Methods and compositions for treating 19q12 autism spectrum disorder

Abstract

Provided herein are methods for decreasing expression and / or activity of one or more pathways associated with neurotransmitter transport and / or synaptic transmission, comprising contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. Further provided herein are methods for treating autism spectrum disorder associated with one or more mutations including the q12 band of chromosome 19 (19q12 ASD) in subjects in need thereof, by administering compositions comprising entrectinib to the subject.

Description

Attorney Docket No: RRBS-008 / 01WO 353016-2020METHODS AND COMPOSITIONS FOR TREATING 19Q12 AUTISM SPECTRUM DISORDERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 761,803, filed February 21, 2025, which is incorporated herein in its entirety for all purposes.TECHNICAL FIELD

[0002] The disclosure relates to methods for decreasing expression and / or activity of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission, comprising contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. The disclosure further relates to treating autism spectrum disorder associated with one or more mutations including the ql2 band of chromosome 19 (19ql2 ASD) in subjects in need thereof, by administering compositions comprising entrectinib.BACKGROUND

[0003] Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired communication, impaired social interaction, and / or behavioral abnormalities. ASD has a strong genetic component, and mutations in certain genetic loci have been associated with ASD. One of these loci is the ql2 band of chromosome 19 (19ql2).

[0004] Genes within the ql2 band of chromosome 19 include ZNF536 and TSHZ3, which encode zinc-finger proteins that act as regulators of transcription of other genes. ZNF536 and TSHZ3 are involved in biological processes that drive neurogenesis and neuronal differentiation.

[0005] New therapeutic strategies are needed for patients suffering from 19ql2 ASD.SUMMARY

[0006] Provided herein are methods for decreasing expression and / or activity of one or more pathways associated with neurotransmitter transport and / or synaptic transmission, comprising contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. Further provided herein are methods of treating autism spectrum disorder (ASD) associated with one or more mutations in the ql2 band ofAttorney Docket No: RRBS-008 / 01WO 353016-2020chromosome 19 (19ql2 ASD) in a subject in need thereof, the method comprising administering an effective amount of entrectinib or a pharmaceutically acceptable salt thereof to the subject.

[0007] In one aspect, the present disclosure provides a method of decreasing the expression and / or activity level, within a cell, of one or more pathways associated with neurotransmitter transport and / or bone or more pathways associated with synaptic transmission. The method comprises contacting the cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

[0008] The cell may comprise one or more mutations, for example in one or more genes. In some embodiments, the cell comprises one or more mutations including the ql2 band of chromosome 19. In some embodiments, the cell comprises one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene. The one or more mutations may comprise one or more types of mutations. For example, the one or more mutations may comprise a deletion, a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, or a mutation that affects an existing splice site. In some embodiments, the one or more mutations result in a mutant ZNF536 gene and / or a mutant TSHZ3 gene encoding a protein with reduced function.

[0009] In some embodiments, the cell has, prior to the contacting of the cell, one or more of the following: reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activity of one or more pathways associated with neurotransmitter transport; increased expression and / or activity of one or more pathways associated with synaptic transmission; reduced expression and / or activity of NTN1; increased expression and / or activity of SLC17A6; reduced glutamate transport; and / or disregulated calcium ion release.

[0010] In some embodiments, the one or more pathways associated with neurotransmitter transport comprise one or more of the following: regulation of release of sequestered calcium ion into cytosol; neurotransmitter receptor activity; neurotransmitter receptor internalization; calcium ion homeostasis; sequestering of calcium ion; regulation of calcium ion transmembrane transport; calcium transmembrane import into cytosol; calcium mediated signaling; calcium ion transport;; dicarboxylic acid transport; inorganic ion homeostasis; passive transmembrane transporter activity; sensory system development; transport vesicle membrane; and transport vesicle.Attorney Docket No: RRBS-008 / 01WO 353016-2020

[0011] In some embodiments, the one or more pathways associated with synaptic transmission comprise one or more of the following: long term synaptic potentiation; positive regulation of synaptic transmission; postsynaptic neurotransmitter receptor activity; postsynaptic signal transduction; postsynaptic membrane; presynaptic modulation of chemical synaptic transmission; regulation of trans synaptic signaling; regulation of long term synaptic potentiation; regulation of synaptic plasticity; regulation of short term neuronal synaptic plasticity; synapse organization; synaptic membrane; and / or synaptic transmission cholinergic.

[0012] In some embodiments, the method results in: increased expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activity of NTN 1 ; reduced expression and / or activity of SLC 17A6; increased glutamate transport; and / or increased regulation of calcium ion release.

[0013] In some embodiments, the method results in reduced expression and / or activity of one or more pathways associated with synaptic transmission in the cell. In some embodiments, the method results in reduced expression and / or activity of one or more pathways associated with neurotransmitter transport in the cell.

[0014] In some embodiments, the method results in an average normalized enrichment score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport in the cell of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0.

[0015] In some embodiments, the cell is a neuron. In some embodiments, the cell has, prior to the contacting, aberrant neuron function characterized by: increased number of spikes; increased number of bursting electrodes; increased number of spikes per burst; and / or increased burst duration.

[0016] In some embodiments, the contact occurs in vivo or ex vivo.

[0017] In one aspect, the present disclosure provides a method of treating or preventing a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof, comprising administering an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

[0018] The cell may comprise one or more mutations, for example in one or more genes. In some embodiments, the subject comprises one or more mutations including the ql2 band of chromosome 19. For example, the subject may comprise one or more mutations in one or both alleles of the ZNF536 and / or TSHZ3 gene. In some embodiments, the one or more mutationsAttorney Docket No: RRBS-008 / 01WO 353016-2020may comprise one or more types of mutations. For example, the one or more mutations may comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion. In some embodiments, the one or more mutations result in a mutant gene encoding a protein with reduced function.

[0019] In some embodiments, the subject is determined to have the one or more mutations by analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence. The analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence may be performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutations canning of select exons, mutation scanning of the entire ZNF536 and / or TSHZ3 coding region, sequence analysis of select exons, sequence analysis of the entire ZNF536 and / or TSHZ3 coding region, and ZNF536 and / or TSHZ3 RNA analysis.

[0020] In some embodiments, the subject has: reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activity of one or more pathways associated with neurotransmitter transport; increased expression of one or more pathways associated with synaptic transmission; reduced expression of NTN1; increased expression of SLC17A6; reduced glutamate transport; and / or disregulated calcium ion release.

[0021] In some embodiments, the subject has aberrant neuron function. Neuron function may be characterized by any one or more of: increased number of spikes; increased number of bursting electrodes; increased number of spikes per burst; and / or increased burst duration.

[0022] In some embodiments, the method results in an average normalized enrichment score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport in the subject. The normalized enrichment score for one or more pathways may be less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0 within the subject.

[0023] In some embodiments, the method results in: reduced expression of NTRK2; increased expression and / or activity of PI3K / AKT signaling pathways; and / or increased expression and / or activity of MAPKZERK signaling pathways.

[0024] In some embodiments, the method results in decreased expression and / or activity of: one or more pathways associated with neurotransmitter transport; and / or one or more pathways associated with synaptic transmission. In some embodiments, the method results in increasedAttorney Docket No: RRBS-008 / 01WO 353016-2020expression of one or more of the following genes: APT6V0C, SNCA, SLC4A1, CCL5,FTH1, HLA-DRB5, MT-ATP6, FTP, ITGB3, TM0D1, MT2A, CPU, GP1BB, SPC25A39, SHISA7, and / or BPC2P

[0025] In some embodiments, the disease or condition is 19ql2 autism spectrum disorder.

[0026] In some embodiments, the effective amount of the composition is about 100 mg to about 2000 mg per week. For example, the effective amount of the composition may be about 200 mg to about 1000 mg per week. In a further example, the effective amount of the composition may be about 560 mg per week.

[0027] The route, dosage regimen, and duration of administration may be as follows. In some embodiments, the composition is administered orally or parenterally. In some embodiments, the composition is administered once or twice per week. In some embodiments, the composition is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0028] In some embodiments, the method results in inhibition of the disease or condition; relief of the disease or condition; and / or reduction or improvement of at least one symptom of the disease or condition. In some embodiments, the at least one symptom comprises impaired ability to follow two-step directions or conversationality.

[0029] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0030] In some embodiments, the composition comprises entrectinib. In some embodiments, the composition additionally comprises a pharmaceutically acceptable carrier.

[0031] In one aspect, the present disclosure provides a method of identifying a subject in need of treatment with an effective amount of a composition comprising entrectinib, comprising: analyzing a genome sequence of the subject; and determining that the subject comprises a mutation of a gene in the ql2 band of chromosome 19 (19q 12).

[0032] In some embodiments, the genome sequence comprises a sequence of a gene in 19ql2, or a portion thereof. For example, the genome sequence may comprise a sequence of ZNF536 and / or TSHZ3. In some embodiments, the subject comprises one or more mutations in one or both alleles of the ZNF536 and / or TSHZ3 gene.Attorney Docket No: RRBS-008 / 01WO 353016-2020

[0033] The method of identifying a subject in need of treatment with an effective amount of a composition comprising entrectinib may further comprise administering to the subject an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

[0034] In one aspect, the present disclosure further provides a composition comprising entrectinib for use in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

[0035] In one aspect, the present disclosure further provides a composition comprising entrectinib for use in treating or preventing a disease or disorder characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof.

[0036] In one aspect, the present disclosure further provides a use of a composition comprising entrectinib in the manufacture of a medicament for in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

[0037] In one aspect, the present disclosure further provides a use of a composition comprising entrectinib in the manufacture of a medicament for treating or preventing a disease or disorder characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1B provide gene expression data for ZNF536 and TSHZ3 during neural maturation (FIG. 1A) and at the neural progenitor (NPC) stage (FIG. IB) of neurons derived from induced pluripotent cell (iPSCs) from a patient with autism spectrum disorder associated with mutations in the ql2 band of chromosome 19 (19ql2 ASD) and a healthy parent.

[0039] FIGS. 2A-2C show regulation of pathways associated with ZNF536 and TSHZ3 and key genes in respective pathways. FIG. 2A provides a heatmap showing the magnitude of regulation. FIGS. 2B and 2C provide volcano plots showing regulation of key genes in these pathways in patient-derived NPCs (FIG. 2B) and day 20 neurons (FIG. 2C).

[0040] FIGS. 3A-3C provide a transcriptomic comparison of patient-derived neurons to healthy control. FIG. 3A shows a co-expression network generated using pairwise pathway correlation across maturation timepoints. FIG.3B provides a heatmap showing fold change ofAttorney Docket No: RRBS-008 / 01WO 353016-2020key genes and FIG. 3C provides normalized enrichment score of excitatory neuron-specific genes.

[0041] FIGS. 4A-4B show pathways associated with SLC17A6 (FIG. 4A) and synaptic transmission (FIG. 4B) that were enriched in patient-derived neurons compared to healthy control.

[0042] FIGS. 5A-5D provide data showing function of patient neurons versus parent control neurons. FIG. 5A provides total number of spikes; FIG. 5B provides number of bursting electrodes; FIG. 5C provides number of spikes per burst; and FIG. 5D provides prolonged burst duration.

[0043] FIGS. 6A-6B show the transcriptomic response of NGN2-derived glutamatergic neurons to low-dose entrectinib treatment. FIG. 6A shows reversal of pathways mis-regulated in patient-derived neurons, estimated using a cosine correlation coefficient. FIG. 6B shows quantification of the response to entrectinib in treated patient-derived neurons.

[0044] FIGS.7A-7B show pathways associated with neurotransmitter transport (FIG.7A) and synaptic transmission / plasticity (FIG. 7B) that were enriched in entrectinib treated versus vehicle-treated neurons.

[0045] FIGS. 8A-8B show cytotoxicity of entrectinib in NGN2-derived neurons as a function of compound concentration measured using percentage dead cells via the lactate dehydrogenase assay (FIG. 8A) and live cell fraction using high-content imaging (FIG. 8B).

[0046] FIGS. 9A-9B show transcription, by whole blood RNA-seq, in a patient administered entrectinib, of its target gene NTRK2, pathways typically inhibited under approved dosage of entrectinib, and signaling pathways (FIG. 9A), and genes involved in synapse transmission and neurotransmitter transport (FIG. 9B).

[0047] FIGS. 10A-10C show transcription, by whole blood RNA-seq, of genes involved in synapse transmission and neurotransmitter transport in a patient prior to treatment compared to a sibling control (FIG. 10A), following 3 months of treatment compared to pre-treatment (FIG. 10B), and following 6 months of treatment compared to pre-treatment (FIG. 10C).

[0048] FIG. 11 provides raw scores for physician reported Vineland Adaptive Behavior Scales (Version 3) sub-domains pre-treatment for evaluation conducted in 2023 (pink) and posttreatment for evaluation conducted in 2025 (orange).Attorney Docket No: RRBS-008 / 01WO 353016-2020DETAILED DESCRIPTION

[0049] The present disclosure provides compositions and methods of decreasing the expression and / or activity level of: a) one or more pathways associated with neurotransmitter transport; and / or b) one or more pathways associated with synapse function; wherein the method comprises contacting a cell compositions with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. The disclosure also provides compositions and methods for treating autism associated with one or more mutations including the ql2 band of chromosome 19 (19ql2 ASD) in subjects in need thereof, by administering compositions comprising entrectinib or a pharmaceutically acceptable salt thereof.Definitions

[0050] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated by reference in their entirety for all purposes.

[0051] The term “a” or “an” refers to one or more of that entity, z.e., can refer to a plural referent. As such, the terms “a” or “an”, and “one or more” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0052] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, z.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specificallyAttorney Docket No: RRBS-008 / 01WO 353016-2020identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0053] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or step.

[0054] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0055] Certain ranges are presented herein with numerical values being preceded by the term “approximately” or “about.” The terms “approximately” and “about” are used interchangeably and mean a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1%, of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length, inclusive of the endpoints. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth, unless otherwise apparent from context that it is impossible to extend the boundary beyond certain points (e.g., below 0% orAttorney Docket No: RRBS-008 / 01WO 353016-2020above 100% in some cases).

[0056] “Treating” or “treatment” as used herein covers the treatment of the disease, injury, or condition of interest, e.g., a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a biological material, e.g., mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease, injury, or condition from occurring in a biological material, e.g., mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) reducing the severity or duration of the disease, injury or condition, e.g., when it occurs, e.g., in a mammal predisposed to the condition; (iii) inhibiting the disease, injury, or condition, i.e., arresting its development; (iv) relieving the disease, injury, or condition, i.e., causing regression of the disease or condition; or (v) relieving the symptoms resulting from the disease, injury, or condition.

[0057] In certain embodiments, as used herein, the term “prevention” includes inhibiting or impeding the onset or progression of a disease, or reducing the amount of damage caused by a disease. As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably.

[0058] The terms “increase” and “increased” are all used herein generally to mean an increase by a statistically significant amount. However, for avoidance of doubt, “increase” and “increased” mean an increase by at least 10% as compared to a reference level, for example an increase by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold, or any increase between 10% and 1000-fold as compared to a reference level.

[0059] As used herein, the term “comparable level” refers to a level that is in between 50% and 200%, inclusive of end points, of the reference level in the control set of conditions or circumstances, unless otherwise apparent from context that it is impossible to extend the boundary beyond certain points. In some embodiments, a comparable level may be preferably between 60% and 170%, more preferably between 70% and 140%, even more preferably between 80% and 125%, or more preferably still between 90% and 110%, of the reference level. Those of ordinary skill in the art will appreciate the appropriate control set in obtaining the reference level. In general, the appropriate control set shares identical features with the testAttorney Docket No: RRBS-008 / 01WO 353016-2020set except the feature(s) of interest, so that the observed results warrant a reasonable conclusion that any changes in the level observed are caused by or indicative of the variation in the feature(s) that are varied.

[0060] As used herein, a “subject” or an “individual” includes animals, such as human (e.g, human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, e.g. non-human primates, and non- mammals, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0061] “Administration,” “administering” and the like, refer to administration to a subject by a medical professional or by self-administration by the subject, as well as to indirect administration, which may be the act of prescribing a composition of the invention. Typically, an effective amount is administered, which amount can be determined by one of skill in the art. Any method of administration may be used. Administration to a subject can be achieved by, for example, oral administration, in liquid or solid form, e.g. in capsule or tablet form; intravascular injection; intramyocardial delivery; or other suitable forms of administration.

[0062] “Effective amount” as used herein refers to an amount of an agent effective in achieving a particular effect, e.g., increasing transcript and / or protein levels of ZNF536 and / or TSHZ3. In certain embodiments, the increase is about 1.1 to about 10-fold, as compared to the amount prior to or without treatment. In the context of therapeutic treatment of a subject, an effective amount may be, e.g., an amount effective or sufficient to reduce one or more disease symptoms in the subject, e.g., a subject with a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3). The amount of a compound or composition of the invention which constitutes an “effective amount” will vary depending on the compound or composition, the disease, injury or condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0063] A “composition” can comprise an active agent and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient. A composition may be a pharmaceutical composition. In particular embodiments, the compositions are sterile,Attorney Docket No: RRBS-008 / 01WO 353016-2020substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.

[0064] “Pharmaceutically acceptable” is employed herein to refer 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 of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0065] As used herein, the term “pharmaceutically acceptable salt” refers to any salt of an acidic or a basic group that may be present in a compound of the present invention (e.g., entrectinib), which salt is compatible with pharmaceutical administration.

[0066] As is known to those of skill in the art, “salts” of compounds may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acid. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0067] Examples of bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is Ci-4 alkyl, and the like.

[0068] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, K+, Ca2+, NH4+, and NW4+(where W can be a Ci-4 alkyl group), and the like.

[0069] For therapeutic use, salts of the compounds of the present invention (e.g., entrectinib)Attorney Docket No: RRBS-008 / 01WO 353016-2020are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0070] As used herein, “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to and / or absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, such as a phosphate buffered saline solution, emulsions (e.g., such as an oil / water or water / oil emulsions), lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates, fatty acid esters, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0071] As used herein, “solid dosage form” means a pharmaceutical dose(s) in solid form, e.g, tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.

[0072] As used herein, the term “mutation” refers to a substitution, a deletion, an insertion, and / or a translocation. The mutation may comprise a missense mutation, a nonsense mutation, a frameshift mutation, a gene fusion, a gain-of-function mutation, a loss-of-function mutation, a duplication mutation, a mutation that introduces a splice site, and / or a mutation that affects an existing splice site. The mutation may be in one or more genes. The mutation may be naturally occurring. Alternatively, the mutation may be induced or engineered.

[0073] “Pharmaceutical compositions” include compositions of one or more compounds disclosed herein and one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0074] “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in two or more boluses, tablets, or injections. In certain embodiments, a dose can be administered in two or more injections to minimize injection site reaction in an individual. Doses can be stated as the amount of pharmaceutical agent per hour, day, week or month.Attorney Docket No: RRBS-008 / 01WO 353016-2020“Dosage amount” may be used interchangeably with “dose”.

[0075] “Dosage regimen” means a schedule according to which doses of a pharmaceutical agent are provided, e.g., daily, weekly, or other schedule capable of being developed by a person of ordinary skill in the art.

[0076] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology techniques, microbiology, biochemistry and immunology, which are within the scope of those of skill in the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et a!.. 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel etal., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated by reference herein.

[0077] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.Methods of Modulating Expression and / or Activity

[0078] In one aspect, the present disclosure provides methods of modulating the expression and / or activity level of one or more pathways in a cell. The term “pathway”, as used herein, refers to a group of two or more genes or gene products involved in an activity or function of the cell. In some embodiments, a pathway involves interactions between molecules that control a cell function.

[0079] In some embodiments, the method increases the expression and / or activity level of oneAttorney Docket No: RRBS-008 / 01WO 353016-2020or more pathways. In some embodiments, the method decreases the expression and / or activity level of one or more pathways. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing.

[0080] In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with neurotransmitter transport. For example, the method may decrease the expression and / or activity level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, or at least 12 pathways associated with neurotransmitter transport. In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with synaptic transmission. For example, the method may decrease the expression and / or activity level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, or at least 12 pathways associated with synaptic transmission. In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with synapse formation. For example, the method may decrease the expression and / or activity level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, or at least 12 pathways associated with synapse formation.

[0081] The one or more pathways associated with neurotransmitter transport may be any known in the art or described herein. In some embodiments, the one or more pathways associated with neurotransmitter transport comprise: regulation of release of sequestered calcium ion into cytosol; neurotransmitter receptor activity; neurotransmitter receptor internalization; calcium ion homeostasis; sequestering of calcium ion; regulation of calcium ion transmembrane transport; calcium transmembrane import into cytosol; calcium mediated signaling; calcium ion transport; dicarboxylic acid transport; inorganic ion homeostasis; passive transmembrane transporter activity; sensory system development; synaptic transmission glutamatergic; transport vesicle membrane; and / or transport vesicle. In some embodiments, the one or more pathways associated with neurotransmitter transport comprise: amino acid transmembrane transporter activity; dicarboxylic acid transport; inorganic ion homeostasis; L alpha amino acid transmembrane transport; L amino acid transmembrane transporter activity; organic acid transport; organic anion transport; passive transmembrane transporter activity; sensory system development; synaptic transmission glutamatergic; transport vesicle membrane; transport vesicle; and / or transporter complex.Attorney Docket No: RRBS-008 / 01WO 353016-2020

[0082] In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with synaptic transmission. The one or more pathways associated with synaptic transmission may be any known in the art or described herein. In some embodiments, the one or more pathways associated with synaptic transmission comprise: long term synaptic potentiation; positive regulation of synaptic transmission; postsynaptic neurotransmitter receptor activity; postsynaptic signal transduction; postsynaptic membrane; presynaptic modulation of chemical synaptic transmission; regulation of trans synaptic signaling; regulation of long term synaptic potentiation; regulation of synaptic plasticity; regulation of short term neuronal synaptic plasticity; synapse organization; synaptic membrane; and / or synaptic transmission cholinergic.

[0083] In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with synaptic transmission. The one or more pathways associated with synapse formation may be any known in the art or described herein. In some embodiments, the one or more pathways associated with synapse formation comprise: long term synaptic potentiation; positive regulation of synaptic transmission; postsynaptic neurotransmitter receptor activity; postsynaptic signal transduction; postsynaptic membrane; presynaptic modulation of chemical synaptic transmission; regulation of trans synaptic signaling; regulation of long term synaptic potentiation; regulation of synaptic plasticity; regulation of postsynaptic membrane potential; regulation of short term neuronal synaptic plasticity; regulation of synaptic transmission glutamatergic; synapse organization; synaptic membrane; synaptic transmission cholinergic; synaptic cleft; and / or synaptic transmission glutamatergic.

[0084] In some embodiments, the method results in any one or more of the following: increased expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activity of NTN1; reduced expression and / or activity of SLC17A6; increased glutamate transport; and / or increased regulation of calcium ion release.

[0085] In some embodiments, the method comprises contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. The effective amount may be any described herein.

[0086] In some embodiments, the method decreases the expression and / or activity level of one or more pathways associated with neurotransmitter transport. In other embodiments, the method comprises contacting a cell with an effective amount of a composition comprisingAttorney Docket No: RRBS-008 / 01WO 353016-2020entrectinib, or a pharmaceutically acceptable salt thereof. In further embodiments, the method decreases the expression and / or activity level of: one or more pathways associated with neurotransmitter transport; and one or more pathways associated with synaptic transmission. The method further comprises contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

[0087] The cell may comprise one or more mutations, for example in one or more genes. In some embodiments, the cell comprises one or more mutations including the ql2 band of chromosome 19. As used herein, “including the ql2 band of chromosome 19” refers to one or more mutations that are, in whole or in part, in the ql2 band of chromosome 19. In some embodiments, the one or more mutations including the ql2 band of chromosome 19 comprise at least one mutation in the ql2 band of chromosome 19.

[0088] The one or more mutations may comprise one or more types of mutations. In some embodiments, the one or more mutations comprise a deletion. In some embodiments, the one or more mutations comprise a duplication. In some embodiments, the one or more mutations comprise an inversion. In some embodiments, the one or more mutations comprise a missense mutation. In some embodiments, the one or more mutations comprise a frameshift mutation. In some embodiments, the one or more mutations comprise a nonsense mutation. In some embodiments, the one or more mutations comprise a mutation that introduces a splice site. In some embodiments, the one or more mutations comprise a mutation that affects an existing splice site.

[0089] In some embodiments, the at least one mutation in the ql2 band of chromosome 19 comprises a deletion of the ql2 band of chromosome 19 or a portion thereof. The at least one mutation in the q 12 band of chromosome 19 (19ql2) may affect additional regions outside of 19ql2. For example, the one or more mutations including the ql2 band of chromosome 19 may comprise at least one mutation in the ql2 band of chromosome 19 and at least one mutation in the q 13 band of chromosome 19. The at least one mutation in the ql2 band of chromosome 19 and at least one mutation in the q 13 band of chromosome 19 may each comprise a deletion. In some embodiments, the deletion comprises the ql2 band of chromosome 19 or a portion thereof and the ql 3 band of chromosome 19 or a portion thereof.

[0090] The one or more mutations including the ql2 band of chromosome 19 may comprise other types of mutation aside from, or in addition to, a deletion. In some embodiments, the at least one mutation in the q!2 band of chromosome 19 and at least one mutation in the q 13 bandAttorney Docket No: RRBS-008 / 01WO 353016-2020of chromosome 19 each comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, and / or a mutation that affects an existing splice site.

[0091] The one or more mutations including the ql2 band of chromosome 19 (19ql2) may comprise a deletion that affects one or more regions outside of 19ql2. For example, the one or more mutations including the ql2 band of chromosome 19 may comprise a deletion that spans the ql2 band and the ql 3 band of chromosome 19. Alternatively, the one or more mutations including the ql2 band of chromosome 19 may comprise a deletion that spans the ql2 band and a portion of the ql3 band of chromosome 19. In a further example, the one or more mutations including the ql2 band of chromosome 19 comprise a deletion that spans a portion of the ql2 band and a portion of the q 13 band of chromosome 19.

[0092] The size of the deletion may be of any size described herein. For example, the deletion amy comprise at least 0.1 Mb, at least 0.2 Mb, at least 0.3 Mb, at least 0.4 Mb, at least 0.5 Mb, at least 0.6 Mb, at least 0.7 Mb, at least 0.8 Mb, at least 0.9 Mb, at least 1.0 Mb, at least 1.1 Mb, at least 1.2 Mb, at least 1.3 Mb, at least 1.4 Mb, at least 1.5 Mb, at least 1.6 Mb, at least 1.7 Mb, at least 1.8 Mb, at least 1.9 Mb, or at least 2.0 Mb. In some embodiments, the deletion comprises about 0.1 Mb to about 10.0 Mb. For example, the deletion may comprise about 0.4 Mb to about 2.0 Mb. The size of the deletion may alternatively or additionally be characterized by a minimum size. In some embodiments, the deletion comprises at least 0.83 Mb.

[0093] In some embodiments, the one or more mutations including the ql2 band of chromosome 19 comprise a duplication comprising the ql2 band of chromosome 19 or a portion thereof. For example, the one or more mutations including the ql2 band of chromosome 19 may comprise a duplication comprising the ql2 band of chromosome 19 or a portion thereof and the ql3 band of chromosome 19 or a portion thereof. The size of the duplication may be of any size described herein. For example, the duplication may comprise at least 0.5 Mb, at least 1.0 Mb, at least 1.5 Mb, at least 2.0 Mb, at least 2.5 Mb, at least 3.0 Mb, at least 3.5 Mb, at least 4.0 Mb, at least 4.5 Mb, at least 5.0 Mb, at least 5.5 Mb, at least 6.0 Mb, at least 6.5 Mb, at least 7.0 Mb, at least 7.5 Mb, at least 8.0 Mb, at least 8.5 Mb, at least 9.0 Mb, at least 9.5 Mb, or at least 10.0Mb. In some embodiments, the duplication comprises about 0.1 Mb to about 10.0 Mb. For example, the duplication may comprise about 2.0 Mb to about 8.0 Mb. In some embodiments, the duplication comprises about 8.17 Mb. In some embodiments, the one or more mutations including the ql2 band of chromosome 19 comprise a deletion comprising a first portion of the ql2 band of chromosome 19 and a duplication comprising a second portionAttorney Docket No: RRBS-008 / 01WO 353016-2020of the ql2 band of chromosome 19.

[0094] In some embodiments, the one or more mutations including the ql2 band of chromosome 19 comprise an inversion comprising the ql2 band of chromosome 19 or a portion thereof. For example, the one or more mutations including the ql2 band of chromosome 19 may comprise an inversion comprising the ql2 band of chromosome 19 or a portion thereof and the ql 3 band of chromosome 19 or a portion thereof. The size of the inversion may be of any size described herein. For example, the inversion may comprise at least 0.5 Mb, at least 1.0 Mb, at least 1.5 Mb, at least 2.0 Mb, at least 2.5 Mb, at least 3.0 Mb, at least 3.5 Mb, at least 4.0 Mb, at least 4.5 Mb, at least 5.0 Mb, at least 5.5 Mb, at least 6.0 Mb, at least 6.5 Mb, at least 7.0 Mb, at least 7.5 Mb, at least 8.0 Mb, at least 8.5 Mb, at least 9.0 Mb, at least 9.5 Mb, or at least 10.0Mb. In some embodiments, the inversion comprises about 0.1 Mb to about 10.0 Mb. For example, the inversion may comprise about 2.0 Mb to about 8.0 Mb. In some embodiments, the inversion comprises about 8.17 Mb.

[0095] Mutations including the ql2 band of chromosome 19 (19ql2) can disrupt expression and / or activity of genes within or adjacent to this region. 19ql2 mutations can be associated with autism spectrum disorder (ASD), and / or with symptoms consistent with ASD (see Caubit et al. 2016. Nat Genet. 48(11): 1359-1369; Nacinovich et al. 2017. Neuropsychiatr. Dis. Treat.13:2545-2550; Adalat et al. 2010. Pediatr. Nephrol. Berl. Ger. 25(11):2247-2255; Chowdhury et al. 2014. Am. J. Med. Genet. A. 164A (1):62— 69; and Kulharya et al. 1998. Am. J. Med. Genet. 77(5): 391-394; each of which is incorporated by reference herein in its entirety). Genes in 19ql2 include ZNF536, TSHZ3, AcO5394.2, UQCRFS1, POP 4, C Worf 12, CCNE1, and UPJ1. Mutations including the ql2 band of chromosome 19 (19ql2) may comprise mutations in any one or more of these genes.

[0096] In some embodiments, the cell comprises one or more mutations in in one or both alleles of the ZNF536 gene (NCBI Gene ID: 9745) and / or the TSHZ3 gene (NCBI Gene ID: 57616). ZNF536 and TSHZ3 encode zinc-finger proteins and act as regulators of transcription of other genes, along with a myriad of other diverse functions. Both ZNF536 and TSHZ3 have been implicated in a network of complex biological processes that drive neurogenesis and neuronal differentiation of progenitors to neurons during development.

[0097] The human ZNF536 gene has 90 transcripts, each of which encode a protein isoform of ZNF36. In some embodiments, the decreased transcript level of ZNF536 comprises transcript NM_014717.3, NM_001352260.2, NM_001376110.1, NM_001376111.1, XM_047439770.1,Attorney Docket No: RRBS-008 / 01WO 353016-2020XM_011527554.3, XM_047439763.1, XM_047439779.1, XM_047439781.1, XM_047439774.1, XM_047439775.1, XM_047439776.1, XM_017027532.2, XM_047439765.1, XM_017027531.2, XM_047439764.1, XM_047439767.1, XM_047439769.1, XM_047439778.1, XM_047439780.1, XM_047439785.1, XM_017027533.2, XM_047439772.1, XM_017027535.2, XM_047439783.1, XM_047439784.1, XM_047439773.1, XM_047439766.1, XM_017027530.2, XM_024451807.2, XM_017027529.2, XM_047439768.1, XM_017027534.2, XM_017027527.2, XM_017027537.2, XM_017027528.2, XM_017027536.2, XM_011527558.3, XM_047439771.1, XM_047439782.1, XM_017027539.2, XM_017027542.2, XM_047439777.1, XM_047439786.1, XM_011527557.3, XM_011527560.3, XM_017027543.3, XM_054322774.1, XM_054322754.1, XM_054322753.1, XM_054322786.1, XM_054322788.1, XM_054322780.1, XM_054322782.1, XM_054322783.1, XM_054322765.1, XM_054322760.1, XM_054322756.1, XM_054322758.1, XM_054322771.1, XM_054322773.1, XM_054322785.1, XM_054322787.1, XM_054322792.1, XM_054322766.1, XM_054322777.1, XM_054322762.1, XM_054322790.1, XM_054322791.1, XM_054322778.1, XM_054322768.1, XM_054322757.1, XM_054322764.1, XM_054322759.1, XM_054322772.1, XM_054322767.1, XM_054322755.1, XM_054322770.1, XM_054322752.1, XM_054322769.1, XM_054322763.1, XM_054322775.1, XM_054322789.1, XM_054322779.1, XM_054322781.1, XM_054322784.1,054322793.1,054322761.1, XM 054322776.1, and / or XM 054322794.1.

[0098] In some embodiments, the decreased protein level ofZNF536 comprises NP_055532.1, NP_001339189.1, NP_001363039.1, NP_001363040.1, XP_011525856.1, XP_011525859.1, XP_011525860.1, XP_011525862.1, XP 016883016.1, XP_016883017.1, XP 016883018.1, XP 016883019.1, XP_016883020.1, XP_016883021.1, XP_016883022.1, XP_016883023.1, XP_016883024.1, XP_016883025.1, XP_016883026.1, XP_016883028.1, XP 016883031.1, XP_016883032.1, XP_024307575.1, XP_047295719.1, XP_047295720.1, XP_047295721.1, XP_047295722.1, XP_047295723.1, XP_047295724.1, XP_047295725.1, XP_047295726.1, XP_047295727.1, XP_047295728.1, XP_047295729.1, XP_047295730.1, XP_047295731.1, XP_047295732.1, XP_047295733.1, XP_047295734.1, XP_047295735.1, XP_047295736.1, XP_047295737.1, XP_047295738.1, XP_047295739.1, XP_047295740.1, XP_047295741.1, XP_047295742.1, XP_054178727.1, XP_054178728.1, XP_054178729.1, XP_054178730.1,Attorney Docket No: RRBS-008 / 01WO 353016-2020XP_054178731.1, XP_054178732.1, XP_054178733.1, XP_054178734.1, XP_054178735.1, XP_054178736.1, XP_054178737.1, XP_054178738.1, XP_054178739.1, XP_054178740.1, XP_054178741.1, XP_054178742.1, XP_054178743.1, XP_054178744.1, XP_054178745.1, XP_054178746.1, XP_054178747.1, XP_054178748.1, XP_054178749.1, XP_054178750.1, XP_054178751.1, XP_054178752.1, XP_054178753.1, XP_054178754.1, XP_054178755.1, XP_054178756.1, XP_054178757.1, XP_054178758.1, XP_054178759.1, XP_054178760.1, XP_054178761.1, XP_054178762.1, XP_054178763.1, XP_054178764.1, XP_054178765.1, XP_054178766.1, XP_054178767.1, XP_054178768.1, and / or XP_054178769.1.

[0099] The human TSHZ3 gene has 5 transcripts, two of which encode a protein isoform of TSHZ3. In some embodiments, the decreased transcript level of TSHZ3 comprises transcript NM_020856.4, NR_138034.2, NR_138035.2, NR_138036.2, and / or XM_047439132.1.

[0100] In some embodiments, the decreased protein level of TSHZ3 comprises NP_065907.2 and / or XP_047295088.1.

[0101] The reduced expression may involve reduced transcript levels, and / or reduced protein levels. In some embodiments, reduced expression of ZNF536 comprises reduced transcript levels of ZNF536. In some embodiments, reduced expression of TSHZ3 comprises reduced transcript levels of TSHZ3. In some embodiments, reduced expression of ZNF536 comprises reduced protein levels of ZNF536. In some embodiments, reduced expression of TSHZ3 comprises reduced protein levels of TSHZ3.

[0102] The one or more mutations may involve mutations in the ZNF536 gene and / or mutations in the TSHZ3 gene. In some embodiments, the one or more mutations comprise a mutation in the ZNF536 gene. In some embodiments, the one or more mutations comprise a mutation in the TSHZ3 gene. In some embodiments, the one or more mutations comprise a mutation in the ZNF536 gene and a mutation in the TSHZ3 gene. In some embodiments, the one or more mutations comprise any mutation described herein.

[0103] The one or more mutations may involve a deletion, a duplication, an inversion, or a combination thereof. In some embodiments, the one or more mutations comprise a deletion of the ZNF536 gene or a portion thereof. In some embodiments, the one or more mutations comprise a deletion of the TSHZ3 gene or a portion thereof. In some embodiments, the one or more mutations comprise a duplication of the ZNF536 gene or a portion thereof. In some embodiments, the one or more mutations comprise a duplication of the TSHZ3 gene or a portion thereof. In some embodiments, the one or more mutations comprise an inversion of the ZNF536Attorney Docket No: RRBS-008 / 01WO 353016-2020gene or a portion thereof. In some embodiments, the one or more mutations comprise an inversion of the TSHZ3 gene or a portion thereof. In some embodiments, the one or more mutations comprise a deletion, duplication, and / or inversion of the ZNF36 gene or a portion thereof; and a deletion, duplication, and / or inversion of the TSHZ3 gene or a portion thereof. In some embodiments, the one or more mutations result in a mutant gene encoding a truncated protein. In some embodiments, the one or more mutations result in a mutant gene encoding an abnormal copy number of a gene.

[0104] In some embodiments, the one or more mutations in the ZNF36 gene correspond to one or more deletions, duplications, inversions, or amino acid substitutions in the ZNF536 protein (Uniprot Accession No: 015090). In some embodiments, the one or more mutations in the TSHZ3 gene correspond to one or more amino acid substitutions or deletions in the TSHZ3 protein (Uniprot Accession No: Q63HK5). Non-limiting examples of ZNF536 and / or TSHZ3 mutations include those described in Caubit et al. 2016. Nat Genet. 48(11): 1359-1369; Nacinovich et al. 2017. Neuropsychiatr. Dis. Treat. 13:2545-2550; Adalat et al. 2010. Pediatr. Nephrol. Berl. Ger. 25(11):2247-2255; Chowdhury et al. 2014. Am. J. Med. Genet. A. 164A (l):62-69; and Kulharya et al. 1998. Am. J. Med. Genet. 77(5): 391-394; each of which is incorporated by reference herein in its entirety.

[0105] In some embodiments, the one or more mutations result in a mutant ZNF536 gene and / or a mutant TSHZ3 gene encoding a protein with reduced function. Functions of ZNF536 and / or TSHZ may be any known in the art and / or disclosed herein. In some embodiments, reduced function of ZNF536 comprises a mutation that demonstrates reduced effectiveness in one or more functions of ZNF536. In some embodiments, reduced function of TSHZ3 comprises a mutation that demonstrates reduced effectiveness in one or more functions of TSHZ3. Non-limiting examples of functions of ZNF536 include regulating transcription of other genes, regulating neurogenesis, regulating differentiation of neural progenitors to neurons during development, and regulating RARA signaling. In some embodiments, RARA signaling is involved in synaptic homeostasis (see, for example, Shruti et al. eLife. 2022; ll:e79863, which is incorporated by reference herein in its entirety). Non-limiting examples of functions of TSHZ3 include regulating transcription of other genes, regulating neurogenesis, regulating differentiation of neural progenitors to neurons during development.

[0106] Prior to the contacting of the cell with entrectinib, the cell may have altered expression and / or activity of one or more pathways and / or genes. In some embodiments, the cell has, prior to the contacting, increased expression and / or activity of one or more pathwaysAttorney Docket No: RRBS-008 / 01WO 353016-2020or genes. In some embodiments, the cell has, prior to the contacting, reduced expression and / or activity of one or more pathways and / or genes. In some embodiments, the cell has, prior to the contacting, reduced expression and / or activity of one or more of a first group of pathways and / or genes; and increased expression and / or activity of one or more of a second group of pathways and / or genes.

[0107] In some embodiments, the cell has, prior to the contacting: reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation. In some embodiments, the cell has, prior to the contacting, increased expression and / or activity of one or more pathways associated with neurotransmitter transport. In some embodiments, the cell has, prior to the contacting, increased expression and / or activity of one or more pathways associated with synaptic transmission. In some embodiments, the cell has, prior to the contacting, reduced expression and / or activity of NTN1. In some embodiments, the cell has, prior to the contacting, increased expression and / or activity of SLC17A6. In some embodiments, the cell has, prior to the contacting, reduced glutamate transport. In some embodiments, the cell has, prior to the contacting, disregulated calcium ion channel activity. As used herein, disregulated calcium ion channel activity refers to calcium ion release / uptake from the cell that differs in amount relative to a control cell (e.g., calcium ion release that is increased or reduced relative to a control cell), or calcium ion release / uptake with different kinetics compared to a control cell (e.g., calcium ion release with different timing, peak, or average release relative to a control cell). In some embodiments, the cell has, prior to the contacting, reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activity of one or more pathways associated with neurotransmitter transport; increased expression and / or activity of one or more pathways associated with synaptic transmission; reduced expression and / or activity of NTN1; increased expression and / or activity of SLC17A6; reduced glutamate transport; and / or dysregulated calcium ion release. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing.

[0108] In some embodiments, the cell has, prior to the contacting, a disease signature characteristic of 19ql2 ASD. The disease signature may comprise altered expression and / or activity of one or more pathways and / or genes as described herein. In some embodiments, the disease signature is measured by pathway enrichment analysis. In some embodiments, theAttorney Docket No: RRBS-008 / 01WO 353016-2020pathway enrichment analysis is based on RNA sequencing data from whole blood from the subject.

[0109] In some embodiments, the method decreases the expression and / or activity level in the cell of one or more genes and / or pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission, as described herein. The expression and / or activity level may be decreased compared to an untreated cell or a cell treated with vehicle control. In some embodiments, the method decreases the expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission, to a level comparable to a control cell. The control cell may be, for example, a cell that does not comprise one or more mutations in the ql2 band of chromosome 19. Alternatively, the control cell may be a cell that does not comprise one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene. In some embodiments, the control cell is a cell from an individual who does not have 19ql2 ASD. The decrease in expression and / or activity level to a level comparable to a control cell may be measured by any suitable method known in the art. For example, the decrease may be measured by cosine correlation coefficient. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. For example, the expression and / or activity level of one or more pathways is measured by RNA sequencing.

[0110] In some embodiments, the method results in a reversal of the disease signature characteristic of 19ql2 ASD in the cell. In some embodiments, the reversal of the disease signature comprises decreasing the expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission, as described herein. For example, the reversal of the disease signature may comprise increasing expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; reducing expression and / or activity of one or more pathways associated with neurotransmitter transport; reducing expression and / or activity of one or more pathways associated with synaptic transmission; increasing expression and / or activity of NTN1; and / or reducing expression and / or activity of SLC17A6. In some embodiments, the reversal of the disease signature comprises producing an expression and / or activity level of one or more genes and / or pathways described herein, wherein the expression and / or activity level of the one or more genes and / or pathways is comparable to the level in a control cell. The reversal of the disease signature may be measured by any suitableAttorney Docket No: RRBS-008 / 01WO 353016-2020method known in the art. For example, the decrease may be measured by cosine correlation coefficient. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing.

[0111] The method may result in, for example, an average normalized enrichment score for one or more pathways. The normalized enrichment score may be calculated based on fold change values from Sleuth for a control and test comparison using Gene Set Enrichment Analysis (GSEA) using the R ssGSEA package (e.g., according to Subramanian et al. PNAS.102(43): 15545-15550). In some embodiments, a modified GSEA approach may be utilized. For example, gene-level fold changes may be ranked for a full concentration series for entrectinib, then a normalized enrichment score may be calculated for each gene-concentration combination using published procedures for GSEA.

[0112] In some embodiments, the method results in an average normalized enrichment score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport in the cell of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0. The enrichment score may be determined by any suitable method known in the art. In some embodiments, the enrichment score is calculated based on gene enrichment analysis.

[0113] In some embodiments, the cell is a neuron. Prior to being contacted with a composition comprising entrectinib, the cell may have aberrant neuron function. Neuron function may be measured by any suitable method in the art. A non-limiting example of a method for measuring neuron function is multi-electrode array (MEA). For example, the cell may have an increased number of spikes, an increased number of bursting electrodes, an increased number of spikes per burst; and / or an increased burst duration. The number of spikes directly measures neuronal firing rate (reflects overall excitability). A burst is a cluster of spikes that shows network activity of neurons communicating with each other. A burst may be defined as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 spikes within 100ms on a single electrode. A burst may be defined as, for example, at least 5 spikes within 100 ms on a single electrode.

[0114] In some embodiments, the contacting occurs in vivo. In some embodiments, the contacting occurs ex vivo.Methods of TreatmentAttorney Docket No: RRBS-008 / 01WO 353016-2020

[0115] In one aspect, the present disclosure provides methods of treating a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof. The method may comprise administering an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof, to the subject.

[0116] In some embodiments, the subject comprises one or more mutations including the ql2 band of chromosome 19. The one or more mutations including the ql2 band of chromosome 19 may comprise any mutations described herein.

[0117] In some embodiments, the subject comprises one or more mutations in in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene. The one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene may comprise any mutations described herein.

[0118] In some embodiments, the one or more mutations result in a mutant gene encoding a protein with reduced function. The reduced function may be any function of ZNF536 and / or TSHZ3 mutations described herein.

[0119] In some embodiments, the subject is determined to have the one or more mutations by analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence. In some embodiments, the analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutation scanning of select exons, mutation scanning of the entire ZNF536 and / or TSHZ3 coding region, sequence analysis of select exons, sequence analysis of the entire ZNF536 and / or TSHZ3 coding region, or ZNF536 and / or TSHZ3 RNA analysis.

[0120] In some embodiments, the subject has at least one ZNF536 and / or TSHZ3 allele encoding a protein with residual function. Residual function of ZNF536 and / or TSHZ3, as used herein, refers to an ability of a ZNF536 protein and / or TSHZ3 protein to perform at least one function of a wild-type ZNF536 and / or TSHZ3 at a level that is at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, of that of the wild-type ZNF536 and / or TSHZ3 protein. In some embodiments, the function ofZNF536 and / or TSHZ3 is a biological activity. The at least one function ofZNF536Attorney Docket No: RRBS-008 / 01WO 353016-2020and / or TSHZ3 may be measured by any suitable method known in the art.

[0121] In some embodiments, the ZNF536 allele encoding a protein with residual function comprises at least one or more zinc finger domains with residual function. In some embodiments, the TSHZ3 allele encoding a protein with residual function comprises at least one or more zinc finger domains with residual function. In some embodiments, the ZNF536 allele encoding a protein with residual function does not comprise a mutation in a zinc finger domain. In some embodiments, the TSHZ3 allele encoding a protein with residual function does not comprise a mutation in a zinc finger domain. In some embodiments, the ZNF536 and / or TSHZ3 allele encoding a protein with residual function does not comprise a mutation described in Caubit et al. 2016. Nat Genet. 48(11): 1359-1369; Nacinovich et al. 2017. Neuropsychiatr. Dis. Treat. 13:2545-2550; Adalat et al. 2010. Pediatr. Nephrol. Berl. Ger.25(11):2247-2255; Chowdhury et al. 2014. Am. J. Med. Genet. A. 164A (l):62-69; or Kulharya et al. 1998. Am. J. Med. Genet. 77(5): 391-394.

[0122] In some embodiments, the subject has altered expression and / or activity of one or more pathways, prior to the administration of entrectinib or a pharmaceutically acceptable salt thereof. In some embodiments, the subject has increased expression and / or activity of one or more pathways and / or genes. In some embodiments, the subject has reduced expression and / or activity of one or more pathways and / or genes. In some embodiments, the subject has reduced expression and / or activity of one or more of a first group of pathways and / or genes; and increased expression and / or activity of one or more of a second group of pathways and / or genes.

[0123] In some embodiments, the subject has reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation. In some embodiments, the subject has increased expression and / or activity of one or more pathways associated with neurotransmitter transport. In some embodiments, the subject has increased expression and / or activity of one or more pathways associated with synaptic transmission. In some embodiments, the subject has increased expression and / or activity of one or more pathways associated with synapse formation. In some embodiments, the subject has reduced expression and / or activity of NTN1. In some embodiments, the subject has increased expression and / or activity of SLC17A6. In some embodiments, the subject has reduced glutamate transport. In some embodiments, the subject has disregulated calcium ion release. In some embodiments, the subject has reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; increased expression and / or activityAttorney Docket No: RRBS-008 / 01WO 353016-2020of one or more pathways associated with neurotransmitter transport; increased expression and / or activity of one or more pathways associated with synaptic transmission; reduced expression and / or activity ofNTNl; increased expression and / or activity of SLC17A6; reduced glutamate transport; and / or disregulated calcium ion release. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing of one or more cells, one or more tissues, or whole blood from the subject. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing of whole blood from the subject.

[0124] The one or more pathways associated with neurotransmitter transport may be any described herein. The one or more pathways associated with synaptic transmission may be any described herein.

[0125] In some embodiments, the subject has, prior to the administration of entrectinib or a pharmaceutically acceptable salt thereof, a disease signature characteristic of 19ql2 ASD. In some embodiments, the disease signature comprises altered expression and / or activity of one or more pathways and / or genes as described herein. In some embodiments, the disease signature is measured by pathway enrichment analysis. In some embodiments, the pathway enrichment analysis is based on RNA sequencing data from whole blood from the subject.

[0126] In some embodiments, the subject has, prior to the administration of entrectinib or a pharmaceutically acceptable salt thereof, aberrant neuron function. Neuron function may be measured by any suitable method in the art (e.g., EEG). For example, the subject may have an aberrant EEG. Alternatively, cells from the subject may have aberrant neuron function as described herein.

[0127] As noted above, the present disclosure provides methods of treating a subject in need thereof, comprising administering an effective amount of entrectinib or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the subject, having received the method of treatment, will have outcomes including, but not limited to, altered gene expression in blood and / or target tissues. In some embodiments, the subject, having received the method of treatment, will have an improvement or reduction in at least one symptom of a disease or condition.

[0128] In some embodiments, the method results in altered expression and / or activity of one or more genes or pathways in the blood and / or one or more target tissues of the subject.Attorney Docket No: RRBS-008 / 01WO 353016-2020In some embodiments, the altered expression and / or activity comprises reduced expression and / or activity of one or more pathways and / or genes. In some embodiments, the altered expression and / or activity comprises increased expression and / or activity of one or more pathways and / or genes. In some embodiments, the altered expression and / or activity comprises reduced expression and / or activity of one or more of a first group of pathways and / or genes; and increased expression and / or activity of one or more of a second group of pathways and / or genes.

[0129] The altered expression and / or activity of one or more genes or pathways may be measured using any suitable method known in the art. For example, expression of one or more pathways may be measured in blood and / or one or more target tissues of the subject. The expression may be determined by, for example, RNA-seq. Having determined expression of the one or more pathways in a subject, whether expression of the one or more pathways is altered may be assessed by calculating the cosine correlation between the fold changes of subject post-treatment vs. pre-treatment (e.g., the difference in expression between a sample from the treated subject compared to a sample from the subject prior to treatment), and the fold changes of a healthy control vs. subject pre-treatment samples (e.g., the difference in expression between a sample from a person without 19ql2 ASD [for example, an unaffected sibling or parent] and a sample from the subject prior to treatment).

[0130] In some embodiments, the method results in a decreased expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission. In some embodiments, the method results in a decreased expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport and / or one or more pathways associated with synapse formation. The genes and / or pathways associated with neurotransmitter transport and the genes and / or pathways associated with synaptic transmission and / or synapse formation may be any described herein.

[0131] In some embodiments, the method results in increased expression of one or more of APT6V0C, SNCA, SLC4A1, CCL5, FTH1, HLA-DRB5, MT-ATP6, FTP, ITGB3, TM0D1, MT2A, CLU, GP1BB, SLC25A39, SHISA7, and / or BLC2 In some embodiments, the method results in increased expression of APT6V0C. In some embodiments, the method results in increased expression of SNCA. In some embodiments, the method results in increased expression of SLC4A1. In some embodiments, the method results in increased expression of CCL5. In some embodiments, the method results in increased expression of SNCA. In someAttorney Docket No: RRBS-008 / 01WO 353016-2020embodiments, the method results in increased expression of FTH1. In some embodiments, the method results in increased expression of HLA-DRB5. In some embodiments, the method results in increased expression of MT-ATP6. In some embodiments, the method results in increased expression of FTL. In some embodiments, the method results in increased expression of ITGB3. In some embodiments, the method results in increased expression of TM0D1. In some embodiments, the method results in increased expression of MT2A. In some embodiments, the method results in increased expression of CLU. In some embodiments, the method results in increased expression of GP1BB. In some embodiments, the method results in increased expression of SLC25A39. In some embodiments, the method results in increased expression of SHISA7. In some embodiments, the method results in increased expression of BLC21. In some embodiments, the method results in increased expression of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, or at least 15 of APT6V0C, SNCA, SLC4A1, CCL5, FTH1, HLA-DRB5, MT-ATP6, FTL, ITGB3, TM0D1, MT2A, CLU, GP1BB, SLC25A39, SHISA7, and BLC21.

[0132] In some embodiments, expression of the one or more genes and / or pathways is altered (e.g., increased or decreased) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

[0133] In some embodiments, expression of the one or more genes and / or pathways is altered (e.g., increased or decreased) within 1 month of beginning administration of entrectinib. In some embodiments, expression of the one or more genes and / or pathways is altered (e.g., increased or decreased) within 2 months of beginning administration of entrectinib.

[0134] In some embodiments, the expression and / or activity level is altered (e.g., increased or decreased) compared to the level in the subject prior to the method. In some embodiments, the expression and / or activity level is altered (e.g., increased or decreased) to a level comparable to a control subject. In some embodiments, the method decreases the expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport, one or more pathways associated with synaptic transmission, and / or one or more pathways associated with synapse formation, compared to the level in the subject prior to the method. In some embodiments, the method decreases the expression and / or activityAttorney Docket No: RRBS-008 / 01WO 353016-2020level of one or more genes and / or pathways associated with neurotransmitter transport, one or more pathways associated with synaptic transmission, and / or one or more pathways associated with synapse formation, to a level comparable to a control subject.

[0135] In some embodiments, the disease or condition associated with reduced expression and / or function of ZNF536 and / or TSHZ3 is autism spectrum disorder. For example, the disease or condition may be 19ql2 ASD.

[0136] The control subject may be any one of the control subjects described herein. For example, the control subject may be a subject that does not comprise one or more mutations in the ql2 band of chromosome 19. In some embodiments, the control subject is a subject that does not comprise one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene. In some embodiments, the control subject is an individual who does not have 19ql2 ASD. In some embodiments, the control subject is biologically related to the subject with 19ql2 ASD. In some embodiments, the decrease in expression and / or activity level to a level comparable to a control subject is measured by cosine correlation coefficient. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activity level of one or more pathways is measured by RNA sequencing from whole blood from the subject.

[0137] In some embodiments, the method results in a reversal of the disease signature characteristic of 19ql2 ASD. In some embodiments, the reversal of the disease signature comprises decreasing the expression and / or activity level of one or more genes and / or pathways associated with neurotransmitter transport, one or more pathways associated with synaptic transmission, and / or one or more pathways associated with synapse formation, as described herein. In some embodiments, the reversal of the disease signature comprises increasing expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation; reducing expression and / or activity of one or more pathways associated with neurotransmitter transport; reducing expression and / or activity of one or more pathways associated with synaptic transmission; increasing expression and / or activity of NTN1; and / or reducing expression and / or activity of SLC17A6. In some embodiments, the reversal of the disease signature comprises producing an expression and / or activity level of one or more genes and / or pathways described herein, comparable to the level in a control subject. In some embodiments, the reversal of the disease signature is measured by cosine correlation coefficient. The expression and / or activity level of one or more pathways may be measured by any suitable method known in the art. In some embodiments, the expression and / or activityAttorney Docket No: RRBS-008 / 01WO 353016-2020level of one or more pathways is measured by RNA sequencing from whole blood from the subject.

[0138] In some embodiments, the method results in an average normalized enrichment score for one or more pathways in the cell. For example, the method may result in an average normalized enrichments score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport in the cell. The normalized enrichment score may be less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -0.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0. In some embodiments, the enrichment score is calculated based on gene enrichment analysis.

[0139] The method may result in reduced expression and / or activity of certain genes or pathways described here. For example, the method may result in reduced expression of NTRK2. In some embodiments, the method results in increased expression and / or activity of PI3K / AKT signaling pathways. In some embodiments, the method results in increased expression and / or activity of MAPK / ERK signaling pathways. In some embodiments, the method results in reduced expression of NTRK2, increased expression and / or activity of PI3K / AKT signaling pathways, and increased expression and / or activity of MAPK / ERK signaling pathways. In some embodiments, the expression of NTRK2, expression and / or activity of PI3K / AKT signaling, and / or expression and / or activity of MAPK / ERK signaling pathways is measured by RNA sequencing from whole blood from the subject.

[0140] In some embodiments, the method results in decreased expression and / or activity of one or more pathways associated with neurotransmitter transport. In some embodiments, the method results in decreased expression and / or activity of one or more pathways associated with synaptic transmission. In some embodiments, the method results in decreased expression and / or activity of one or more pathways associated with synapse formation. In some embodiments, the method results in decreased expression and / or activity of: a) one or more pathways associated with neurotransmitter transport; b) one or more pathways associated with synaptic transmission; and c) one or more pathways associated with synapse formation. In some embodiments, the decreased expression and / or activity of: a) one or more pathways associated with neurotransmitter transport; b) one or more pathways associated with synaptic transmission; and c) one or more pathways associated with synapse formation is measured by RNA sequencing from whole blood from the subject.

[0141] In some embodiments, the method results in increased expression of one orAttorney Docket No: RRBS-008 / 01WO 353016-2020more of the following genes: APT6V0C, SNCA, SLC4A1, CCL5,FTH1, HLA-DRB5, MT-ATP6, FTL, ITGB3, TM0D1, MT2A, CLU, GP1BB, SLC25A39, SHISA7, and / or BLC21. In some embodiments, the increased expression is measured by RNA sequencing from whole blood from the subject.

[0142] In some embodiments, the subject, having received the method of treatment, will show inhibition of the disease or condition, relief of the disease or condition, and / or relief, improvement, or reduction of at least one symptom of the disease or condition. In some embodiments, the disease or condition is 19ql2 ASD. In some embodiments, the subject, having received the method of treatment, will have an improvement or reduction in at least one symptom of 19ql2 ASD.

[0143] Subjects with 19ql2 ASD often have one or more associated symptoms including, but not limited to, impaired communication, impaired social interaction, speech delay, repetitive behaviors, stereotyped behaviors, impaired social-emotional reciprocity, deficits in nonverbal communicative behaviors, echolalia, restricted interests, hyperreactivity to sensory input, hyporeactivity to sensory input, impaired ability to follow two-step directions, impaired conversationality, feeding difficulties, intellectual disability, epilepsy or seizure disorder, and / or renal tract abnormalities. Severity of individual symptoms or overall disease burden ( / .< ., the cumulative effect of the symptoms affecting a subject with 19ql2 ASD) may be measured by any method known in the art. Non-limiting examples of methods to measure severity of symptoms and / or disease burden in a subject with 19ql2 ASD include those described in Yu et al. Assessment. 2023; 31(1):24-41; and Santos et al. Clinics (Sao Paulo).2024; 14(79): 100343, each of which are incorporated by reference herein in their entirety. Nonlimiting examples of methods to measure severity of symptoms and / or disease burden in a subject with 19ql2 ASD include the Autism Diagnostic Observation Schedule (ADOS-2); the Modified Checklist for Autism in Toddlers (M-CHAT); the M-CHAT, Revised with Follow-Up (M-CHAT-R / F); the Social Communication Questionnaire (SCQ); the Screening Tool for Autism in Toddlers and Young Children (STAT); the Childhood Autism Rating Scale-2 (CARS-2); the Autism Diagnostic Interview-Revised (ADI-R); the Brief Observation of Symptoms of Autism (BOS A); the Parents’ Evaluation of Developmental Status (PEDS); and the Ages and Stages Questionnaire (ASQ).

[0144] In some embodiments, the method results in improvement or reduction of one or more symptoms of 19ql2 ASD. For example, the method may result in improvement of one or more of: communication, social interaction, speech, repetitive behaviors, stereotypedAttorney Docket No: RRBS-008 / 01WO 353016-2020behaviors, social-emotional reciprocity, nonverbal communicative behaviors, echolalia, restricted interests, hyperreactivity to sensory input, hyporeactivity to sensory input, ability to follow two-step directions, conversationality, feeding, intellectual disability, epilepsy or seizure disorder, and / or renal tract abnormalities. In some embodiments, the method results in improved ability to follow two-step directions and / or improved conversationality.

[0145] In some embodiments, the method results in improvement of the subject’s score in any one or more methods used to measure severity of 19ql2 ASD severity of symptoms and / or disease burden. Non-limiting examples of such methods used to measure severity of 19ql2 ASD symptoms and / or disease burden include the DAYC-2 (Developmental Assessment of Young Children, Second Edition) for cognitive functioning, and the caregiver reported Vineland Adaptive Behavior Scales (VABS) for adaptive functioning. The DAYC-2 is a measure of developmental functioning in young children, and it is completed using information obtained through direct observation / assessment as well as caregiver report. The DAYC-2 helps identify developmental delays, determine strengths, and track progress over time. The VABS-3 is based on parental report and is a standardized measure of adaptive behavior - the things that people do to function in their everyday lives.

[0146] In some embodiments, improvement of the subject’s score in one or more method used to measure severity of 19ql2 ASD severity of symptoms and / or disease burden is observed within a certain number of months. For example, improvement of the subject’s score may be observed within 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, or 21 months.

[0147] Improvement of the subject’s score in the DAYC-2 may comprise an increase in any one or more of the following subcategories: cognitive, communication, social-emotional, physical development, and adaptive behavior. For example, the method may result in improvement of the subject’s cognitive score. In some embodiments, improvement of the subject’s score in the DAYC-2 may comprise an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, improvement of the subject’s score in the DAYC-2 may result in a raw score above a particular number. For example, improvement of the subject’s score in the DAYC-2 may result in a raw score of at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 95.

[0148] Improvement of the subject’s score in the VABS may comprise an increase inAttorney Docket No: RRBS-008 / 01WO 353016-2020any one or more of the following categories: communication, daily living skills, socialization, and motor skills. Improvement of the subject’s score in the VABS may comprise an increase in any one or more of the following sub-categories: expressive communication, written communication, personal daily living skills, domestic daily living skills, community daily living skills, interpersonal relationships, play and leisure, coping skills, gross motor skills, and fine motor skills. In some embodiments, improvement of the subject’s score in the VABS may comprise an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in one or more categories and / or subcategories. In some embodiments, improvement of the subject’s score in the VABS may result in a raw score above a particular number in one or more categories or subcategories. For example, improvement of the subject’s score in the VABS may result in a raw score of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50.Methods of Identifying a Subject in Need of Treatment

[0149] In one aspect, the present disclosure provides methods of identifying a subject in need of treatment with an effective amount of a composition comprising entrectinib. In some embodiments, the method comprises analyzing a genome sequence of the subject and determining that the subject comprises a mutation of a gene in the ql2 band of chromosome 19 (19q 12). The genome sequence may be determined using any method disclosed herein. The genome sequence of the subject may comprise a sequence of a gene in 19ql2. For example, the genome sequence may comprise sequence of ZNF536 and / or TSHZ3.

[0150] In some embodiments, the subject comprises one or more mutations including the ql2 band of chromosome 19. The one or more mutations including the ql2 band of chromosome 19 may comprise any mutations described herein. For example, the subject may comprise one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene.

[0151] In some embodiments, the method further comprises administering to the subject an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof. The effective amount may be any effective amount described herein.Compounds

[0152] In some embodiments, the subject is administered an effective amount of entrectinib or a pharmaceutically acceptable salt thereof. Entrectinib (CAS number 1108743-Attorney Docket No: RRBS-008 / 01WO 353016-202060-7), also known as Rozlytrek, and / or RXDX-101 has the chemical structure of formula (I), as depicted below:(I).

[0153] As noted above, the present disclosure provides methods of treatment comprising administering an effective amount of entrectinib or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the method comprises administering entrectinib to the subject orally or parenterally. In some embodiments, the method comprises administering entrectinib to the subject orally. In some embodiments, the method comprises administering entrectinib to the subject parenterally, e.g., intravenously, rectally, or by injection. In some embodiments, the method comprises administering entrectinib to the subject locally, e.g., topically or intramuscularly. In some embodiments, the method comprises administering entrectinib to target tissues. The skilled artisan can determine an appropriate site and route of administration based on factors including, but not limited to, the disease or condition being treated.

[0154] The dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or condition, the characteristics of the subject, and the subject's history. In some embodiments, the effective amount of entrectinib is about 100 mg to about 2000 mg per week. In some embodiments, the effective amount of entrectinib is about 200 mg to about 1000 mg per week. In some embodiments, the effective amount of entrectinib is about 200 mg, about 300 mg, about 400 mg, about 500 mg per day, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg,Attorney Docket No: RRBS-008 / 01WO 353016-2020about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg per week. In some embodiments, the effective amount of entrectinib is about 400 to about 800 mg per week. In some embodiments, the effective amount of entrectinib is about 500 to about 600 mg per week. In some embodiments, the effective amount of entrectinib is about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg per week. In some embodiments, the effective amount of entrectinib is at least 400 mg per week. In some embodiments, the effective amount of entrectinib is no more than 1500 mg per week. In some embodiments, the effective amount of entrectinib is about 560 mg per week.

[0155] In some embodiments, entrectinib is administered once or twice per week. In some embodiments, entrectinib is administered once every 3 days, once every 4 days, once every 5 days, or once every 6 days. For example, entrectinib may be administered once every 5 days. In some embodiments, entrectinib is administered as a first or prime dose on a first day, followed by a second or boost dose on a latter day. For example, entrectinib may be administered every 5 days (e. .3prime dose every 5 days) with a boost dose administered 2 days after each prime dose.

[0156] In some embodiments, the effective amount of entrectinib is measured per dose rather than per week. For example, the effective amount of entrectinib may be about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg per dose. In some embodiments, the effective amount of entrectinib is about 300 mg to about 600 mg per dose. For example, the effective amount of entrectinib may be about 400 mg per dose.

[0157] In embodiments in which a prime dose and a boost dose are administered, the prime dose and the boost dose may be the same or may be different. For example, the prime boost may be about 300 mg to about 600 mg and the boost dose may be about 300 mg to about 600 mg. In some embodiments, the prime boost may be about 400 mg and the boost dose may be about 400 mg. The skilled artisan can determine an appropriate dosage regimen based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0158] In some embodiments, entrectinib is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months,Attorney Docket No: RRBS-008 / 01WO 353016-2020at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.. The skilled artisan can determine an appropriate duration of treatment based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0159] In some embodiments, the method further comprises administering an additional therapeutic agent.

[0160] In some aspects, the present disclosure provides a composition comprising entrectinib for use in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

[0161] In some aspects, the present disclosure provides a composition comprising entrectinib for use in treating or preventing a disease or disorder disclosed herein.

[0162] In some aspects, the present disclosure provides use of a composition comprising entrectinib in the manufacture of a medicament for in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

[0163] In some aspects, the present disclosure provides use of a composition comprising entrectinib in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.Pharmaceutical Compositions

[0164] In various embodiments, a compound of the present invention (e.g., entrectinib) or a pharmaceutically acceptable salt thereof is administered to the subject in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, diluents, or reagents. In some embodiments, the compound is combined with one or more pharmaceutically acceptable carriers, diluents, excipients, and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.

[0165] Examples of carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Solutions or suspensions used for the formulations can include a sterile diluent such as water for inj ection,Attorney Docket No: RRBS-008 / 01WO 353016-2020saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

[0166] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some embodiments, the composition is sterile and may be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In certain embodiments, the pharmaceutical composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0167] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent disclosed herein.

[0168] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV)Attorney Docket No: RRBS-008 / 01WO 353016-2020administration, intramuscular (IM) administration, and intranasal administration. In certain embodiments, the pharmaceutical compositions described herein are administered orally.

[0169] The pharmaceutical compositions described herein may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

[0170] In certain embodiments, the pharmaceutical compositions provided herein are administered to the subject as a liquid dosage form. In some embodiments, the liquid dosage form is a syrup, suspension, solution, emulsion, elixir, or drops. In certain embodiments, the pharmaceutical compositions provided herein are administered to the subject as a solid dosage form. In certain embodiments, the solid dosage form is a capsule, a tablet, a lozenge, a powder, or granules.

[0171] In some aspects, the present disclosure provides a composition for use in treating or preventing a disease or disorder disclosed herein. In some aspects, the present disclosure provides use of a composition of the present disclosure in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.

[0172] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation. General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21sted., Lippincott Williams & Wilkins, 2005.Attorney Docket No: RRBS-008 / 01WO 353016-2020EXAMPLESEXAMPLE 1. IDENTIFICATION OF A GENE SIGNATURE ASSOCIATED WITH 19Q12 ASD

[0173] Mutations including the ql2 band of chromosome 19 have been associated with autism spectrum disorder (19ql2 ASD). These mutations lead to multiple gene-level changes in the ql2 band of chromosome 19. Induced pluripotent stem cells (iPSCs) were generated from a 7-year-old female diagnosed with autism spectrum disorder. Briefly, iPSCs were generated using modified 2D cortical glutamatergic protocol based on the STEMdiff neural system. iPSCs were treated with neural induction media containing SMAD inhibitors and basic fibroblast growth factor and replated onto Matrigel-coated plates on day 5. Over the next several days, media was gradually switched to N2 / B27 media without Vitamin A, Neurobasal medium, N2-B supplement, B27 supplement without Vitamin A, Glutamine, Non-Essential Amino Acids, and beta-mercaptoethanol, supplemented with SMAD inhibitors (LDN193189 (250nM), SB431542 (10pM)), and ascorbic acid (200pM). Neural rosettes formed and were selectively replated on day 12 onto Matrigel-coated plates following trypsinization, cultured for 4 days in N2 / B27 media without Vitamin A, and cryopreserved.

[0174] Optical genome mapping (OGM) showed changes in various regions of the ql2 arm of chromosome 19, as shown in Table 1. Gene-level fold changes are based on RNA extracted from frozen lymphocytes from patient vs. sibling. OGM in the region spanning 27,904,110-31,334,862 showed that at least one uninterrupted copy of every gene existed. This region encodes genes AC005394.2, UQCRFS1, POP4, C19orfl2 and CCNE1. Genomic segments 5 and 6, spanning genes URI1 and ZNF536, were found to be inverted and duplicated and deleted, respectively. Finally, genomic segment 10 encodes TSHZ3 and is deleted. While URI1 has no clear disease association, both ZNF536 and TSHZ3 have been implicated in autism spectrum disorder.Table 1. Results from optical genome mapping of the 19ql2 region in patient.Attorney Docket No: RRBS-008 / 01WO 353016-2020

[0175] iPSCs were also generated from a healthy parent according to the method described above, and the iPSC lines were differentiated to produce neural progenitor cells (NPCs). Hereafter, the iPSCs and other cells derived from the healthy parent are referred to as “control” cells.

[0176] The NPC lines were matured into neurons (using BrainPhys™ Neuronal Medium) for 20 days and gene expression was profiled at multiple time points during maturation using RNA sequencing. Differential expression analysis showed significant downregulation of both ZNF536 and TSHZ3 in affected patient-derived cells, compared to the control cells, starting at the NPC stage and persisting at all neuronal timepoints sequenced (FIG. 1A). The down-regulation of ZNF536 showed significant correlation (p-value = 0.017, calculated using the student’s T-test on days time points 7, 14 and 20) with maturation time point in derived neurons, such that ZNF536 down-regulation increased as neurons matured. The down-regulation of TSHZ3 and ZNF536 was further validated by qPCR (normalized to housekeeping gene glyceraldehyde-3 -phosphate dehydrogenase) at the NPC stage and showed significant (calculated using Student’s T-test between parent and patient) down-regulation in NPCs from the patient with 19ql2 ASD when compared to parent NPCs (FIG. IB). ZNF536 and TSHZ3 levels in the patient were reduced by 40% (p-value = 0.015) and 48% (p-value = 8.07E-05), respectively.

[0177] ZNF536 and TSHZ3 are zinc-finger proteins. ZNF536 plays an important role in neuronal differentiation during early brain development. TSHZ3 represents a hub gene in a module of co-expressed genes that control early cortical development. The mis-regulation of ZNF536 and TSHZ3 during development induces a complex dynamic of gene-level changes that drive and control early neuronal differentiation. Analysis of gene ontology (GO) using gene set enrichment analysis (GSEA) revealed that the top pathways commonly associated withAttorney Docket No: RRBS-008 / 01WO 353016-2020these regulatory genes shared a common theme of being associated with neurogenesis and neuron differentiation (FIG. 2A). The genes involved in these pathways show significantly decreased expression compared to wildtype neurons (FIG. 2B-C) as neurons mature.

[0178] The pathways identified above showed overwhelmingly that neurogenesis in the patient with 19ql2 ASD was prematurely down-regulated. However, since most neurogenesis happens prenatally, these pathways may not be representative of current neurodevelopmental status in the patient.

[0179] To reveal neuronal pathways co-regulated with neurogenesis pathways, we built a co-expression network using pairwise pathway correlation across maturation timepoints. Community detection using a greedy modularity algorithm partitioned the network into eight modules, as shown in FIG.3A. Module 1 (shown as medium-gray dots clustered in the center) contained 389 pathways and was enriched for neurogenesis and neurodevelopmental pathways. In addition, two other neuron-relevant pathway groups were identified, related to excitatory neurotransmission and synapses.

[0180] The first group of pathways represented processes involved in the transport of glutamate, the most abundant excitatory neurotransmitter, and therefore a core function of these neurons. A central figure in this function is SLC17A6 (Solute Carrier Family 17 Member 6). SLC17A6 (Solute Carrier Family 17 Member 6) encodes the Vesicular Glutamate Transporter 2 (vGLUT2) protein, a key marker gene for excitatory neurons. An overexpression of vGLUT2 has been previously linked with an autism spectrum disorder due to MEF2C haploinsufficiency (showing a similar phenotype to the 19ql2 ASD, including reduced neurogenesis and lower neuronal proliferation rates) leading to overexcitability and synaptic dysfunction (Tu et al. Nat. Comm. 2017; 8(1): 1488). As shown in FIG. 3B, SLC17A6 was significantly (q-value < 5E-11) over-expressed in patient-derived neurons during maturation relative to control, indicating dysregulated neurotransmission.

[0181] The second group of pathways focused on synaptic transmission. Neurons in the brain function via the formation and transmission of synapses between each other to perform a wide variety of complex tasks. As shown in FIG.3B, NTN1 was significantly down-regulated (q-value < 1.5E-38) in patient-derived neurons duration maturation relative to control. Down-regulation of NTN1 appeared to be an early indicator of dysregulated synaptic transmission, among the leading edge genes of neurogenesis pathways. As used herein, “leading edge” refers to those genes that most contribute to a pathway being up-regulated orAttorney Docket No: RRBS-008 / 01WO 353016-2020down-regulated. NTN1 or Netrin-1 is implicated in neurodevelopmental processes and inactivation of NTN1 leads to disruption of excitatory synaptic connectivity (Cline et al. eLife.2023; 12:e83760).

[0182] Pathways associated with the gene SLC17A6 were also assessed. Since SLC17A6 is an important marker gene for glutamatergic neurons, its mis-regulation indicates a key disease-specific marker for 19ql2 ASD. Pathway enrichment analysis revealed that SLC17A6 was a top ranked gene for pathways associated with neurotransmitter and anion (glutamate) transport as well as glutamatergic synaptic transmission and sensory system development (FIG. 4A). The protein vGLUT2 encoded by SLC17A6 is known to play an important role in synaptic transmission and plasticity by filling vesicles with the neurotransmitter glutamate (Gilmore et al., Nat. Rev. Neurosci. 2018, 79(3): 123-137; Sorrells et al. Nature 2018, 555(7696):377-381). SLC17A6 imbalance has also been implicated in some sensory processing impairments in SLC17A6 KO mice (Sorrells et al. Nature 2018.555(7696):377-381).

[0183] A majority of these pathways also appeared in Module 1, indicating coregulation between neurogenesis and changes in the SLC17A6-pathways. The movement of glutamate is intricately linked with that of calcium (Ca2+) ions during excitatory synaptic transmission. We therefore also observed that of the 12 pathways in the network associated with the keyword “calcium”, 8 belonged to module 1 and shared ~ 70% genes with the leading edge geneset of SLC17A6-related pathways. A majority of all significant pathways associated with SLC17A6 were primarily up-regulated similar to up-regulation of SLC17A6 observed in the patient (FIG. 4A).

[0184] Pathways associated with synaptic transmission were also assessed. Similar to the results observed with the pathways associated with SLC17A6, a majority of the synaptic pathways also showed higher activity in the patient neurons (FIG.4B). This increased synaptic transmission activity agrees with previously published results showing that increase in vGLUT2 (SLC17A6) eventually leads to enhanced excitatory neurotransmission in mice with MEF2C haploinsufficiency (Tu et al, Nat. Commun. 2017, 5(1): 1488).

[0185] Further, over-activity of excitatory neuron-specific gnees that were derived previous from a single-cell RNA-seq characterization of the human embryonic cerebral cortex (Fan et al. Cell Res. 2018; 28(7):730-745) was observed (FIG. 3C).

[0186] Additional pathways associated with SLC17A6 (Table 2A) and thoseAttorney Docket No: RRBS-008 / 01WO 353016-2020associated with synaptic transmission (Table 2B) were assessed in the patient neurons. Tables 2A-2B show regulation of these additional relevant pathway sets associated with the neuronal phenotype of 19ql2 ASD as a function of neuron maturation stage. Table 2A shows pathways associated with neurotransmitter export and trafficking associated with key glutamatergic marker gene SLC17A6 and Table 2B shows pathways associated with synapse transmission and organization.Table 2A. Pathways associated with neurotransmitter export and trafficking associated with SLC17A6Table 2B. Pathways associated with synapse transmission and organizationAttorney Docket No: RRBS-008 / 01WO 353016-2020

[0187] Both classes of pathways were significantly up-regulated in the patient. The two identified classes of pathways aberrantly regulated in 19ql2 ASD — pathways associated with synaptic transmission and pathways associated with neurotransmission — highlight misregulated neuronal function in the patient. Combined, these pathways can be utilized as a disease signature for 19ql2 ASD. Table 3 shows the two main classes of pathways identified in the disease signature for 19ql2 ASD, the average overall direction of regulation, and the number of individual pathways constituting each class.Attorney Docket No: RRBS-008 / 01WO 353016-2020Table 3. Main classes of pathways identified in 19ql2 ASD disease signature

[0188] To further validate the functional consequences of the 19ql2 transcriptional disease signature, we performed multi-electrode array (MEA) recordings on differentiating neurons derived from patient and parent NPCs between day 13 and day 22 in vitro (the same time frame used for the RNA-seq data). A burst was defined as > 5 spikes within 100 ms on a single electrode; no network-level bursts or synchrony were detected in either group during this period. While the total number of spikes did not differ between patient and parent cultures (FIG. 5 A), patient-derived neurons exhibited clear evidence of hyperexcitability at the level of single-electrode activity. Specifically, patient-derived neurons showed a significantly greater number of bursting electrodes (FIG. 5B), more bursts per electrode (FIG. 5C), and prolonged burst duration (FIG. 5D) compared with controls. These changes occurred without an increase in overall spike counts, indicating that spiking activity was redistributed into more robust bursting events rather than globally elevated firing rates. Taken together, these data indicate that patient neurons display a hyperexcitable bursting phenotype during early in vitro development, characterized by a higher propensity for bursts and extended intra-burst activity in the absence of network-wide synchronization.EXAMPLE 2. HIGH-THROUGHPUT SCREENING OF SMALL MOLECULE MODULATORS OF THE 19Q12 ASD DISEASE SIGNATURE

[0189] A collection of bioactive, FDA approved molecules were profiled in NGN2-derived glutamatergic neurons similar to the familial parent control neurons described above. Neurons were matured for 18 days on a 384-well plate followed by treatment with each respective drug in 10-point concentration responses for 72 hours and sequenced to profile the transcriptomic response to each respective compound-concentration. Parallel to sequencing, a high content imaging assay was used to generate phenotypic profiles of the neurons under drug treatment.

[0190] Following data analysis, the candidate compound entrectinib was identified for further validation. Entrectinib is an FDA approved potent inhibitor of tropomyosin receptorAttorney Docket No: RRBS-008 / 01WO 353016-2020kinases (TRKs) and R0S1 and is approved for use in used for treating neurotrophic tyrosine receptor kinase gene (NTRK)-fusion positive solid tumors.

[0191] Transcriptomic response of NGN2-derived glutamatergic neurons to low-dose entrectinib treatment (entrectinib concentration < 14 nM) showed reversal of pathways in the disease signature for 19ql2 ASD identified in Example 1. The extent of reversal of signature pathways was estimated using a cosine correlation coefficient of the response in 19ql2 ASD patient-derived neurons vs. control and the response in the screened glutamatergic neuron line under entrectinib treatment (FIG. 6A). The cosine correlation coefficient was calculated using the normalized enrichment scores of each class of signature pathways in Table 3 between patient-derived day 20 neurons compared to a parental control line and NGN2-derived neurons treated with entrectinib compared to DMSO vehicle control. A cosine correlation was used since it provides a simple way to identify effects that are most opposite of the input (correlation coefficient < 0). The magnitude of correlation is the extent of this effect, for example a coefficient of 1 indicates a perfectly similar effect and that of -1 indicates a perfectly opposite effect.

[0192] Reversal of signature pathways was also assessed by average enrichment score of each class of signature pathways in Table 3. Average enrichment score for both the synaptic transmission pathways and the neurotransmitter transport pathways were regulated in the opposite direction (normalized enrichment score [NES] <0) during entrectinib treatment, compared to that observed in the patient neuronal model (NES > 0) (FIG. 6B).

[0193] The negative correlation shows that the effect of entrectinib on the 19ql2 ASD disease signature pathways is opposite of the observed activity in 19ql2 ASD patient neurons. Entrectinib normalized the disease signature pathway activity in 19ql2 ASD neurons. Further, the effect of entrectinib on disease signature pathways was dose-dependent such that the magnitude of correlation increased as the dose increased, up to 14 nM.

[0194] Pathways associated with neurotransmitter transport and synaptic transmission and transmission were all regulated in the opposite direction from what was observed in patient neurons. Tables 4A-4B provide normalized enrichment scores of additional pathways associated with neurotransmitter transport (Table 4A) and synaptic transmission / plasticity (Table 4B) in entrectinib treated versus vehicle-treated neurons. The average enrichment scores were reduced in entrectinib-treated neurons, whereas they were increased for both pathways in patient neurons (FIGS. 7A-7B).Attorney Docket No: RRBS-008 / 01WO 353016-2020Table 4A. Normalized enrichment score of pathways associated with neurotransmitter transportTable 4B. Normalized enrichment score of pathways associated with synaptic transmission / plasticityAttorney Docket No: RRBS-008 / 01WO 353016-2020

[0195] Cytotoxicity of Entrectinib during the screen was assessed using the lactate dehydrogenase (LDH) assay (FIG.8A) and live cell fraction was calculated using high-content imaging (FIG. 8B) Entrectinib was only mildly cytotoxic to the cells at concentrations between 14 nM to 3.33 pM and caused almost complete cell death at concentrations above that. No cytotoxicity was recorded at concentrations below 14 nM.

[0196] Concentrations that exhibited signature reversal were much lower than the steady state plasma concentration of 2 pM observed in patients being prescribed the FDA approved dosage for the treatment of cancer (Fischer et al. 2020. Neuro Oncol. 6:819-829). These results suggested that Entrectinib at low concentrations could normalize the disease gene expression profile in 19ql2 ASD.EXAMPLE 3. ADMINISTRATION OF ENTRECTINIB TO A SUBJECT WITH 19Q12 ASD

[0197] A patient with 19ql2 ASD was administered entrectinib (200 mg / doseweek,Attorney Docket No: RRBS-008 / 01WO 353016-2020administered every 4 days, orally) for two months, then administered a higher dose of entrectinib (400 mg / doseweek, administered every 5 days orally) for four additional months, then administered entrectinib at increased frequency (400 mg twice a week / dose twice a week, with a prime dose administered every 5 days followed with a boost dose 2 days after the prime dose). Finally, the patient was administered 400 mg / dose every 5 days. The dosing schedule is shown in Table 5 below. The patient had been determined to have de-novo copy number variation on chromosome 19, as described in Example 1.Table 5. Dosing schedule of entrectinib in patient

[0198] A pre-dose blood collection was performed before treatment started, and two post-dose collections at approximately 3 and 6 months after treatment started were performed (Table 6) Post-dose collections were performed approximately 24 hours after drug intake. Entrectinib dose was doubled between the first and second post-dose collection, as shown in Table 5. A blood sample was also collected from an unaffected age-matched sibling to serve as a control.Table 6: Dates of blood collections used for whole blood RNA sequencing.

[0199] In line with entrectinib’s known mechanism of action, a small but significant down-regulation of its target gene NTRK2 was observed (FIG.9A). This validated that marker genes of the pharmacodynamic effect of entrectinib were captured in whole blood RNA-seq from the patient. Interestingly, pathways normally inhibited under approved dosage ofAttorney Docket No: RRBS-008 / 01WO 353016-2020entrectinib, such as MAPKZERK signaling, signaling by phospholipase-y and PI3K / AKT signaling, were activated in the patient (FIG. 9A).

[0200] In a study of entrectinib-resistant cancer lines, a similar biological activity was observed, with increased PI3K / AKT and MAPKZERK signaling conferring resistance towards entrectinib. This phenomenon is known as the paradoxical drug effect. Various other studies have demonstrated similar paradoxical effects for other drugs at low concentrations compared to higher FDA-approved concentrations. Without being bound by theory, the effect of entrectinib treatment observed in this patient with 19ql2 ASD may be a compensatory mechanism specific to the low dosage. This activity is inversely related to dosage, such that pathway activity decreases at higher doses (e.g., at month 6) (FIG. 9A).

[0201] The leading causative genes associated with the two classes of dysregulated pathways in the patient were examined pre-treatment in the blood of the patient compared to a sibling control using whole blood RNA sequencing. Fold changes were calculated for the sibling control and each of the two post-treated samples compared to an untreated blood collection. A cosine correlation coefficient was calculated for the fold changes of the leading genes in the disease pathways between the sibling control and the post-treatment samples to assess if Entrectinib showed reversal in the patient similar to its activity in the neuronal model (FIG. 9B) Cosine correlation between sibling and treated samples increased with treatment time showing normalization of gene expression profile in the patient towards sibling control levels with entrectinib treatment. Unlike the pharmacodynamic effect shown in FIG. 9A, a significant dose-specific decrease was not observed for the cosine correlation.

[0202] In the patient pre-treatment blood samples, a large number of the identified genes from pathways in the 19ql2 ASD disease signature (identified in Example 1) were down-regulated compared to the sibling control (FIG. 10A). Both 3-month and 6-month post-treated samples showed that entrectinib reversed the expression pattern of these genes (FIGS. 10B-10C) These results indicate that administration of entrectinib was able to reverse the disease signature associated with 19ql2 ASD.

[0203] To measure therapeutic changes post-treatment cognitive, behavioral, and developmental changes were monitored using virtual neuropsychology evaluations with a trained provider. The patient had two neuro-psychology evaluations, one on 6-18-2023 and another on 1-24-2025. The patient was ~ 5 years 11 months at her first evaluation and 7 years 7 months during the second. Both evaluations were additionally performed at the same time ofAttorney Docket No: RRBS-008 / 01WO 353016-2020day to minimize any differences due to time of testing.

[0204] At the evaluations, two tests were administered, the DAYC-2 (Developmental Assessment of Young Children, Second Edition) for cognitive functioning and caregiver reported Vineland Adaptive Behavior Scales (VABS) for adaptive functioning. All assessments were conducted by a single, licensed pediatric neuropsychologist who was trained and certified in its administration. The patient’s primary clinical challenges were in adaptive functioning, specifically, communication and daily living skills. The VABS-II is the gold standard for measuring these exact behaviors. Our therapeutic goal was to improve real-world functional abilities, which VABS is designed to capture. Unlike diagnostic instruments, which measure stable, core autistic traits and are less sensitive to short-term behavioral changes, adaptive behavior scales like the Vineland are well-suited for measuring functional gains over the course of an intervention.

[0205] The DAYC-2 is a measure of developmental functioning in young children. Information is obtained through direct observation / assessment as well as caregiver report. It helps identify developmental delays, determine strengths, and track progress over time. To understand functional skills VABS-3 was administered. This is a measure based on parental report and is a standardized measure of adaptive behavior - the things that people do to function in their everyday lives. In addition to the aforementioned physician-performed evaluations, daily evaluations were also conducted by trained teaching staff at the patient’s school.

[0206] Nineteen months after the pre-treatment evaluation, the clinician reported that the patient presented to be calmer, had dramatically improved communication with caregivers, and did not exhibit any frustration-related disruptive behaviors that had been observed during the baseline evaluation. This was in alignment with the observed reduction in challenging behaviors post-treatment by the parents.

[0207] Quantitatively, the patient’s DAYC-2 overall cognitive score was consistent with a 23 month old child, which was increased from the 17 month old cognitive level that was noted at the baseline evaluation (Table 7). The patient’s cognitive developmental level had plateaued around that of a 2 year old child, however a total of 6 months of progress within 2 years is noteworthy compared to 17 months of progress made within her first 5 years, suggesting at least some acceleration in cognitive development post-treatment.Table 7. DAYC-2 scores at pre- and post-treatment evaluations of patientAttorney Docket No: RRBS-008 / 01WO 353016-2020<

[0208] Significant qualitative improvements were also demonstrated by caregiver reported Vineland measures (FIG. 11). Raw scores for communication all showed growth, with remarkable improvement in written scores that nearly doubled post-treatment, as well as in daily living skills. For example, at the follow-up evaluation the patient was reportedly toilet trained, which had been a major challenge for caregivers. Additionally, the patient made an extraordinary 6 months worth of progress in her personal domain. Caregivers also reported changes in the child’s ability to navigate and function within the community and although assistance and supervision were still required, there was notable progress reported in her ability to communicate her needs and tolerate frustration. Socially, the patient made meaningful qualitative progress, i.e. engaging with other children outside of her parental circle.

[0209] The patient was also independently evaluated at her school by both special education teachers and a speech therapist who evaluated her on her ability to follow two-step directions as well as conversationality but were blinded to the treatment to maintain impartiality. Both metrics measure cognitive development and functional skills. The evaluation began at the end of August just after the dose was decreased. Within a few months, both teachers and the speech therapist reported marked improvement in both metrics.

[0210] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0211] All references, issued patents, and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entireties, for all purposes.EXEMPLARY EMBODIMENTS:Exemplary embodiments of the invention are provided below:Attorney Docket No: RRBS-008 / 01WO 353016-2020Embodiment 1. A method of decreasing the expression and / or activity level of:a) one or more pathways associated with neurotransmitter transport; and / orb) one or more pathways associated with synapse formation;wherein the method comprises contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.Embodiment 2. The method of embodiment 1, wherein the cell comprises one or more mutations including the ql2 band of chromosome 19.Embodiment s. The method of embodiment 1 or embodiment 2, wherein the cell comprises one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene.Embodiment 4. The method of embodiment 2 or embodiment 3, wherein the one or more mutations comprise a deletion, a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, or a mutation that affects an existing splice site.Embodiment 5. The method of any one of embodiments 2 -4, wherein the one or more mutations result in a mutant ZNF536 gene and / or a mutant TSHZ3 gene encoding a protein with reduced function.Embodiment 6. The method of any one of embodiments 1-5, wherein the cell has, prior to the contacting:a) reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation;b) increased expression and / or activity of one or more pathways associated with neurotransmitter transport;c) increased expression and / or activity of one or more pathways associated with synapse formation;d) reduced expression and / or activity of NTN1; and / ore) increased expression and / or activity of SLC17A6.Attorney Docket No: RRBS-008 / 01WO 353016-2020Embodiment 7. The method of any one of embodiments 1-6, wherein the one or more pathways associated with neurotransmitter transport comprise:a) amino acid transmembrane transporter activity;b) dicarboxylic acid transport;c) inorganic ion homeostasis;d) L alpha amino acid transmembrane transport;e) L amino acid transmembrane transporter activity;f) organic acid transport;g) organic anion transport;h) passive transmembrane transporter activity;i) sensory system development;j) synaptic transmission glutamatergic;k) transport vesicle membrane;l) transport vesicle; and / orm) transporter complex.Embodiment 8. The method of any one of embodiments 1-7, wherein the one or more pathways associated with synapse formation comprise:a) long term synaptic potentiation;b) positive regulation of synaptic transmission;c) postsynaptic neurotransmitter receptor activity;d) postsynaptic signal transduction;e) postsynaptic membrane;f) presynaptic modulation of chemical synaptic transmission;g) regulation of trans synaptic signaling;h) regulation of long term synaptic potentiation;Attorney Docket No: RRBS-008 / 01WO 353016-2020i) regulation of synaptic plasticity;j) regulation of postsynaptic membrane potential;k) regulation of short term neuronal synaptic plasticity;l) regulation of synaptic transmission glutamatergic;m) synapse organization;n) synaptic membrane;o) synaptic transmission cholinergic;p) synaptic cleft; and / orq) synaptic transmission glutamatergic.Embodiment 9. The method of any one of embodiments 1-8, wherein the method results in an average normalized enrichment score for synaptic transmission pathways and / or neurotransmitter transport pathways in the cell of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0.Embodiment 10. The method of any one of embodiments 1-9, wherein the cell is a neuron.Embodiment 11. The method of any one of embodiments 1-10, wherein the contact occurs in vivo or ex vivo.Embodiment 12. A method of treating or preventing a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof, comprising administering an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.Embodiment 13. The method of embodiment 12, wherein the subject comprises one or more mutations including the ql2 band of chromosome 19.Embodiment 14. The method of embodiment 12 or embodiment 13, wherein the subject comprises one or more mutations in one or both alleles of the ZNF536 and / or TSHZ3 gene.Attorney Docket No: RRBS-008 / 01WO 353016-2020Embodiment 15. The method of embodiment 14, wherein the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion.Embodiment 16. The method of embodiment 14 or embodiment 15, wherein the one or more mutations result in a mutant gene encoding a protein with reduced function.Embodiment 17. The method of any one of embodiments 13-16, wherein the subject is determined to have the one or more mutations by analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence.Embodiment 18. The method of embodiment 17, wherein the analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutations canning of select exons, mutation scanning of the entire ZNF536 and / or TSHZ3 coding region, sequence analysis of select exons, sequence analysis of the entire ZNF536 and / or TSHZ3 coding region, or ZNF536 and / or TSHZ3 RNA analysis.Embodiment 19. The method of any one of embodiments 12-18, wherein the subject has:a) reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation;b) increased expression and / or activity of one or more pathways associated with neurotransmitter transport;c) increased expression of one or more pathways associated with synapse formation; d) reduced expression of NTN1; and / ore) increased expression of SLC17A6.Embodiment 20. The method of any one of embodiments 12-19, wherein the method results in an average normalized enrichment score for synaptic transmission pathways and / or neurotransmitter transport pathways in the cell of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0.Embodiment 21. The method of any one of embodiments 12-20, wherein the method results in:Attorney Docket No: RRBS-008 / 01WO 353016-2020a) reduced expression of NTRK2;b) increased expression and / or activity of PI3K / AKT signaling pathways; and / or c) increased expression and / or activity of MAPKZERK signaling pathways.Embodiment 22. The method of any one of embodiments 12-21, wherein the method results in decreased expression and / or activity of:a) one or more pathways associated with neurotransmitter transport; and / orb) one or more pathways associated with synapse formation.Embodiment 23. The method of any one of embodiments 12-22, wherein the method results in increased expression of one or more of the following genes: APT6V0C, SNCA, SLC4A1, CCL5,FTH1, HLA-DRB5, MT-ATP6, FTP, ITGB3, TM0D1, MT2A, CPU, GP1BB, SPC25A39, SHISA7, and / or BPC2PEmbodiment 24. The method of any one of embodiments 12-23, wherein the disease or condition is 19ql2 autism spectrum disorder.Embodiment 25 The method of any one of embodiments 12-24, wherein the effective amount of the composition is about 100 mg to about 2000 mg per week.Embodiment 26. The method of embodiment 25, wherein the effective amount of the composition is about 200 mg to about 1000 mg per week.Embodiment 27. The method of embodiment 26, wherein the effective amount of the composition is about 200 mg, about 300 mg, about 400 mg, about 500 mg per day, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg per week.Embodiment 28. The method of any one of embodiments 12-27, wherein the composition is administered orally or parenterally.Embodiment 29. The method of any one of embodiments 12-28, wherein the composition is administered once or twice per week.Embodiment 30. The method of any one of embodiments 12-29, wherein the composition is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months,Attorney Docket No: RRBS-008 / 01WO 353016-2020at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.Embodiment 31. The method of any one of embodiments 12-30, wherein the method results in reduction or improvement of at least one symptom of the disease or condition.Embodiment 32. The method of embodiment 31, wherein the at least one symptom comprises impaired ability to follow two-step directions or conversationality.Embodiment 33. The method of any one of embodiments 12-32, wherein the subject is a mammal.Embodiment 34. The method of embodiment 33, wherein the subject is a human.Embodiment 35. The method of any one of embodiments 12-34, wherein the composition comprises entrectinib.Embodiment 36. The method of embodiment 35, wherein the composition additionally comprises a pharmaceutically acceptable carrier.

Claims

Attorney Docket No: RRBS-008 / 01WO 353016-2020WHAT IS CLAIMED IS:

1. A method of decreasing the expression and / or activity level of:a) one or more pathways associated with neurotransmitter transport; and / orb) one or more pathways associated with synaptic transmission;wherein the method comprises contacting a cell with an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the cell comprises one or more mutations including the ql2 band of chromosome 19.

3. The method of claim 1 or claim 2, wherein the cell comprises one or more mutations in one or both alleles of the ZNF536 gene and / or the TSHZ3 gene.

4. The method of claim 2 or claim 3, wherein the one or more mutations comprise a deletion, a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, or a mutation that affects an existing splice site.

5. The method of any one of claims 2-4, wherein the one or more mutations result in a mutant ZNF536 gene and / or a mutant TSHZ3 gene encoding a protein with reduced function.

6. The method of any one of claims 1-5, wherein the cell has, prior to the contacting: a) reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation;b) increased expression and / or activity of one or more pathways associated with neurotransmitter transport;c) increased expression and / or activity of one or more pathways associated with synaptic transmission;d) reduced expression and / or activity of NTN1;e) increased expression and / or activity of SLC17A6;f) reduced glutamate transport; and / org) disregulated calcium ion release.

7. The method of any one of claims 1-6, wherein the one or more pathways associated with neurotransmitter transport comprise:Attorney Docket No: RRBS-008 / 01WO 353016-2020a) regulation of release of sequestered calcium ion into cytosol;b) neurotransmitter receptor activity;c) neurotransmitter receptor internalization;d) calcium ion homeostasis;e) sequestering of calcium ion;f) regulation of calcium ion transmembrane transport;g) calcium transmembrane import into cytosol;h) calcium mediated signaling;i) calcium ion transport;j) dicarboxylic acid transport;k) inorganic ion homeostasis;l) passive transmembrane transporter activity;m) sensory system development;n) transport vesicle membrane; and / oro) transport vesicle.

8. The method of any one of claims 1-7, wherein the one or more pathways associated with synaptic transmission comprise:a) long term synaptic potentiation;b) positive regulation of synaptic transmission;c) postsynaptic neurotransmitter receptor activity;d) postsynaptic signal transduction;e) postsynaptic membrane;f) presynaptic modulation of chemical synaptic transmission;g) regulation of trans synaptic signaling;h) regulation of long term synaptic potentiation;i) regulation of synaptic plasticity;j) regulation of short term neuronal synaptic plasticity;k) synapse organization;l) synaptic membrane; and / orm) synaptic transmission cholinergic.Attorney Docket No: RRBS-008 / 01WO 353016-20209. The method of any one of claims 1-8, wherein the method results in:a) increased expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation;b) increased expression and / or activity of NTN 1 ;c) reduced expression and / or activity of SLC17A6;d) increased glutamate transport; and / ore) increased regulation of calcium ion release.

10. The method of any one of claims 1-9, wherein the method results in reduced expression and / or activity of one or more pathways associated with synaptic transmission in the cell.

11. The method of any one of claims 1-10, wherein the method results in reduced expression and / or activity of one or more pathways associated with neurotransmitter transport in the cell.

12. The method of any one of claims 1-11, wherein the method results in an average normalized enrichment score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport in the cell of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0.

13. The method of any one of claims 1-12, wherein the cell is a neuron.

14. The method of any one of claims 1-13, wherein the cell has, prior to the contacting, aberrant neuron function characterized by:a) increased number of spikes;b) increased number of bursting electrodes;c) increased number of spikes per burst; and / ord) increased burst duration.

15. The method of any one of claims 1-14, wherein the contact occurs in vivo or ex vivo.

16. A method of treating or preventing a disease or condition characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof, comprising administering an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.Attorney Docket No: RRBS-008 / 01WO 353016-202017. The method of claim 16, wherein the subject comprises one or more mutations including the ql2 band of chromosome 19.

18. The method of claim 16 or claim 17, wherein the subject comprises one or more mutations in one or both alleles of the ZNF536 and / or TSHZ3 gene.

19. The method of claim 18, wherein the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion.

20. The method of claim 18 or claim 19, wherein the one or more mutations result in a mutant gene encoding a protein with reduced function.

21. The method of any one of claims 17-20, wherein the subject is determined to have the one or more mutations by analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence.

22. The method of claim 21, wherein the analysis of the subject’s ZNF536 and / or TSHZ3 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutations canning of select exons, mutation scanning of the entire ZNF536 and / or TSHZ3 coding region, sequence analysis of select exons, sequence analysis of the entire ZNF536 and / or TSHZ3 coding region, or ZNF536 and / or TSHZ3 RNA analysis.

23. The method of any one of claims 16-22, wherein the subject has:a) reduced expression and / or activity of one or more pathways associated with neurogenesis and / or neurodifferentiation;b) increased expression and / or activity of one or more pathways associated with neurotransmitter transport;c) increased expression of one or more pathways associated with synaptic transmission; d) reduced expression of NTN1;e) increased expression of SLC17A6;f) reduced glutamate transport; and / org) disregulated calcium ion release.

24. The method of any one of claims 16-23, wherein the subject has aberrant neuron function characterized by:Attorney Docket No: RRBS-008 / 01WO 353016-2020a) increased number of spikes;b) increased number of bursting electrodes;c) increased number of spikes per burst; and / ord) increased burst duration.

25. The method of any one of claims 16-24, wherein the method results in an average normalized enrichment score for one or more pathways associated with synaptic transmission and / or neurotransmitter transport of less than 0, less than -0.1, less than -0.2, less than -0.3, less than -0.4, less than -.5, less than -0.6, less than -0.7, less than -0.8, less than -0.9, or less than -1.0 within the subject.

26. The method of any one of claims 16-25, wherein the method results in:a) reduced expression of NTRK2;b) increased expression and / or activity of PI3K / AKT signaling pathways; and / or c) increased expression and / or activity of MAPKZERK signaling pathways.

27. The method of any one of claims 16-26, wherein the method results in decreased expression and / or activity of:a) one or more pathways associated with neurotransmitter transport; and / orb) one or more pathways associated with synaptic transmission.

28. The method of any one of claims 16-27, wherein the method results in increased expression of one or more of the following genes: APT6V0C, SNCA, SLC4A1, CCL5,FTH1, HLA-DRB5, MT-ATP6, FTP, ITGB3, TM0D1, MT2A, CPU, GP1BB, SPC25A39, SHISA7, and / or BPC2P29. The method of any one of claims 16-28, wherein the disease or condition is 19ql2 autism spectrum disorder.

30. The method of any one of claims 16-29, wherein the effective amount of the composition is about 100 mg to about 2000 mg per week.

31. The method of claim 30, wherein the effective amount of the composition is about 200 mg to about 1000 mg per week.Attorney Docket No: RRBS-008 / 01WO 353016-202032. The method of claim 31, wherein the effective amount of the composition is about 560 mg per week.

33. The method of any one of claims 16-32, wherein the composition is administered orally or parenterally.

34. The method of any one of claims 16-33, wherein the composition is administered once or twice per week.

35. The method of any one of claims 16-34, wherein the composition is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

36. The method of any one of claims 12-35, wherein the method results in:a) inhibition of the disease or condition;b) relief of the disease or condition; and / orc) relief, reduction or improvement of at least one symptom of the disease or condition.

37. The method of claim 36, wherein the at least one symptom comprises impaired ability to follow two-step directions or conversationality.

38. The method of any one of claims 16-37, wherein the subject is a mammal.

39. The method of claim 33, wherein the subject is a human.

40. The method of any one of claims 16-39, wherein the composition comprises entrectinib.

41. The method of claim 40, wherein the composition additionally comprises a pharmaceutically acceptable carrier.

42. A method of identifying a subject in need of treatment with an effective amount of a composition comprising entrectinib, comprising:a) analyzing a genome sequence of the subject; andb) determining that the subject comprises a mutation of a gene in the ql2 band of chromosome 19 (19ql2).Attorney Docket No: RRBS-008 / 01WO 353016-202043. The method of claim 42, wherein the genome sequence comprises a sequence of a gene in 19ql2, or a portion thereof.

44. The method of claim 42 or claim 43, wherein the genome sequence comprises a sequence of ZNF536 and / or TSHZ3.

45. The method of any one of claims 42-44, wherein the subject comprises one or more mutations in one or both alleles of the ZNF536 and / or TSHZ3 gene.

46. The method of any one of claims 42-45, wherein the method further comprises administering to the subject an effective amount of a composition comprising entrectinib, or a pharmaceutically acceptable salt thereof.

47. A composition comprising entrectinib for use in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

48. A composition comprising entrectinib for use in treating or preventing a disease or disorder characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof.

49. Use of a composition comprising entrectinib in the manufacture of a medicament for in decreasing the expression and / or activity level of one or more pathways associated with neurotransmitter transport and / or one or more pathways associated with synaptic transmission.

50. Use of a composition comprising entrectinib in the manufacture of a medicament for treating or preventing a disease or disorder characterized by reduced expression and / or function of ZNF536 and / or TSHZ3 in a subject in need thereof.

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