data analysis according to genotype and experimental condition. Randomization of individual samples was performed by numbering. Statistical tests were performed using GraphPad Prism 10.0 software. Data distribution was assumed to be normal, but this was not formally tested. A Mann-Whitney t / -test or t test (appropriate for small sample sizes) was used for two-group comparisons between WT and 16p11 ,2-deficient ECs isolated from mice for metrics including mitochondrial density and fragmentation, mitochondrial function, Western blot quantifications. A two-way ANOVA (e.g. ‘genotype x condition’) and Sidak’s multicomparisons post-hoc test was used for in vitronetwork formation assay. A one-way ANOVA and Tukey’s multicomparison post-hoc test was used for steady state Ca2+readouts and Ca2+transients, behavioral assays as well as ex vivo vascular reactivity assessment. P<0.05 was considered significant.Example 8: P2Y2 receptors are localized at the endothelial barrier in vivo

[0180] Electron microscopy with immunogold labeling confirmed that P2Y2 receptors were localized at the luminal membrane. This method allows for precise localization, at subcellular resolution, of proteins labeled with an antibody coupled to gold nanoparticles. Using this, P2Y2 was detected at the brain endothelial barrier, including at the luminal endothelial membrane (FIG. 9). This confirmed that ASD patients treated with DQS via an administration that results in DQS being delivered through the blood stream will successfully stimulate the P2Y2 receptor in the brain endothelium, thereby treating ASD. An advantage in treating endothelial cell function is that these cells line the vessel wall and are the main line of defence of the blood brain barrier. As such, the drugs do not need to cross the blood brain barrier for an effect.

[0181] Methods:

[0182] P14 16 p 11 .2df / + and WT mice were anesthetized using a cocktail of xylazine (10 mg / kg, IP) and ketamine (100 mg / kg IP). The brain was extracted and initially fixed by immersion in fixative solution (0.5% glutaraldehyde, 4% paraformaldehyde, and 0.1 M sodium-cacodylate) for 1 h at room temperature (RT). Tissue was then transferred to fresh fixative solution and incubated for 5 hrs at RT and then rinsed overnight with 0.1 M sodiumcacodylate at 4°C. Coronal vibratome free-floating sections of 50 pm were collected and immersed in 0.1 % sodium borohydride / PBS for 30 min at room temperature. Samples were thoroughly rinsed in PBS and blocked with 10% goat serum / 0.5% gelatin / PBST (0.01 % Triton X-100), for 2 h at room temperature. Sections were then incubated with rabbit anti-P2Y2 (1 :200, Abeam: AB272891) diluted in blocking solution (without Triton X-100) overnight at room temperature. After thoroughly rinsing sections in PBS, sections were then incubated with gold-labeled goat anti-rabbit IgG (1 :50, Nanoprobes: 2005- 1 ML), diluted in blocking solution (without Triton X-100), overnight at roomtemperature. Sections were washed with PBS followed by 3% sodium acetate rinse (3 x 5 min). Sections were subjected to silver-enhancement, following manufacturer’s instructions (Nanoprobes: 2012-45ML). Sections were then thoroughly rinsed in 3% sodium acetate and extensively washed in 0.1 M PB pH 7.4 prior to post-fixation. Following staining, sections were post-fixed in a solution containing 1% osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated by increasing ethanol concentrations followed by propylene oxide. Afterward, post-fixed sections were embedded in Durcupan ACM Epoxy resin (Electron Microscopy Sciences: 14040). Ultrathin sections (80 nm) were then cut from the block surface, using a Leica EM UC6 ultramicrotome, and collected on copper grids. Samples were examined under JEM-1400Flash transmission electron microscope operating at 80kV and equipped with a 16MP digital camera (GATAN One View).Example 9: Diquafosol treatment rescues or improves behavior defects in 16p11.2 ASD mouse modelBehavioral assessment

[0183] Mice harboring the 16p11.2 deletion (ASD mouse model) received an intraperitoneal (i.p.) injection of Diquas® (available ophthalmic solution of Diquafosol tetrasodium (DOS)), 20mg / mL (volume injected ~1 % of animal weight) daily for 1 -week prior to testing. Dosage may range between 10- 30 mg / ml. The week-long treatment resembled the prolonged treatment recommended for this solution in the clinic for treating patients with Dry Eye Syndrome. On the 8th day, mice were assessed for selected behavior via assays directly associated with ASD-related behaviors including repetitive movement, hyperactivity and recognition memory (cognition).

[0184] Before behavioral testing, all animals were left to habituate to an inverted light cycle housing room for 7 days. Mice were then handled and injected intraperitoneally (i.p.) with 5 mg / ml of Diquafosol tetrasodium (DOS, Sigma Aldrich cat # SML305) or 1X sterile PBS as control at a volume of 1 % of body weight once a day for 1 week prior to testing and until completion of behavior assessment (FIG. 10). Behavioral tests were completed between 9:00 and 17:00 under dim red light. On the testing day, before the task, animals werehabituated to the testing room for 60 minutes. Behavior tests were performed with all mice in the following order: marble burying test 1 (1 day), novel-object recognition (2 days), marble burying test 2 (1 day).Marble Burying

[0185] As described above in Example 5, the marble burying test assesses repetitive behaviors which are increased in ASD. DQS treatment decreased marble burying behaviors in the ASD mouse model improving repetitive phenotype identified in this ASD model. A marble burying test was performed as a proxy for repetitive behavior assessment using ASD mice 1 week and 2 weeks after DQS injection. ASD mice injected with PBS (saline) displayed an increased number of buried marbles compared to non-ASD mice (FIGs. 11A and 11C), suggesting that these mice have increased repetitive behaviors as found in the human condition. Following DQS injections, ASD mice buried less marbles compared to PBS (vehicle) treated ASD mice suggesting that DQS injection improves autism-related repetitive behaviors which was also maintained following 2 weeks of injection.

[0186] Beam break assay

[0187] The beam break assay is a quantification of activity, where the number of beam breaks represents a measure of hyperactivity. The ASD mice treated with DQS show reduced hyperactivity (decreased beam breaks) compared to vehicle (saline) treated ASD mice.

[0188] Novel Object Recognition

[0189] The Novel object recognition test is for assessment of recognition memory (cognition). DQS treated mice showed an increased ability to distinguish familiar and novel objects suggestive of improved recognition memory compared to vehicle (saline) treated ASD mice.

[0190] A 2-day novel-object recognition test was performed. On day one, each animal was habituated to an empty open-field arena (45x45x45 cm) for 30 minutes and returned to their home cage. On the experimental day, mice were habituated for a second time to the open field for 10 minutes. Following habituation, each mouse was removed from the open field and placed in a cleanholding cage for 2 minutes. Two identical objects (red cup or white funnel) were placed in the arena and the mouse returned to the arena for a 10-minute familiarization period. The mouse was removed from the arena and placed in a clean holding cage for 1 hour. After the one hour, the object-recognition test consisted of one cleaned familiar object and one cleaned novel object (red cup or white funnel switched). The mouse was returned to the arena for a 5-minute recognition period. All interactions with the objects were recorded using Ethovision 17.5 XT software (Noldus). Object recognition was scored as the time during which the nose of the animal was located within 2 cm of the object. A discrimination index was calculated as follows: [time spent interacting with novel object / (time spent interacting with novel object + time spent interacting with familiar object)].

[0191] ASD mice injected with PBS (saline) displayed an impaired recognition memory compared to non-ASD mice. Following DOS injections, ASD mice had improved recognition memory compared to PBS (vehicle) treated ASD mice suggesting that DQS injection improves autism-related impaired recognition memory (cognition) (FIG. 11 B).Example 10: Cerebral blood flow dynamics in Diquafosol treated mice

[0192] As the endothelium controls cerebral blood flow, measuring cerebral blood flow dynamics in the ASD mouse model is an indicator of successful activation of the P2Y2 receptor.

[0193] The 16p11.2df / + mouse model is associated with dysregulated cerebral blood flow dynamics, revealing elevated baseline cerebral blood flow in the cerebral cortex as well as altered activity-dependent cerebral blood flow regulation in these ASD mice. Altered cerebral blood flow dynamics following exposure to vasomodulators were shown to be attributed to endothelial cell dysfunction in these ASD mice and accordingly, ruling out the involvement of vascular smooth muscle cells in mediating these deficits. Both endothelial cells and vascular smooth muscle cells are mediators of vascular tone and in turn cerebral blood flow. As endothelial dysfunction contributes to altered cerebral blood flow dynamics in ASD mice, activating P2Y2 receptors in endothelial cellswill restore endothelial cell function in ASD mice, and thus will improve and / or restore cerebral blood flow regulation. Cerebral blood flow dynamics measurement will confirm activation of P2Y2 receptors reinstate cerebral blood flow dynamics in ASD mice (reinstate = returning to control, non-ASD, mouse dynamics).

[0194] Four techniques were used to assess this:

[0195] Technique 1- Activity dependent regulation of cerebral blood flow (neurovascular coupling) was assessed using laser doppler flowmetry (LDF) coupled with whisker stimulation (increased neuronal activity). In this assay, DQS treatment improved hemodynamic responses by normalizing response kinetics and amplitude of response compared to vehicle (saline) treated ASD mice.

[0196] Mice were injected intraperitoneally (i.p.) with 5 mg / ml of Diquafosol tetrasodium (DQS, Sigma Aldrich cat # SML305) or 1X sterile PBS as control at a volume of 1 % of body weight once a day for 1 week prior to testing and on the day of testing. LDF measurements (Transonic Systems) of evoked cerebral blood flow (CBF) in response to sensory stimulation were carried out in anesthetized mice (ketamine given at 100mg per kg i.p.) fixed in a stereotaxic frame. In brief, CBF was recorded over the contralateral somatosensory cortex before, during and after unilateral stimulation of the right whiskers. Recordings from four to six stimulations (every 60s) were acquired and averaged for each mouse. Quantifications of cerebral blood flow increase was measured. ASD mice treated with DQS demonstrated an improvement in evoked cerebral flow responses (FIG. 12).

[0197] Technique 2- Resting baseline cerebral blood flow in large superficial arteries was assessed using laser speckle imaging. The ASD mouse model did not have an impairment in superficial arterial brain blood supply (FIG. 13B). Further, DQS treatment did not impact general brain blood supply in these large superficial arteries in this ASD mouse model (FIG. 13B).

[0198] Mice were injected intraperitoneally (i.p.) with 5mg / ml of Diquafosol tetrasodium (DQS, Sigma Aldrich cat # SML305) or 1X sterile PBSas control at a volume of 1 % of body weight once a day for 2 weeks prior to testing and on the day of testing. Laser speckle imaging (LSI) measurements (RWD Life Science) of cerebral blood flow (CBF) were carried out in anesthetized mice (ketamine given at 100mg per kg i.p.) fixed in a stereotaxic frame. In brief, CBF was recorded over the somatosensory cortex. Quantification of the brain blood supply was measured. This showed that DQS does not affect general blood supply to the brain (FIG. 13B).

[0199] Technique 3 - (vascular permeability, cadaverine leakage assay): Mice were injected intraperitoneally (i.p.) with 5 mg / ml of Diquafosol tetrasodium (DQS, Sigma Aldrich cat # SML305) or 1X sterile PBS as control at a volume of 1 % of body weight once a day for 2 weeks prior to testing and on the day of testing. Vascular permeability was assessed using a cadaverine leakage assay. Mice were anesthetized (ketamine given at 100mg per kg i.p.) and retro-orbitally injected with 200pg / 25g of cadaverine-Alexa Fluor 555 (Invitrogen, A-30677). Cadaverine-Alexa Fluor 555 was left to circulate for 2 hours post-injection. Mice were then perfused with 50m L of cold 1X PBS and brain cortices were dissected. Brain cortices were weighed and homogenized into 1 mL of 1 % triton X-100 buffer in 1X PBS. Homogenates were centrifuged for 20 minutes at 4°C at maximum speed. A total of 200pL of the supernatant was transferred for each sample to a 96-well black plate in duplicates. Fluorescence was read using appropriate filters (Excitation: 540 / 25 nm, Emission: 590 / 20 nm). This vascular permeability assessment showed that DQS does not affect blood-brain barrier leakage in ASD mice (FIG. 13A).

[0200] Technique 4- Vascular reactivity was assessed using pressure myography in cortical parenchymal arterioles in response to vasodilatory adenosine. DQS treatment improved the impaired vessel relaxation by adenosine in this ASD mouse model compared to vehicle (artificial cerebrospinal fluid, aCSF) treated ASD mice (FIG. 14).

[0201] Following euthanasia and decapitation, mouse brains were removed and placed into cold (4°C) MOPS-buffered saline (composition: 135 mM NaCI, 5 mM KCI, 1 mM KH2PO4, 1 mM MgSQ4, 2.5 mM CaCI2, 5 mM glucose, 3 mM MOPS, 0.02 mM EDTA, 2 mM pyruvate, 10 mg / mL bovineserum albumin, pH 7.3). Cortical parenchymal arterioles (PAs) originating from the middle cerebral artery (MCA) were dissected free of surrounding tissue. In an organ chamber (University of Colorado IDEA Core), PAs were then cannulated on borosilicate glass micropipettes with one end occluded, tied at both ends, and pressurized at 40mmHg using an arteriography system (Living Systems Instrumentation, Inc., St. Albans, VT, USA). PAs were perfused (4 mL / min) with prewarmed (36.5°C ± 1 °C) and gassed (5% CO2, 20 % 02, 75 % N2) artificial cerebrospinal fluid (aCSF; 125 mM NaCI, 3 mM KCI, 26 mM NaHCOa, 1 .25 mM NaH2PO4, 1 mM MgCl2, 4 mM glucose, 2 mM CaCl2, pH 7.3 at room temperature with gas aeration) for at least 30 min to allow development of myogenic tone. PA lumen diameter was continuously measured using a CCD camera and edge-detection software (lonOptix, Westwood, MA, USA). Passive PA lumen diameter was obtained in aCSF free of Ca2+(0 mM [Ca2+]0with 5 mM EGTA). PA tone was calculated with the following equation: [(passive diameter - active diameter) / (passive diameter)] x 100. Only viable PAs, defined as those that developed pressure-induced myogenic tone greater than 15% at 40 mmHg, were used in the experiments. Diameter changes of PAs, in response to bath perfusion of drug, were calculated as a percent of the maximum dilation using the following equation [(drug-induced diameter - active diameter) / (passive diameter - active diameter)]. The percent of maximum dilation in response to 10 pM bath perfusion of adenosine was calculated before and after 30-minute bath perfusion of 100 pM Diquafosol tetrasodium (DQS) in aCSF.Example 11 : Denufosol treatment ‘

[0202] Brain endothelial cells (ECs) isolated from the ASD mouse model were assessed for their ability to generate a network of tubes (branches) in culture. Denufosol tetrasodium treatment increased tube formation (increased angiogenic capacity) of ECs compared to ASD non-treated ECs (FIG. 15).

[0203] Primary ECs were treated with 10 pM of DNS (Denufosol Tetrasodium, Biosynth, Cat #: ND45968) following cell isolation, throughout culturing and during the tube formation assay. DNS was directly added to EC specific culture media (Lonza, CC-3202) and was replaced every 48hrs until required confluency was reached (90-100%).

[0204] EC network-formation assays were performed using growth- factor-reduced Matrigel (BD Bioscience, cat. no. C354230). Growth-factor- reduced condition included: EGF < 0.5 ng ml— 1 , PDGF < 5 pg ml— 1 , IGF1 = 5 ng ml-1 and TGF-p = 1.7 ng ml-1. In brief, each well of a 96-well plate was coated with 50 pL of Matrigel and incubated at 37 °C for 30 min to promote polymerization. ECs were collected with TryplE (Gibco, 12604013) and counted. A total of 2 x 104cells were seeded in each well with 150 pL EC specific media (Lonza, CC-3202) or EC media supplemented with DNS. TIFF images of capillary-like networks were captured using a Zeiss Axio Image M2 microscope equipped with a digital camera at 4, 8, and 24h post-seeding (mouse ECs). Images were processed using the Angiogenesis function of Imaged.

[0205] ECs from ASD mice demonstrated impaired tube formation (FIG. 15, middle), which was improved by DNS treatment (FIG. 15, right).Example 12: Safety of P2Y2 AgonistsDiquafosol tetrasodium (DQS, Sigma Aldrich cat # SML305) was injected daily at 5mg / ml with an injection volume of 1 % of body weight for a total of 14 days. Animal survival (FIG. 13C), and body weight (FIG. 13D) were not affected, demonstrating safety and tolerability of DQS treatment.

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Claims

CLAIMS:

1. A method of treating or preventing a neurodevelopmental disorder comprising administration of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof to a subject in need thereof.

2. The method of claim 1 , wherein the neurodevelopmental disorder is Autism Spectrum Disorders (ASD).

3. The method of claim 2, wherein the ASD comprise a mutation in a gene comprising SHANK1, SHANK3, SYNGAP1, SYNAPSIN, NCAM, MY016, MECP2, ADNP, FMR1, KCTD13, ASTN2, PTEN, CHD2, CHD4, CHD8, NLGN3, NLGN4X, CNTN4, CNTNAP2, and / or NRXN.

4. The method of claim 2, wherein the ASD comprise a mutation in a gene selected from: SHANK3, SYNGAP1, SYNAPSIN, NCAM, MY016, MECP2, ADNP, FMR1, and KCTD13.

5. The method of any one of claims 2 to 4, wherein the ASD comprise a genomic copy number variation (CNV) in 16p11 .2.

6. The method of any one of claims 1 to 5, wherein the P2Y2 agonist is Diquafosol, Denufosol, MRS2698, MRS2768, PSB1114, ATP, ATP derivatives, UTP, UTP derivatives, 2-((ethyl(4-fluorobenzyl)amino)methyl)-7,8- dimethylquinolin-4(1 / 7)-one , or a pharmaceutically acceptable salt, solvate or prodrug thereof.

7. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is Diquafosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

8. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is Denufosol or a pharmaceutically acceptable salt, solvate or prodrug thereof.

9. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is MRS2698 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

10. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is MRS2768 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

11. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is adenosine triphosphate (ATP), a derivative thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

12. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is uridine triphosphate (UTP), a derivative thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

13. The method of any one of claims 1 to 6, wherein the P2Y2 agonist is 2- ((ethyl(4-fluorobenzyl)amino)methyl)-7,8-dimethylquinolin-4(1 / 7)-one, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

14. The method of any one of claims 1 to 13, wherein the P2Y2 agonist is administered parenterally or orally.

15. The method of any one of claims 1 to 14, wherein the P2Y2 agonist is administered daily, optionally the P2Y2 agonist is administered daily for one week.

16. The method of any one of claims 1 to 15, wherein the P2Y2 agonist is permeable to the blood-brain barrier.

17. The method of any one of claims 1 to 15, wherein the P2Y2 agonist is impermeable to the blood-brain barrier.

18. The method of any one of claims 1 to 17, wherein the P2Y2 agonist is administered as a pharmaceutical composition comprising the P2Y2 agonist and at least one pharmaceutically acceptable carrier, excipient or diluent.

19. The method of claim 18, wherein the pharmaceutically acceptable carrier, excipient or diluent is a nanoparticle.

20. The method of any one of claims 1 to 19, wherein the P2Y2 agonist is administered as a combination therapy.

21. The method of claim 20, wherein the combination therapy comprises a behavioral therapy, a physical exercise, a speech and language pathology therapy, an ergotherapy, a pediatric occupational therapy, or a psychotherapy.

22. The method of any one of claims 1 to 21 , wherein the subject is an infant or a child, wherein the infant or child has not been diagnosed with the neurodevelopmental disorder.

23. The method of any one of claims 1 to 21 , wherein the subject is an infant, a child, a teenager, or an adult, wherein the infant, child, teenager or adult has been diagnosed with the neurodevelopmental disorder.

24. A use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof for treating or preventing a neurodevelopmental disorder in a subject in need thereof.

25. A use of a therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a neurodevelopmental disorder in a subject in need thereof.

26. A therapeutically effective amount of a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof for use for treating or preventing a neurodevelopmental disorder in a subject in need thereof.

27. A composition for use in treating or preventing a neurodevelopmental disorder in a subject in need thereof, the composition comprising a P2Y2 agonist or a pharmaceutically acceptable salt, solvate or prodrug thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

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