Compositions and methods for treating autism spectrum disorder using granulocyte-macrophage colony-stimulating factor (GM-CSF)

Abstract

Embodiments of the instant disclosure relate to intervention and / or treatment of autism spectrum disorder (ASD). Certain embodiments relate to treating a subject having ASD with one or more proinflammatory cytokines. In some embodiments, the one or more proinflammatory cytokines include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) or derivative thereof, or fusion polypeptide thereof, or recombinant thereof. Other embodiments include a mouse model for assessing efficacy of treatment of ASD in humans and other mammals.

Description

COMPOSITIONS AND METHODS FOR TREATING AUTISM SPECTRUM DISORDER AND SIDE EFFECTS THEREOF USING GRANULOCYTEMACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)PRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 687,276 filed August 26, 2024. This application is incorporated herein by reference in its entirety for all purposes.FIELD

[0002] Embodiments of the instant disclosure relate to intervention and / or treatment of autism spectrum disorder (ASD). Certain embodiments relate to treating a subject having ASD with one or more proinflammatory cytokines. In some embodiments, the proinflammatory cytokine includes, but is not limited to, granulocyte-macrophage colonystimulating factor (GM-CSF) or derivative thereof.BACKGROUND

[0003] Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficits, repetitive / restricted behaviors, and cognitive impairments, affecting 1 in 16 children in the U. S. and 1 in 100 children in the globally, with males being diagnosed approximately 3-4-fold times more frequently than females. Although some clinical and behavioral phenotypes have been well-studied, the corresponding relationship with brain pathologies remains poorly understood. A need exists for new therapeutic interventions in ASD to improve side effects of these conditions.SUMMARY

[0004] Embodiments of the instant disclosure relate to intervention and / or treatment of autism spectrum disorder (ASD). Certain embodiments relate to treating a subject having ASD with one or more proinflammatory cytokines. In some embodiments, the proinflammatory cytokine includes, but is not limited to, granulocyte-macrophage colonystimulating factor (GM-CSF) or derivative or analog thereof. In certain embodiments, male and female subjects having ASD can be treated with one or more pro-inflammatory cytokine to improve brain pathology, improve social or other behaviors, improve communications, improve social interactions, and / or improve cognition in the subject. In other embodiments, male and female subjects having ASD can be treated with one or more pro-inflammatory cytokine including, but not limited to, a composition, or aqueous formulation containing GM-CSF, a biologically active derivative thereof, an analog thereof, or recombinant thereof,or formulated version thereof, or microparticle containing GM-CSF to improve brain pathology and cognition in the subject.

[0005] It is known that range and severity of symptoms of a subject having ASD can vary widely. Common symptoms in a subject having ASD can include, but are not limited to, difficulty with communication, difficulty with social interactions, obsessive interests, uncontrollable behaviors and outbursts, and repetitive behaviors among other symptoms. It is contemplated that compositions disclosed herein can be used to reduce these tendencies / behaviors and improve social interactions in the subject.

[0006] In certain embodiments and further to paragraphs

[0004] -

[0005] above, GM-CSF, or a biologically active derivative or analog thereof is the only therapeutically effective compound administered to the subject. In certain embodiments, the GM-CSF, or a biologically active derivative or analog thereof is part of a combination therapy or combination composition with at least one other agent used to treat ASD in a subject. In some embodiments, the GM-CSF, or a biologically active derivative or analog thereof is the only therapeutically effective agent administered to the subject in a sufficient amount to improve brain pathology, improve social or other behaviors, improve communications, improve social interactions, and / or improve cognition in the subject. In certain embodiments, cognition includes, but is not limited to, learning, memory, knowledge and learning, attention, working memory, judgment and evaluation, reasoning and computation, problem solving and decision making, comprehension and production of language, recognition memory executive function, ability to learn and execute activities of daily living, and / or ability to recognize and respond appropriately to social clues from other people.

[0007] In certain embodiments and further to paragraphs

[0004] -

[0006] above, GM-CSF or a biologically active derivative, a recombinant thereof, a formulated version thereof, or analog thereof is active as a hematopoietic growth factor. In other embodiments, GM-CSF or a biologically active derivative, a recombinant thereof, a formulated version thereof, or analog thereof is at least GM-CSF selected from the group consisting of sargramostim, molgramostim, and any methylated, C-amidated, N-acetylated, glycosylated, deglycosylated, partially glycosylated, PEGylated and / or partially PEGylated analogue, or a fusion polypeptide or derivative thereof. In certain embodiments, the fusion polypeptide can include, but is not limited to, a GM-CSF -transferrin fusion polypeptide. In certain embodiments, the GM-CSF of the GM-CSF-transferrin fusion polypeptide can be a full length or biologically active fragment thereof. In some embodiments, the agents of use herein are in oral form. In yet other embodiments, the biologically active derivative oranalogue is sargramostim. In yet other embodiments, the dose of sargramostim administered to a subject is about 250 pg / m2per day, 125 pg / m2per day, or 50 pg / m2per day or about 10 pg / m2per day to about 400 pg / m2per day. In some embodiments, administration of compositions disclosed herein can be for a predetermine period (e.g., a week, a month, several months, 6 months, a year etc.) or for the remainder of the subject’s life. In certain embodiments, treatment can include multiple times per day, 2-3 times per day, daily, every other day, bi-weekly, weekly, couple times per month, monthly or other appropriate schedule.

[0008] In some embodiments and further to paragraphs

[0004] -

[0007] above, administration of a treatment to a subject can be performed by at least one route of administration including, but not limited to, subcutaneous, inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, otic, intraocular, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. In certain embodiments, the subject is a human. In some embodiments, the human subject is a young child, a child, an adolescent, a teenager, a young adult, a middle-aged adult or an older adult.

[0009] In some embodiments and further to paragraphs

[0004] -

[0008] above, kits are contemplated herein for example for packaging, transport, and use. In other embodiments, kits can include instructions for administering a composition disclosed herein with or without performing one or more tasks and / or assessing outcome of an ASD treatment regimen before and / or after performing one or more tasks.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0011] FIG. 1 represents an exemplary bar graph of control (saline injected) versus treated (GM-CSF treated) populations of an animal model of ASD and outcomes of testing Radial Arm Water Maze (RAWM) performance according to certain aspects of the present disclosure.

[0012] FIGS. 2A-2D represent exemplary bar graphs of control versus treated populations of an animal model of ASD and outcome of various testing 2A rotarod performance, 2B holeboard performance, 2C social novelty performance; and 2D RAWM performance according to certain aspects of the present disclosure.

[0013] FIGS. 3A-3F are exemplary images of samples derived from control (WT) animals not having ASD (3 A, 3D) compared to samples derived from two different acceptable mouse models for ASD, BTBR and Fmrl KO mice, treated with a placebo (3B, 3E respectively) and an agent (3C and 3F, respectively) mice where the samples are harvested cerebellum cells stained for presence or absence of a representative binding protein linked to full neuronal function according to certain aspects of the present disclosure.DEFINITIONS

[0014] Terms, unless defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0015] Unless otherwise indicated, all numbers expressing quantities of agents and / or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that can vary from about 10% to about 15% plus and / or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

[0016] As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0017] As used herein, “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0018] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, pets, livestock, horses or other animals.

[0019] As used herein, “treat,” “treating” or “treatment” can refer to treating, reversing, ameliorating, or inhibiting onset or inhibiting progression of a health condition or disease or a symptom of the health condition or disease.DETAILED DESCRIPTION

[0020] In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details can be modified through routine experimentation. In some cases, well-known methods, or components have not been included in the description.

[0021] In accordance with embodiments disclosed herein, autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficits, repetitive and / or restricted behaviors, and cognitive impairments, affecting 1 in 16 children in the United States and 1 in 100 children globally. It is understood from various studies that males are being diagnosed with ASD about 3-4-fold more frequently than females. Although some of the clinical and / or behavioral phenotypes are well-studied, relationship within brain pathologies remains poorly understood. It is known that the cerebellum is involved in ASD, yet its role as a target in treatment has been mostly underexplored. Composition and methods disclosed herein target brain of ASD patients in both male and female subjects to reduce ASD clinical and behavioral issues as acute, intermittent, or long-term or chronic therapies. It has been discovered that GM-CSF, a mutant or derivative or analog thereof, a construct thereof, a recombinant thereof treatment rescues behavioral and / or social deficits in a treated mouse model of autism spectrum disorder (ASD) representative of human disease. In addition, these treatments rescue or preserve neuronal cell numbers and attenuate neuronal arborization abnormalities in the brain of autism spectrum disorder (ASD) in the representative mouse mode. In accordance with these embodiments, these observations support use of these treatments in human having ASD to treat these conditions.

[0022] In certain embodiments and further to paragraph

[0021] above, granulocytemacrophage colony-stimulating factor (GM-CSF), an FDA approved immune system modulator for other purposes, has been demonstrated to improve brain pathology and cognition in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and Down syndrome (DS) in animal models and clinical trials. It was unknown until these studies to have had any effect on ASD. As disclosed herein, GM-CSF treatment of ASD was demonstrated to improve task performance and improve outcomes in social, behavioral, and cognitive deficit in acceptable ASD mouse testing models which support human outcomes regarding the same. These observations demonstrate that GM-CSF's and any derivative, or analog, or fragment having biological activity thereof, has the ability to restore ASD-associated social, behavioral, and cognitive deficits, as well as pathology of the underlying disorders.

[0023] In certain embodiments and further to paragraphs

[0021] -

[0022] above, efficacy of interventions to treat ASD or side-effect thereof in ASD subjects can be enhanced and early intervention are possible as provided herein. In certain embodiments, compositions and methods disclosed herein can be used to observe social and behavioral improvements over a lifetime of a subject having ASD, if early intervention is achieved. In certain embodiments, timing and frequency of interventions and efficacy of therapeutics can also be determined; for example, using interactive testing to design an appropriate treatment regimen for a more personalized approach in treating ASD in the subject.

[0024] In certain embodiments and further to paragraphs

[0021] -

[0023] above, methods can further include administering a treatment to a subject to treat declining neuronal cell integrity, to treat increasing neuronal cell damage, and / or to treat age-related neuronal cell damage and stabilize and / or reverse progression and / or effects of increasing neuronal cell damage. Suitable treatments are described further below but can include any treatment, process or procedure known in the art to ameliorate or reverse declining neuronal cell integrity and / or increasing neuronal cell damage or age-related neuronal cell damage and stabilize and / or reverse progression and / or effects of increasing neuronal cell damage. In certain embodiments, GM-CSF or analog or derivative or recombinant thereof can be combined with other standard treatments to treat declining neuronal cell integrity, to treat increasing neuronal cell damage, and / or to treat age-related neuronal cell damage and stabilize and / or reverse progression and / or effects of increasing neuronal cell damage.

[0025] As disclosed herein and further to paragraphs

[0021] -

[0024] above, GM-CSF is a hematopoietic growth factor that stimulates proliferation and differentiation of hematopoietic progenitor cells. Commercially available forms of GM-CSF exist in the market, such as recombinant human GM-CSF (LEUKINE® / sargramostim) which has been approved by the FDA since 1991 and safely used worldwide for various conditions. In certain embodiments, recombinant human GM-CSF (sargramostim) can be used for treating ASD in a subject to reduce adverse ASD conditions in the subject. It is further contemplated herein that any analog or derivative of GM-CSF having the same biological activity can be used as an alternative. Non-limiting examples of analogs or derivatives of GM-CSF include, but are not limited to, sargramostim and molgramostim. Other examples include, but are not limited to, methylated, C-amidated, N-acetylated, glycosylated, de-glycosylated, partially glycosylated analogs or derivatives thereof including derivatives that can include transferrin, for example,to aid oral availability or ability to cross the blood brain barrier. In some embodiments, a GM-CSF molecule can include a fusion polypeptide (e.g., a transferrin-GM-CSF fusion polypeptide of full-length or biologically active fragment of GM-CSF and / or transferrin forming the fusion polypeptide). Certain examples of analogs can include constructs of GM- CSF fragments of biologically active regions which can include repeated biologically active fragments as found in PCT / US18 / 36736 filed June 08, 2018, incorporated herein by reference in its entirety for all purposes. Other examples include, but are not limited to, analogs of GM-CSF that have been modified by addition of polyethylene glycol chains, including GM-CSF derivatives in which the amino acid cysteine has been added in one or more locations or used in place of one or more natural-occurring amino acids to provide stabilization and / or to permit crossing the blood brain barrier, or which have been used as the site(s) of addition of poly ethylene glycol. In certain embodiments, transferrin or peptides derived therefrom can be used to create and use GM-CSF fusion polypeptides. For example, THR, HAI and CRT derived from transferrin can be used. These peptides, derived from the serum protein transferrin, bind to the transferrin receptor (TfR), which is highly expressed on brain capillary endothelial cells, facilitating transcytosis across the BBB. TfR-mediated transport is a widely researched and promising strategy for drug delivery to the CNS. These peptides have been successfully used to transport various cargo, including liposomes, nanoparticles, and antibodies into the brain. In other embodiments, a blood-brain barrier penetrating molecule can include, angiopep-2. This peptide has a high affinity for low- density lipoprotein receptor-related proteins (LRPs) (LRP-1 and LRP-2) and has demonstrated higher transcytosis capacity than transferrin in in vitro assays known in the art. In this example, Angiopep-2 agent can transport large polypeptides across the BBB. In other embodiments, Glutathione (y-L-Glutamyl-L-cysteinylglycine) can be used which is a naturally occurring tripeptide that possesses antioxidant properties and utilizes glutathione transporters to enter the brain. GSH-targeted nanoparticles have successfully delivered anticancer drugs into mouse brain tumors, highlighting its potential in brain drug delivery. Other known BBB transporting molecules are contemplated of use herein to generate and use GM-CSF or fragments thereof for generating a fusion polypeptide. Other embodiments include using Cell-Penetrating Peptides (CPPs) such as Tat peptide, Penetratin (which traverses the BBB via adsorptive-mediated transcytosis and can deliver various cargos); SynBl (SynBl enhances the brain uptake of various drugs); PepH3 (PepH3 has shown promising BBB penetration via adsorptive-mediated transcytosis): RGD (arginine-glycine- aspartate) where this tri-amino acid sequence targets avP3 integrin, which is overexpressedin brain tumors, and has been utilized to carry drugs and diagnostic agents to the brain. RGD peptide-modified nanoparticles are thought to mediate indirect endocytosis by entering leukocytes that are recruited to the brain in response to inflammation. Dipeptides are also contemplated for use in BBB-adapted fusion polypeptides and / or cell penetrating polypeptides; for example, small dipeptides, like Gly-Sar, Gly-Pro, and Tyr-Pro, can also cross the BBB and accumulate in the brain parenchyma.

[0026] GM-CSF as a monomeric glycosylated polypeptide signaling molecule is typically secreted by immune cells such as macrophages, T cells, mast cells, natural killer (NK) cells, as well as normal tissue cells such as endothelial cells and fibroblasts. In the bone marrow, GM-CSF functions as a leukocyte growth factor, and stimulates hematopoietic progenitor cells to differentiate into monocytes and granulocytes. In addition to its growth factor function, GM-CSF also acts as an important modulator of immune responses. Upon stimulation, many types of immune cells produce and secrete GM-CSF where it can act both locally to enhance maturation and antigen presentation function of macrophages and dendritic cells, as well as in a paracrine fashion to recruit circulating neutrophils, monocytes, and lymphocytes to areas of infection and inflammation.

[0027] In certain embodiments and further to paragraphs

[0021] -

[0026] above, where described and contemplated for therapeutic treatment disclosed herein, GM-CSF can include any form of GM-CSF or biologically active fragment thereof or mimetic thereof (e.g., GM- CSF mimetic). In some embodiments, GM-CSF can include, but is not limited to, recombinant GM-CSF. In certain embodiments, the GM-CSF includes, but is not limited to, human recombinant GM-CSF. In other embodiments, GM-CSF can include human recombinant GM-CSF, including, but not limited to, sargramostim, molgramostim, or regramostim or other recombinant GM-CSF. In other embodiments, exogenous administration of a viral or plasmid vector or mRNA construct designed to encode GM-CSF can be administered and expressed in the subject. In some embodiments, the GM-CSF includes, but is not limited to other non-human mammalian recombinant GM-CSF (e.g., for a pet, a companion animal or livestock or other animal). In some embodiments, compositions including, but not limited to, GM-CSF can be administered to a subject diagnosed with a neurodegenerative condition. In other embodiments compositions including, but not limited to, GM-CSF can be administered to a subject at risk of an age- associated neuronal change.

[0028] In some embodiments and further to paragraphs

[0021] -

[0027] above, compositions and methods disclosed herein can include administration of about 50pg / m2 / day to about 1000 pg / m2 / day; about 100 pg / m2 / day to about 750 pg / m2 / day; about 150 pg / m2 / day to about 600 pg / m2 / day; about 200 pg / m2 / day to about 500 pg / m2 / day; about 200 pg / m2 / day to about 400 pg / m2 / day; or about 250 pg / m2 / day of GM-CSF, recombinantly produced molecule thereof or fragment or analog thereof in a single treatment or multiple treatments per day. It is contemplated herein that a recombinant or derivative or conjugate of GM-CSF concentration of use herein can be reduced depending on potency of the molecule compared to a wild-type control or another standard treatment (e.g., one-half or less, one-quarter or less, one tenth or less, or any concentration in between compared to another treatment). In other embodiments, a subject can be treated every other day, 2 times per week, once a week or other dosing regimen such as a periodic regimen. In some embodiments, compositions and methods herein can include administration of about 250 pg / m2 / day of GM-CSF, recombinantly produced molecule thereof, or fragments, or analogs thereof in a single dose or multiple doses. In some embodiments, two doses can be provided to the subject where the total concentration of GM-CSF or analog thereof is about 10 pg / m2 / day to about 1000 pg / m2 / day or about 20 pg / m2 / day to about 500 pg / m2 / day or about 50 pg / m2 / day to about 300 pg / m2 / day or about 100 pg / m2 / day to about 250 pg / m2 / day or more or any concentration in between. In some embodiments, a single dose of about 100 pg / m2 / day to about 250 pg / m2 / day can be administered on a daily basis for about 3 days to about 2 weeks; or about 150 pg / m2 / day to about 200 pg / m2 / day on a daily basis for about 3 days to about 2 weeks (e.g., 178.57 pg / m2 / day daily (e.g., for about a week)) or a single dose of about 100 pg / m2 / day to about 250 pg / m2 / day can be administered on a daily basis for up to about 24 weeks or more depending on the ASD to be treated and response of the subject to the treatment as assessed by a health professional. In certain embodiments, recombinant GM-CSF can be sargramostim, molgramostim, or regramostim or other recombinant GM-CSF. In some embodiments, a recombinantly produced molecule or fragment thereof can be administered in a composition at a significantly lower concentration such as about 2.5 pg / m2 / day to about 500 pg / m2 / day to a subject in need thereof having ASD for up to 24 weeks or more depending on the ASD to be treated and response of the subject to the treatment as assessed by a health professional.

[0029] In other embodiments and further to paragraphs

[0021] -

[0028] above, compositions and methods can include a composition including, but not limited to, GM-CSF, a recombinantly produced molecule or fragment thereof, or an analog thereof formulated in a pharmaceutical composition, which can further include a pharmaceutically acceptable carrier or excipient. In certain embodiments, the GM-CSF can be recombinantly produced or be afusion polypeptide for facilitating brain access by crossing the blood-brain barrier (e.g., a transferrin chimeric molecule or fusion polypeptide thereof). In certain embodiments, the recombinantly produced GM-CSF can be sargramostim, molgramostim, or regramostim or other recombinantly produced GM-CSF. In some embodiments, GM-CSF is delivered in a particle, such as a lipid nanoparticle or any other delivery system. In accordance with these embodiments, the pharmaceutical composition can be administered to the subject by any means known in the art. In other embodiments, the pharmaceutical composition can be administered to the subject by at least one of oral administration, by inhalation, subcutaneous, intravenous, intranasal, intra-arterially, by slow-release microparticles or timed-released formulation by any mode (e.g., oral administration), by targeted deposit directly to the central nervous system. In yet other embodiments, the pharmaceutical composition can be administered to the subject by inhalation, intranasal and / or subcutaneous administration. In some embodiments, the subject can be treated one time, two times, or three times daily for a predetermined period to reduce and / or prevent neuronal damage and improved side effects of ASD in the subject. In certain embodiments, combination administrations routes can be used to treat a subject depending on a particular subject and dosing regimen such as by inhalation and subcutaneous or intravenous administration, or by oral in combination with other modes of administration. In yet other embodiments, the pharmaceutical composition can be administered to the subject alone, in combined treatment regimens or in combination with other agents or treatments for treating, preventing, or ameliorating viral infections or viral infection-related conditions in the subject. In some embodiments, compositions and methods disclosed herein can be used in combination treatments to reduce onset, prevent, reduce progression of and / or treatment of neurodegeneration where other treatments can include any standard treatment for the condition. For example, it is contemplated to be used in combination with other neurodegenerative treatment agents which will be beneficial to improve outcome.

[0030] In yet other embodiments and further to paragraphs

[0021] -

[0029] above, testing efficacy of a potential therapeutic for treating ASD in a subject is provided. In some embodiments, the method can include (a) measuring a protein or agent related to neuronal function in a subject having ASD, (b) administering a target therapeutic to the subject, and (c) measuring the protein or agent related to neuronal functional in at least a second sample obtained from the subject after administration of the target therapeutic, where efficacy of the target therapeutic can be determined by a restoration of the at least one protein or agent related to neuronal function. In some embodiments, the first sample and / or at least thesecond sample can each include brain-related sample or neuronal sample obtained from another region.

[0031] In some embodiments and further to paragraphs

[0021] -

[0030] above, a process or task contemplated herein can include at least one of a behavioral test or a social indicator test.

[0032] In certain embodiments and further to paragraphs

[0021] -

[0031] above, GM-CSF or the like agent treatment disclosed herein improves certain social and cognitive behavioral phenotypes using an acceptable animal model. For example, animal models of ASD are well known in the art. ASD is a complex disorder in humans with multiple and varied manifestations between subjects, and no animal model replicates the complete human disease. It is known that both BTBR T+Itpr3tf / J (BTBR) and Fmrl knockout (KO) mouse model exhibit several social cognitive phenotypes associated with ASD and represent reliable animal models to test new treatments for ASD. As disclosed herein, several behavioral tasks in juvenile BTBR and Fmrl KO mice were performed and compared to performance of wild-type (WT) mice. In accordance with these embodiments, specific tests of rodent behavior designed to mimic ASD phenotypes in some human ASD patients were performed including, but not limited to, Rotarod, Hole Board, Three Chamber Social test, and Radial Arm Water Maze (RAWM) in BTBR mice, Fmrl KO mice and WT mice to assess efficacy of treatments disclosed herein.

[0033] As disclosed herein and further to paragraphs

[0021] -

[0032] above, a rotarod test can be used to detect motor coordination and motor learning. Motor dysfunction is a common dystonia-like behavior in ASD and it correlates with cerebellar abnormalities in ASD. Both mouse models as referenced above had a significant deficit in motor coordination / functions compared to the WT mice. As demonstrated herein, after GM-CSF treatment, both ASD mouse models demonstrated an improvement in the motor coordination and strength. In accordance with these embodiments, these GM-CSF treatments or FM-CSF analog or recombinant disclosed herein can affect motor coordination and / or strength in a subject having ASD.

[0034] In other embodiments and further to paragraphs

[0021] -

[0033] above, a Hole Board Task can be used to assess other ASD-related side effects and treatments thereof. As disclosed herein, a Hole Board Task is a test for repetitive behavior which is a common, but not a universal phenotype found in humans with ASD and in animal models of ASD. In accordance with these embodiments, both animal models which are reflective of human ASD conditions demonstrated significant repetitive behavior (stereotypical) phenotypeassessed by the hole board test. As demonstrated herein, number of repeat hole pokes were significantly higher in placebo treated (e.g., saline) mice and the KO mice compared to the WT mice. In some embodiments, GM-CSF treatment of the animal models described herein led to a trend in reduction in the number of repeat hole pokes. In accordance with these embodiments, GM-CSF, conjugate thereof or fragment or recombinant thereof or other GM- CSF type agent can be used to treat ASD and reduce repetitive behaviors in the subject.

[0035] In some embodiments and further to paragraphs

[0021] -

[0034] above, a Three Chamber Social Interaction Task (TCSIT) was used to assess social novelty preference as a measure of social behavioral deficits. As disclosed herein, tests using TCSIT in both animal models were compared to WT mice. In accordance with these embodiments, the TCSIT demonstrated that BTBR mouse model animals spent less sniffing-time compared to sniffing-times of WT mice, which demonstrates a strong trend in social deficits (p=0.07) in these BTBR mice prior to treatment. In certain embodiments, after GM-CSF treatment, the BTBR mice demonstrated a significant increase in sniffing time with novel mice. Therefore, GM-CSF treatment plays a significant role in returning an ASD subject having social novelty preference deficits to a more control, normal or wild-type control behavior. In contrast, the Fmrl KO mice animal model did not demonstrate deficits in social novelty preference and as such indicate this mouse model is not a reliable model for analyzing this ASD variant in subjects having such deficits and instead, reflects subjects having ASD that do not demonstrate such deficits.

[0036] In certain embodiments and further to paragraphs

[0021] -

[0035] above, a radial arm water maze (RAWM) task can be used to assess and analyze animal models for effects of a target agent disclosed herein to affect the RAWM task outcome in a subject having ASD. In accordance with these embodiments, hippocampal -based spatial learning and memory performance were evaluated in the animal models and compared to wild-type animals using the RAWM task. A RAWM task was performed and working memory errors were analyzed and compared. It was observed that placebo-treated Fmrl KO mouse model demonstrated a significant deficit compared to placebo-treated WT mice, and placebo- treated BTBR mice demonstrated a trend deficit compared to placebo-treated WT mice. In other embodiments and as disclosed herein, GM-CSF-treated Fmrl KO animals demonstrated a strong improvement in learning and memory behavior compared to placebo- treated mice. In contrast to this analysis, the BTBR animal model did not demonstrate effects of GM-CSF treatment. This is not unexpected. It has been demonstrated that juvenile animal models used in the studies of other health conditions when performing the RAWM task hadfew total and working memory errors, while older animals of animal models used, exhibit errors in the RAWM. Therefore, the RAWM task appears to be an age-dependent test. In certain embodiments, GM-CSF or analog or derivative, or fragment repeats thereof, or recombinant molecules thereof, can be used to improve spatial learning and memory performance in subjects demonstrating these impairments, for example, older ASD subjects. In certain embodiments, GM-CSF treatment in older subjects can improve these repetitive behaviors.

[0037] In other embodiments and further to paragraphs

[0021] -

[0036] above, neuronal cell abnormalities in animal models of ASD can be used to compare to other mammalian equivalents or even human subjects. Cerebellar abnormalities and loss of Purkinje cells are some common pathologies observed in post-mortem brains of people with ASD, and similar phenotypes have also been reported in animal models of ASD. Furthermore, recent studies have reported altered neuronal signaling in lobules IV / V of the cerebellum in BTBR mice, an ASD mouse model used herein. It is understood that this defect in the brain of a subject can be associated with defects in social behavior. In certain embodiments, restoration of Purkinje cells in the cerebella of BTBR and Fmrl KO mice were assessed in control and GM-CSF treated animals. As disclosed herein, immunohistochemical staining with an anti- Calbindin-28 antibody in the cerebella of 3 -month-old BTBR and Fmrl KO mice after saline or GM-CSF treatment were compared. In these experiments, it was demonstrated that both BTBR and Fmrl KO mice injected with saline exhibit a significant reduction in numbers of calbindin-positive Purkinje cells and a loss of neuronal arborization / processes compared to WT mice (e.g., treated with saline). After treatment with GM-CSF, both BTBR and Fmrl KO mice demonstrated increased numbers and arborization of Purkinje cells compared to saline-injected mice. It was demonstrated that GM-CSF treatment restores normal behavior and normal neuronal numbers and structure in two mouse models of ASD and supports GM- CSF use in the treatment of ASD in humans. In accordance with these embodiments, GM- CSF treatment restored neuronal numbers, morphology, and function, and thus behavior in ASD mice supporting use in humans.Pharmaceutical Compositions

[0038] In certain embodiments and further to paragraphs

[0021] -

[0037] above, pharmaceutical compositions are contemplated. In accordance with these embodiments, pharmaceutical compositions can include GM-CSF or GM-CSF analog, recombinant, fragment or conjugate thereof. In some embodiments, pharmaceutical compositions herein can include GM-CSF and at least one pharmaceutically acceptable excipient or carrier. Asused herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of a subject without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. As used herein, the term “pharmaceutically acceptable carrier” can refer to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use herein, include, but are not limited to, buffers that are well known in the art, and can be phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. In certain embodiments, GM-CSF, derivative thereof, recombinant thereof, fragment thereof or construct thereof can be used alone or in combination with other agents to treat ASD-associated brain or behavioral disorders. In some embodiments, these agents can be used in combination with therapies such as standard ASD treatments including, but not limited to, anti-depressants, anti-psychotics, anxiolytics and ASD medications and / or behavior interventions. In accordance with these embodiments, ASD medications and / or behavior interventions can include, but are not limited to, behavior and communication therapies. For example, programs address a range of social, language and behavioral difficulties linked with ASD. In certain embodiments, a program can focus on reducing challenging behaviors and teaching new skills. Other programs can focus on teaching subjects such as young subjects or children how to act in social situations or communicate better with others. Applied behavior analysis can assist young subjects or children to learn new skills and adapt these skills to many situations by motivating them with rewards. Other embodiments can include combining treatments disclosed herein with educational therapies. For example, children with ASD often respond well to highly structured educational programs. Successful programs can include a team of specialists and various activities to improve social skills, communication and behavior. In some embodiments, preschool children who get intensive, individualized behavioral treatments often demonstrate improved progress. In yet other embodiments, family therapies can assist with treating a subject having ASD. For example, parents and other family members can learn how to play and interact with children who have ASD in ways that support social interaction skills,manage challenging behaviors, and teach daily living skills and communication. In other embodiments, therapies can be specific to the subject being treated. For example, depending on need of a subject such as a young subject or child, speech therapy to facilitate communication skills, occupational therapy to teach activities of daily living, and physical therapy to make movement and balance better can improve outcome of treatment with compositions disclosed herein. In certain embodiments, a subject can be observed or treated by a psychologist for managing any problem behavior.

[0039] In some embodiments and further to paragraphs

[0021] -

[0038] above, pharmaceutical compositions for use herein can be formulated for parenteral administration, such as intravenous, intracerebroventricular injection, intra-ci sterna magna injection, intra- parenchymal injection, intra-renal, intradermal, subcutaneous, direct introduction to the brain or central nervous system (e.g., CSF) or a combination thereof. In some embodiments, pharmaceutical compositions for use herein can be formulated for local delivery to the brain or cerebral spinal fluid (CSF). In some embodiments, pharmaceutical compositions for use herein can be formulated for nasal delivery. In some embodiments, pharmaceutical compositions for use herein can be formulated for parenteral administration and can include pharmaceutically acceptable carriers including sterile liquids, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. In other embodiments, pharmaceutical compositions for use herein can further include additional agents, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. In some embodiments, pharmaceutical compositions described herein can be packaged in single unit dosages or in multi -dosage forms.

[0040] In some embodiments and further to paragraphs

[0021] -

[0039] above, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. In accordance with some embodiments herein, aqueous solutions can be suitably buffered (preferably to a pH of from 3 to 9). The preparation of suitable parenteral formulations for use herein under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0041] In some embodiments and further to paragraphs

[0021] -

[0040] above, pharmaceutical compositions herein can further include one or more pharmaceutically acceptable salts. Non-limiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid, or an organic acid. In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base, or an organic base. In some embodiments, any of the pharmaceutical compositions herein can be used in therapeutic applications which are also disclosed herein.

[0042] In certain embodiments and further to paragraphs

[0021] -

[0041] above, kits are contemplated of use herein. In accordance with these embodiments, kits can include at least one composition including, but not limited to, GM-CSF, mutant thereof, chimeric molecule thereof, fusion polypeptide thereof, recombinant thereof, or similar agent thereof or fragment thereof or mimetic thereof. In yet other embodiments, kits can include combinations of agents to treat or reduce ASD affiliated disorders including but not limited to, behavioral disorders. In other embodiments, kits can include one or more directives for a cognitive, social and / or behavioral test. In other embodiments, kits can further include a pamphlet for administering compositions disclosed herein in conjunction with standard therapies for complementary treatment of a subject having ASD (e.g., human subject).

[0043] Practice of embodiments of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature.EXAMPLES

[0044] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made in some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1

[0045] In one exemplary method, GM-CSF treatment was demonstrated to improve cognitive behavioral phenotypes in the Fmrl KO mouse model of ASD (which is a representative of human ASD), measured by the RAWM which reflects hippocampal -basedspatial learning and memory performance. This test was used to evaluate 3 -month-old male Fmrl KO and WT mice for two days using the RAWM task. A two-day RAWM task was performed in which a submerged (hidden) platform was placed in Arm 6 for Day 1 and then moving the platform to Arm 2 on Day 2 to evaluate both leaming / memory and learning flexibility. Each mouse was exposed to 10 trials (60 sec each). Learning and memory were assessed by the mean time to reach the platform in last trial (trial #10) of Day 2 and compared the results for the three groups, including Fmrl KO mice treated with GM-CSF (n=4) or injected with saline (n=3), and WT mice injected with saline (n=4). It was observed that treatment with GM-CSF (e.g. a recombinant) restored the mice being treated to near control / wild type compared to untreated mice. (See for example FIG. 1)Example 2

[0046] GM-CSF treatment improves some of the social and cognitive behavioral phenotypes in mouse models of ASD.

[0047] In one exemplary method, it is understood that ASD is a complex disorder in humans with multiple and varied manifestations between individuals. It is understood that no animal model perfectly replicates human disease but there are reliable animal models for aspects of studying ASD in human. Both the BTBR and Fmrl KO mice exhibit several social cognitive phenotypes associated with ASD and thus represent animal models to test proposed new treatments for ASD in humans. Several behavioral tasks were assessed in 3- month-old male (male or female mice are appropriate for studying ASD in humans but epidemiological studies have historically estimated ASD incidence of a male-to-female ratio is about 4: 1.) BTBR and Fmrl KO mice and the outcomes were compared with the performance of WT mice. Certain specific tests of rodent behavior designed to mimic ASD phenotypes experienced by some but not all ASD patients were used: Rotarod, Hole Board, Three Chamber Social test, and Radial Arm Water Maze (RAWM) in 3 -month-old male BTBR mice, Fmrl KO mice and WT mice.

[0048] A first test to assess these behaviors, the Rotarod Test was used. The rotarod test detects motor coordination and motor learning. Motor dysfunction is one of the common dystonia-like behaviors in ASD and it correlates with cerebellar abnormalities in ASD. Both mouse models demonstrated a significant deficit in motor coordination / functions compared to the WT mice (See for example FIG. 2A). After 17 days of GM-CSF treatment, both ASD mouse models demonstrated an improvement in the motor coordination / strength (FIG. 2A).

[0049] Another test, the Hole Board Task is used to represent repetitive behaviors. Repetitive behavior is one of the common, but not universal phenotypes in both people withASD and in animal models of ASD. In these studies, it was demonstrated that both BTBR and Fmrl KO mice demonstrated a significant repetitive behavior (stereotypical) phenotype assessed by the hole board test. Mice were placed on a board that had 16 holes, 3 cm in diameter. Hole poke activity was scored by an individual blind to the genotype of the mice. It was observed that the number of repeat hole pokes are significantly higher in saline- injected BTBR mice (p= <0.0001) and Fmrl KO mice (p=0.05) compared to the WT mice (FIG. 2B). After GM-CSF treatment, both mouse models demonstrated reduction in the number of repeat hole pokes (p= 0.18 and 0.13, respectively), with some variability (FIG. 2B).

[0050] In yet another test, a Three Chamber Social Interaction Task (TCSIT) was used to assess social behaviors. To assess the social novelty preference as a measure of social behavioral deficits, the TCSIT was performed in both BTBR and Fmrl KO mice and the outcome was compared to WT mice. It was demonstrated that TCSIT using a BTBR mice model that they spent less sniffing time compared to the sniffing time of WT mice, which correlates with a strong trend in social deficits (p=0.07) in the BTBR mice (FIG. 2C). After GM-CSF treatment, BTBR mice demonstrated a significant increase in sniffing time suggesting that GM-CSF treatment assists in the reversal of social novelty preference deficits toward the normal behavior of WT mice (FIG. 2C). Fmrl KO mice did not demonstrate any deficits in social novelty preference (FIG. 2C), indicating that it is not a reliable animal model for studying the ASD variant in people who have such deficits and reflects people or is an animal model for people with ASD that do not have such deficits.

[0051] Using yet another test, radial arm water maze (RAWM) Task which reflects hippocampal-based spatial learning and memory performance as presented above. This test was used to evaluate 3 -month-old male BTBR, Fmrl KO, and WT mice for two days using the RAWM task. A two-day RAWM task was performed in which a submerged (hidden) platform was placed in Arm 4 throughout testing, and each mouse was exposed to 10 trials (60 sec each). The working memory errors were analyzed and compared. For the baseline pre-treatment assessment, data from Day 2 was used by averaging the number of working memory errors from 10 trials for each individual mouse and compared the mean number of errors between BTBR and WT control mice and between Fmrl KO and WT control mice and mice treated with saline of GM-CSF.

[0052] It was observed that saline-treated Fmrl KO mice demonstrated a significant deficit compared to saline-treated WT mice (n=17) (p=0.04), and saline-treated BTBR mice demonstrated a trend deficit compared to saline-treated WT mice (p=0.22) (See FIG. 2D).These experiments demonstrate that GM-CSF-treated Fmrl KO mice (n=8) had a strong trend toward improvement in learning and memory behavior after the platform arm is switched to test learning flexibility, compared to saline-treated Fmrl KO mice (n=6) (p=0.09) on post-treatment Day 22. In contrast, BTBR mice did not change with GM-CSF treatments. This is not unexpected. It has been shown that younger aged mouse models, for example, Down Syndrome (DS) performed the RAWM task as well as WT mice with few total and working memory error, while older DS mice exhibited errors in the RAWM task test. Therefore, the RAWM task is likely an age-dependent test.Example 3

[0053] Neuronal cell abnormalities in animal models of ASD. Cerebellar abnormalities and loss of Purkinje cells are some of the common pathologies observed in post-mortem brains of people with ASD, and similar phenotypes have also been reported in animal models of ASD. Furthermore, recent studies have reported altered neuronal signaling in lobules IV / V of the cerebellum in BTBR mice, a defect that is associated with social behavior.

[0054] In certain exemplary methods, GM-CSF treatment was tested to see if it would restore Purkinje cell abnormalities / numbers in the cerebella of BTBR and Fmrl KO mice. A successful outcome in these experiments would indicate that GM- treatment restores neuronal numbers, morphology, and function, and therefore, behavior in ASD mice reflective of treatment of ASD in humans.

[0055] In these studies, immunohistochemical staining with an anti-Calbindin-28 antibody was used in the cerebella of 3 -month-old BTBR and Fmrl KO mice after saline or GM-CSF treatment and compared to those from saline-injected WT mice (FIG. 3). It was demonstrated that both BTBR and Fmrl KO mice injected with saline exhibit significantly reduced numbers of calbindin-positive Purkinje cells and a loss of neuronal arborization / processes compared to WT mice (treated with saline) (FIG. 3 A-3B and FIG. 3D-3E, respectively). After treatment with GM-CSF, both BTBR and Fmrl KO mice demonstrated increased numbers and arborization of Purkinje cells compared to saline- injected mice (FIG. 3B-3C and 3E-3F, respectively). Together, these results demonstrate that GM-CSF treatment restores normal behavior and normal neuronal numbers and structure in two mouse models of ASD and is a potential treatment for ASD in humans.

[0056] FIGS. 2A-2D illustrates use of GM-CSF treatment in animal models identified a trend or significant change towards normalcy in some of the social and cognitive behavior phenotypes in mouse models of ASD. Three-month old male BTBR, Fmrl KO and WT micewere used to perfume the rotorod, hole board, three chamber social behavior, and radial arm water maze (RAWM) tasks. (2A) Rotorod tests were performed for evaluating the motor coordination and motor learning in male (saline injected), BTBR and Fmrl KO (saline and GM-CSF treated) mice. (2B) Hole Board tasks were performed for detecting the repetitive behaviors of male WT (saline injected), BTBR and Fmrl KO (saline and GM-CSF treated) mice. (2C) The three chamber social behavior task was performed for detecting the social behavior deficits in male WT (saline injected) and BTBR and Fmrl KO (saline and GM- CSF treated) mice. (2D) Post treatment RAWM tasks were performed on treatment day 22 in WT and both BTBR and Fmrl KO mice. For each bar, data are represented as mean + / - SEM for separate groups of mice. Statistical significance was determined by the unpaired Student’s t-test for comparison between groups, left: WT saline; middle: ASD saline, right: ASD GM-CSF.

[0057] FIGS. 3A-3F illustrates GM-CSF treatment restored number and morphological abnormalities of Purkinje cells in the cerebellum of mouse models of ASD. Immunofluorescence staining was performed with anti-Calbindin-28 antibody in the cerebella of 3-month-old BTBR, Fmrl KO and WT mice. Immunofluorescence microscopy images demonstrated the expression of Calbindin-positive Purkinje cells in the cerebella of WT and BTBR mice (3A-3C) and in the cerebella of WT and Fmrl KO mice (3D-3F). Both BTBR and Fmrl KO mice injected with saline demonstrate a relatively lower number of calbindin-positive Purkinje cells and loss of arborization / processes compared to WT mice (3 A and 3B; 3D and 3E, respectively). After treatment with GM-CSF, both BTBR and Fmrl KO mice demonstrated increased numbers of Purkinje cells and increased arborization compared to saline-injected control mice (3B and 3C and 3E and 3F, respectively).All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.

Claims

What is Claimed is:

1. A method for treating autism spectrum disorder (ASD) in a subject comprising: administering to the subject a therapeutically effective amount of GM-CSF, or a biologically active derivative, or recombinant molecule, or fusion polypeptide thereof, or analog thereof.

2. The method according to claim 1, wherein the subject is administered a behavioral test before, during, or after the treatment.

3. The method according to claim 1 or 2, wherein the subject is administered a social interaction test before, during, or after treatment.

4. The method according to any one of claims 1-3, wherein the subject is a human.

5. The method according to any one of claims 1-3, wherein the subject is a child.

6. The method according to any one of claims 1-5, wherein a protein or other agent affiliated with neuronal function is measured in a sample from the subject before, during or after the treatment; optionally, wherein the sample comprises a blood sample.

7. The method according to any one of claims 2-6, wherein treatment of the subject is modified based on outcome of a behavior test, social test, or protein or other agent level affiliated with neuronal function in the subject.

8. The method according to any one of claims 1-7, wherein the subject does not have “chemobrain” and is not afflicted with Alzheimer’s Disease (AD).

9. The method according to any one of claims 1-8, wherein the GM-CSF or a biologically active derivative, or recombinant molecule, or fusion polypeptide thereof, or analog thereof is the only therapeutically effective agent administered to the subject.

10. The method according to any one of claims 1-8, wherein the GM-CSF or a biologically active derivative, or recombinant molecule, or fusion polypeptide thereof, or analog thereof is combined with at least one other agent or separately administered to the subject to treat ASD in the subject.

11. The method according to any one of claims 1-10, wherein the biologically active derivative, or recombinant molecule, or fusion polypeptide thereof, or analog thereof is sargramostim.

12. The method according to any one of claims 1-10, wherein the fusion polypeptide thereof comprises a first polypeptide comprising GM-CSF or fragment thereof; and a second polypeptide comprising a molecule for crossing the blood-brain barrier.

13. The method according to claim 12, wherein the second polypeptide comprising a molecule for crossing the blood-brain barrier comprises at least one of transferrin or fragment thereof, angiopep-2 or fragment thereof or cell -penetrating peptide or fragment thereof; optionally, wherein the second polypeptide comprises a GM-CSF-transferrin fusion polypeptide.

14. A method for testing efficacy of a targeted therapeutic agent, process, or task for treating ASD in a subject, the method comprising:(a) administering the targeted therapeutic agent, process or task to the subject; and(b) analyzing a sample obtained from the subject at least one of before, after or during administration of a) for a protein or other agent affiliated with neuronal function.

15. The method according to claim 14, wherein the analyzing a sample obtained from the subject at least one of before, after or during administration of the test agent comprises analyzing a sample before and after a).

16. The method according to claim 14 or 15, further comprising assessing the targeted therapeutic agent of a) for efficacy in treating the ASD in the subject.

17. The method according to any one of claims 14 to 16, comprising administering at least one of a: behavioral and social interaction test to the subject at least one of: before, during and after administering the test agent to the subject.

18. The method according to claim 14, wherein the subject is an animal model.

19. The method according to claim 18, wherein the subject is a mouse model.

20. A composition comprising: a sample obtained from a subject having ASD; and an agent for measuring, detecting or binding to a protein or other agent affiliated with neuronal function of the subject having ASD.

21. The composition according to claim 20, wherein the protein comprises calbindin-28 or equivalent protein thereof, and the agent comprises a fluorescent binding compound or antibody.

22. A method for at least one of restoring number of or restoring morphological abnormalities of Purkinje cells in a subject having ASD, comprising treating the subject with GM-CSF, or a biologically active derivative, or recombinant molecule, or fusion polypeptide thereof, or analog thereof and at least one of restoring number of, or restoring morphological abnormalities of Purkinje cells in the subject.

23. A kit comprising a composition according to claim 20 or 21; and at least one container.

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