Gene therapy for treatment of psychiatric disorders

Abstract

Provided herein are methods for treating psychiatric disorders by gene therapy (e.g. AAV-directed gene therapy) to deliver and express the human SP4 gene in neuronal cells. Treatable psychiatric disorders include schizophrenia, anxiety, obsessive compulsive disorder, or mood disorder.

Description

GENE THERAPY FOR TREATMENT OF PSYCHIATRIC DISORDERSFEDERAL FUNDING

[0001] This invention was made with government support under grant number R01NS135620 awarded by the National Institutes of Health and under grant number I01BX005209 awarded by the Veterans Affairs (VA) Mental Illness Research, Education and Clinical Center (MIRECC). The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No.63 / 710,300 filed October 22, 2024, which is incorporated herein in its entirety.INCORPORATION OF SEQUENCE LISTING

[0003] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on October 21, 2025, is named P15349WOOO.xml and is 8,136 bytes in size (measured in MS-Windows®).BACKGROUND

[0004] Psychiatric disorders are a source of significant morbidity and mortality worldwide. Psychiatric disorders not only can dramatically alter the quality of life for patients but also can increase risk for comorbid diseases and reduce life span of patients. Despite continuous research and advances in medical treatment for various psychiatric conditions, many patients remain unresponsive to current approaches. Current and developing therapies include delivery of biologic agents such as cell therapies or gene therapy. Development of effective gene therapy requires identification of specific molecular targets to modulate with genetic intervention. Gene therapy has the benefit of combining focal surgical modulation of a specific brain region with the biologic specificity of regulating a particular intracellular pathway that may underlie the psychiatric disorder. Given that most psychiatric illnesses are multifactorial in nature, with contributions from multiple susceptibility genes, identification of specific molecular targets for gene therapy can be difficult. Therefore, there is a need for identifying molecular targets for gene therapy of psychiatric disorders.SUMMARY OF THE INVETION

[0005] Restoration of Sp4 gene expression in the neuronal cells is demonstrated to reverse symptoms of psychiatric disorders such as schizophrenia. Compositions and methods described herein provide treatments for psychiatric disorders through administering a therapeutically effective amount of a composition comprising a polynucleotide sequence encoding a human SP4 gene to a patient, wherein the SP4 gene is expressed in the neuronal cells. Compositions include a recombinant adeno-associated viral (AAV) vector that encodes the polynucleotide sequence encoding the human SP4 gene, wherein the AAV vector is able to cross the blood brain barrier. Delivery and expression of the SP4 gene to neuronal cells is useful for increasing SP4 protein in patients that have a truncated or otherwise nonfunctional SP4 gene, which results in a high risk for developing schizophrenia and phenotypes related to schizophrenia. The disclosure therefore relates to using compositions comprising the human SP4 gene that target neuron cells for treating psychiatric disorders.DESCRIPTION OF THE DRAWINGS

[0006] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0007] FIGs. 1A and B illustrate the body weight and PPI of Sp4 hypomorphic mice from the F2 generation with S129 and C57 background. FIGs. 1A-B are a schematic illustrating a cohort of F2 generation mice on mixed S129 and C57 background contains 17 wildtype females, 17 Sp4 hypomorphic females, 25 wildtype males, and 14 Sp4 hypomorphic males.FIG. 1A shows there were significant Sp4 gene effect (F(l,68) = 49.6, p = 1.2e-09) and sex effect (F(l,68) = 131.2, p < 2e-16) on mouse body weight at 2-2.5 months old. FIG. IB shows before virus injection, PPI tests were conducted at 3 different prepulse (Pre_P) levels (pp04, ppO8, ppl6). At 2-2.5 months old, PPI deficits started to emerge in Sp4 hypomorphic mice (Sp4 effect on PPI: F(1 ,68) = 2.38, p = 0.12). Sex effect: F(l,68) = 2.13, p = 0.15.

[0008] FIGs. 2A and B illustrate rescue of PPI deficits in Sp4 hypomorphic mice 3 months after injection of AAV / PHP.eB-Cre virus. PPI was re-tested in wild type and Sp4 hypomorphic mice 3 months after virus injection. FIG. 2A shows that PPI deficits became robust with a Sp4 X prepulse intensity interaction (F(2,l 16) = 5.18, p = 0.007). In verification of the specific hypothesis, significant PPI deficits were confirmed in Sp4 hypomorphic mice at pp04 (one-tailed t-test: p = 0.006, 95 % confidence interval: (7.84, Inf)) and pp08 (one-tailed t-test: p = 0.016, 95 % confidence interval: (4.69, Inf)). Occasionally, PPI could become negative due to facilitation of the prepulse for subsequent pulse startle or become > 100 % due to ppi 6 great suppression of subsequent pulse startle to a level below the background level ofstartle (normal movement of mice in the absence of sound stimuli). A significant interaction between Sp4 X Sex X AAV / PHP.eB-Cre was observed (F(l,58) = 3.83, p = 0.05). Therefore, male and female mice were separately analyzed. FIG. 2B shows that in males, there is a significant Sp4 X prepulse intensity interaction (F(2,60) = 5.26, p = 0.008). To verify Sp4 gene effect, planned comparisons were conducted. Significant PPI deficits were found in male Sp4 hypomorphic mice at pp04 (one-tailed t-test: p = 0.005, 95 % confidence interval: (12.6, Inf)), ppO8 (one-tailed t-test: p = 0.034, 95 % confidence interval: (2.77, Inf)), and a trend at ppl6 (one-tailed t-test: p = 0.095, 95 % confidence interval: (- 4.86, Inf)). There was a significant Sp4 X AAV / PHP.eB-Cre interaction (F(l,30) = 4, p = 0.05). **p < 0.01; *p < 0.05; 11 < 0.1.

[0009] FIGs. 3A to 3C illustrates PPI rescue in individual Sp4 hypomorphic mice inj ected with the AAV / PHP.eB-Cre before and after the rescue. PPI was tested in Sp4 hypomorphic mice before AAV / PHP.eB-Cre virus injection (Before) and 3 months after the virus injection (After). One-tailed paired Student t-test revealed a trend of higher PPI at pp04 (p = 0.09, 95 % confidence interval: (-Inf, 3.06)). FIG.3A shows no PPI difference at pp08. FIG.3B shows a trend of higher PPI at ppl6 (p = 0.08, 95 % confidence interval: (-Inf, 5.54)). FIG.3C shows prepulse levels in Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre virus.

[0010] FIGs. 4A and B illustrate rescue of ketamine hypersensitivity in Sp4 hypomorphic mice 3 months after injection of AAV / PHP.eB-Cre virus. The mice were habituated in VT chambers for 30 min before ketamine injection (50 mg / kg). Mouse locomotion was recorded for a further 60 min after ketamine injection. Due to sex effect, male and female mice were analyzed separately. FIG. 4A shows in male mice, mouse locomotion was recorded in different time blocks (10 min / block). Injection of ketamine significantly increased locomotor activity in all mice. A significantly larger increase of locomotor activity (F(l,30) = 5.2, p = 0.029) was observed in Sp4 hypomorphic mice following ketamine injection. Significant Sp4 effects were observed in block 4 (F(l,60) = 4.68, p = 0.035), block 5 (F(l,60) = 7.61, p = 0.0077), and block 6 (F(l,60) = 6.04, p = 0.017). There was a trend (F(l, 180) = 3.39, p = 0.06) of Sp4 X AAV / PHP.eB-Cre X Ketamine interaction. FIG. 4B shows total distance traveled from block 4 to 8 after ketamine injection. A significant Sp4 effect (F(l,60) = 5.62, p = 0.021) was detected on total distance traveled. After ketamine administration, there was a reduction of total distance traveled in Sp4 hypomorphic mice injected with the AAV / PHP.eB (group mean: 8604.50 cm) in comparison with the control Sp4 hypomorphic mice (group mean: 11699.91 cm), but the reduction was not significant (one-tailed t-test, p = 0.22, 95 %confidence interval: (- 3819.51, Inf)). Error bar: standard error of the mean. **p < 0.01; *p < 0.05.

[0011] FIGs. 5A and B illustrates no rescue of contextual memory deficits in Sp4 hypomorphic mice. There were no main effects of sex or interactions with sex on contextual memory, thus sexes were combined. One mouse died before the fear conditioning tests, which resulted in the following group sizes: Wildtype-Vehicle (n = 20), wildtype- AAV / PHP.eB-Cre (n = 19), Sp4 hypomorphic-Vehicle (n = 12), Sp4 hypomorphic- AAV / PHP.eB-Cre (n = 15). After Day 1 fear acquisition with foot shock, contextual fear memory was assessed on Day 2.FIG. 5A Freezing percentages were calculated for the first two blocks, 2 min for each block. A shows significant Sp4 effect (F(l, 58) = 9.01, p = 0.004) was observed on context memory in MinO-2 (F(l,58) = 9.75, p = 0.003) and Min2-4 (F(l,58) = 6.52, p = 0.013) blocks. FIG. 5B Freezing percentages were calculated for the combined two blocks (MinO-4). There was no increase of context memory in Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre (group mean: 43.4 %) in comparison with the control Sp4 hypomorphic mice (group mean: 46.4 %) (one-tailed t-test: p = 0.38). Error bar: standard error of the mean. **p < 0.01; *p < 0.05.

[0012] FIG. 6 Illustrates transduction of adult mouse brains by AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE virus. The AAV / PHP.eB virus was injected via mouse tail vein. Behavioral tests were conducted 3 months post-injection. After the behavioral tests, neuronal cells transduced by the AAV / PHP.eB virus were analyzed using immunohistochemical staining of EGFP proteins in different brain regions of both control and the AAV / PHP.eB-Cre mice. For transduction efficiency in this cohort of mice: Thalamus > Striatum > Cortex > Hippocampus. Bar: 100 pm.

[0013] FIG 7. illustrates the Sp4 gene effect (F(l,68) = 0.06, p = 0.81) or sex effect (F( 1 ,68) = 0.35, p = 0.56) on startle or startle habituation in a cohort of the F2 generation mice.

[0014] FIG 8. illustrates the balanced means of the 2 subgroups to avoid PPI differences that could be randomly generated during splitting.

[0015] FIGs. 9A and B illustrate that a significant Sp4 gene effect on PPI (F( 1 ,28) = 2.06, p = 0.16) or a significant interaction between Sp4 gene and prepulse intensity (F(2,56) = 0.72, NS) was not observed in female Sp4 hypomorphic mice (FIG. 9A). FIG. 9B shows that significant PPI deficits were observed in female Sp4 hypomorphic mice from different cohorts of the F2 generation mice on S129 and C57 background.

[0016] FIGs. 10A to 10C PPI rescue in individual Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre before and after the rescue. FIG 10A does not show significant Sp4gene effects on PPI (F(l ,28) = 2.06, p = 0.16) or a significant interaction between Sp4 gene and prepulse intensity (F(2,56) = 0.72, NS). FIG 10B shows significant PPI deficits were observed in female Sp4 hypomorphic mice from different cohorts of the F2 generation mice on S129 and C57 background.

[0017] FIG. 11 illustrate that in female mice, there was a significant Sp4 gene effect (F(l,29) = 5.76, p = 0.023) on locomotion.

[0018] FIGs. 12A and B illustrate the body weight and PPI of Sp4 hypomorphic mice from the F2 generation with S129 and C57 background. FIGs. 12A-B are a schematic illustrating a cohort of F2 generation mice on mixed S129 and C57 background contains 20 wildtype females, 10 Sp4 hypomorphic females, 19 wildtype males, and 22 Sp4 hypomorphic males. FIG. 12A shows there were significant Sp4 gene effect (F(l,68) = 91.3, p = 3.4e-14) and sex effect (F(l,68) = 67.9, p < 8.12e-12) on mouse body weight at 2-3 months old. FIG.12B shows before virus injection, PPI tests were conducted at 3 different prepulse (Pre_P) levels (pp04, ppO8, ppi 6). At 2-3 months old, PPI deficits started to emerge in Sp4 hypomorphic mice (Sp4 effect on PPI: F(1 ,68) = 8.54, p = 0.0047). Sex effect: F(l,68) = 0.161, p = 0.69.DETAILED DESCRIPTION

[0019] Compositions and methods are described herein for making and using compositions comprising the human SP4 gene that target neuron cells for treating psychiatric disorders. Sp4, a homolog of the Drosophila buttonhead (btd) gene, belongs to the Spl family of transcription factors. Spl, a ubiquitous transcription factor, recognizes the DNA sequence 5'-GGGCGG-3' termed GC-box that is often found -40-100 nucleotides upstream of the transcription start site of a variety of genes and activates their expression. Both Spl and Sp4 bind the same GC-box with the same affinity. In the brain, however, Sp4 and a majority of other schizophrenia-risk genes are specifically expressed in neurons, but not glia. The Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) Consortium, the largest exome sequencing project, identified 10 truncated genes above the exome-wide significance that confer very high risk for schizophrenia (odds ratios 3-50).6Among these top 10 risk genes, truncation of one copy of the SP4 gene has an odds ratio of 9.37 (3.38-29.7) for schizophrenia (SCZ). Importantly, SP4 and GRIN2A (encoding NMDAR2A subunit) are the only two genes shared between the top 10 risk genes from the SCHEMA and the 69 single genes prioritized by parallel GWAS studies.7This not only enhances the confidence of the two risk genes; but also implies that more subtle common variants of these two genes with a partial loss-of-function are likely involved in the pathogenesis of schizophrenia in the general population of patients. Recently, SP4 was suggested to act as a hub of network in the regulation of many other SCZ-risk genes.10Therefore, SP4 could be an excellent target gene for schizophrenia drug development.

[0020] Sp4 hypomorphic mice were generated where Sp4 expression was greatly reduced in mouse brain and found that the Sp4 hypomorphic mice display several schizophrenia-related behavioral abnormalities, among which deficient prepulse inhibition (PPI), deficient context memory, and hypersensitivity to NMD AR antagonists are the most robust phenotypes.3,8,11'12Genetic restoration of Sp4 gene expression in forebrain GABAergic inhibitory, but not forebrain excitatory, neurons during embryogenesis rescues hypersensitivity to ketamine, an NMDAR antagonist.2Investigating whether restoration of Sp4 expression in young adult Sp4 hypomorphic mice may rescue these schizophrenia-related phenotypes is, however, needed for drug development.

[0021] The AAV / PHP.eB vector can efficiently cross blood-brain barriers to transduce the whole brain of C57 mice after intravenous injectionl but less efficiently on B6C3 genetic background.5Whether Sp4 restoration by Cre in young adult Sp4 hypomorphic mice may rescue PPI deficits, ketamine hypersensitivity, and context memory deficits was investigated by intravenously injecting AAV / PHP.eB virus carrying Cre recombinase into young Sp4 hypomorphic mice. Such rescue studies are necessary for evaluation of Sp4 as a drug target gene for schizophrenia patients after diagnosis. If validated, small molecule drugs increasing Sp4 expression could be developed.

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0023] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0024] As used herein, the terms “comprises”, “comprising”, “includes”, “including”, “has”, “having”, “contains”, “containing”, “characterized by”, or any other variation thereof, are intended to encompass a non-ex elusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a protein, a pharmaceuticalcomposition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the protein, pharmaceutical composition, and / or method.

[0025] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0026] When introducing elements of the present invention or the preferred embodiment s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0027] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0028] It is understood that aspects and embodiments of the invention described herein include “consisting of’ and / or “consisting essentially of’ aspects and embodiments.

[0029] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about”another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0030] As used herein, and unless otherwise specified, the term "subject" or “patient” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In specific embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.

[0031] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.

[0032] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient asseparate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0033] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0034] As used herein the term “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which demethylation compound(s), is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0035] As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0036] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms (e.g. reversal) associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition. For example, symptoms of schizophrenia can include positive or negative symptoms. Positive symptoms of schizophrenia include delusions, visual and / or audible hallucinations, disorganized speech, disorganized behavior, deficient prepulse inhibition (PPI), and / or hypersensitivity to N-Methyl-D-aspartate receptor (NMD AR) antagonists (e.g. ketamine). Negative symptoms of schizophrenia include flat affect, apathy, social withdrawal, and / or cognitive impairment. Treatment of positive and / or negative symptoms of schizophrenia in a patient can be a full or partial reversal (also referred to as an improvement of symptoms) of the symptoms in that patient.

[0037] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0038] The pharmaceutical compositions of the disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired. The pharmaceutical compositions are typically suitable for parenteral administration, wherein administration includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intranasal, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraocular, intradermal, or infusions. In some embodiments, the compositions of the present disclosure comprise intravenous administration. In some embodiments, the compositions of the present disclosure comprise injection of the composition into an area of the brain during a brain surgery.

[0039] Human SP4 is a high-risk gene for schizophrenia and potentially regulates many other schizophrenia-risk genes in the pathogenesis of schizophrenia. Sp4 hypomorphic mice recapitulated several behavioral phenotypes related to schizophrenia. Among these phenotypes, deficient PPI, ketamine hypersensitivity, and deficient context memory are the most robust ones and have been used in previous genetic rescue studies2. Herein, it was investigated whether restoration of Sp4 expression in young adult Sp4 hypomorphic mice can rescue the schizophrenia-related phenotypes in adults. AAV / PHP.eB virus carrying the Cre gene was used to cross blood-brain barriers and restore Sp4 expression in transduced neuronal cells in young adult Sp4 hypomorphic mice. The results demonstrate that Sp4 partial restoration ameliorates PPI deficits and ketamine hypersensitivity in adult Sp4 hypomorphic mice. However, improvement of deficient context memory in Sp4 hypomorphic mice transduced with AAV / PHP.eB virus carrying the Cre gene was not observed. Since overall virus transduction was less efficient, particularly in hippocampal CAI, such poor transduction may contribute to lack of robust rescue of the schizophrenia-related phenotypes in Sp4 hypomorphic mice.

[0040] In previous studies, Sp4 hypomorphic mice were generated as the Fl generation between S129 and Black Swiss background2,3. These Fl Sp4 hypomorphic mice were healthier and only slightly smaller than their wildtype siblings with little genetic segregation, and no sex effect was observed on PPI, ketamine hypersensitivity, and context memory. However, it was unknown whether AAV / PHP.eB virus can efficiently transduce the brains of S129 or Black Swiss mice. Therefore, Sp4 hypomorphic mice on mixed S129 and C57 background were bred since C57 background is known to be more permissive for efficient transduction of adult mouse brains by the AAV / PHP.eB5. PPI deficits in this F2 generation of mice on mixed SI 29 and C57 background were less robust than PPI deficits in the Fl mice on SI 29 and Black Swiss background2,3,11. Genetic segregation of the F2 generation of mice likely caused large variations in behavioral phenotypes and sex effects were observed, which decreased thestatistical power despite having a large cohort. In the future, a larger cohort of Sp4 hypomorphic mice on pure C57 background will be helpful to replicate the rescue of schizophrenia-related phenotypes at young adult stage. Additionally, Sp4 hypomorphic mice on C57 pure genetic background could be more efficiently transduced by the AAV / PHP.eB virus5than the F2 generation of mice that had segregation of C57 background in individual mice.

[0041] The AAV / PHP.eB-Cre virus was intravenously injected into wildtype and Sp4 hypomorphic mice at 2-2.5 months old. At this stage, PPI deficits started to emerge. Partial restoration of Sp4 expression appeared to attenuate PPI deficits in adult Sp4 hypomorphic mice.

[0042] SP4 is a high-risk schizophrenia gene, and its common variants are associated with schizophrenia in the general population of patients. SP4 also appears to regulate many other risk genes for schizophrenia. Rescue of schizophrenia-related behavioral phenotypes that are developing or developed in adult Sp4 hypomorphic mice is needed for schizophrenia drug discovery. Success of such rescue experiments will validate Sp4 as a drug target gene. High-throughput screening of chemical libraries for small molecules enhancing Sp4 expression may provide potential drug candidates. The Lacz gene controlled by endogenous Sp4 promoter will provide a reporter for such screening in primary neuronal culture from Sp4 hypomorphic mice. In the future, with the development of AAV serotypes that can cross blood-brain barriers in humans, AAV-based gene therapy may be feasible to increase Sp4 expression in the brains of patients with schizophrenia to ameliorate disease phenotypes.

[0043] In various embodiments, the compositions described herein relate to gene therapy vectors or constructs comprising a human Sp4 gene (SEQ ID NO: 1), or derivatives and / or mutants thereof, which are operably linked to at least a promoter element that is capable of being expressed in a tissue of the central nervous system, such as neuronal cells. In certain embodiments, the promoter can be a neuronal-specific calmodulin promoter or derivative thereof. In certain embodiments, the promoter can be a neuronal-specific human Synapsin I promoter (SYN). In other embodiments, the promoter can be a strong hybrid promoter of CMV enhancer (E) and human Synapsin I promoter (eSYN). In other embodiments, the promoter can be a constitutive promoter, e.g., an EFla constitutive promoter or derivative thereof, which is capable of being expressed in neuronal tissues. As demonstrated by a reduction to practice using accepted schizophrenia mouse models, the gene therapy vectors of the present invention were effective in treating schizophrenia symptoms.

[0044] In various embodiments, the nucleic acid constructs may be delivered into the brain using a nonviral vector such as cationic polymers that efficiently condense nucleic acids into nanoparticles, termed “polyplexes,” by self-assembly via electrostatic interactions. An attractive feature of many polymeric vectors is that they contain functional groups that can be modified with ligands such as cell-targeting molecules. Examples of cationic polymers for gene delivery are provided in D. W. Pack, A. S. Hoffman, S. Pun, and P. S. Stayton. Design and development of polymers for gene delivery. Nat. Rev. Drug. Discov.4:581-593 (2005), which is incorporated herein by reference.

[0045] In various embodiments, the nucleic acid constructs may be delivered into the brain using cationic lipids. The various commercially-available cationic lipid formulations are typically composed of some combination of a cationic lipid such as l,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-methyl-4-(dioleyl)methylpyridinium (SAINT-2), 3P-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), or GS1, as well as the neutral lipid di oleoylphosphatidylethanolamine (DOPE) and cholesterol (see (30) for a recent review describing cationic lipids and lipoplexes for gene delivery). The simple addition of polyanionic nucleic acids to mixtures of cationic lipids or liposomes results in their selfassembly into particles termed “lipoplexes.” Examples of cationic lipids for gene delivery are provided in L. Wasungu, and D. Hoekstra. Cationic lipids, lipoplexes and intracellular delivery of genes. J. Control. Release.116:255-264 (2006), which is incorporated herein by reference.

[0046] In various embodiments, the nucleic acid constructs may be engineered into any suitable gene therapy viral vector, such as a retrovirus, lentivirus adenovirus or adeno-associated virus (AAV) vector, nucleic acid such as plasmid DNA, peptide nucleic acids, or mRNA, including mRNAs that contain modified bases to enhance in vivo expression. All forms of nucleic acids can be delivered without further modification, such as naked DNA, or packaged into nanoparticles or lipid nanoparticles and delivered in an appropriate fashion to produce Sp4 expression. In a particular embodiment, the background gene therapy vector is an adeno-associated viral (AAV) vector.

[0047] In some aspects, a recombinant adeno-associated viral (rAAV) vector of the disclosure comprises a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence of a Sp4 gene, or a fragment thereof. In embodiments, the Sp4 gene is a human Sp4 gene.

[0048] In some aspects, the rAAV viral vector or the pharmaceutical composition is administered to the subject at a dose ranging from about l.OxlO12vg / kg to about 2.5x1014vg / kg. In some aspects, the rAAV viral vector or the pharmaceutical composition is administered to the subject at a dose ranging from about l.OxlO12vg / kg to about 5.0x1013 vg / kg.

[0049] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.EXAMPLESExample 1. Materials and Methods.Mouse strains.

[0050] Sp4 heterozygous mice on S129 background were bred with C57 mice to produce Fl generation of Sp4 heterozygous mice on mixed S129 and C57 background. After breeding the Fl generation of Sp4 heterozygous mice with each other, F2 generation of Sp4 hypomorphic mice were produced on mixed and segregated S129 and C57 background. Seventy-three mice were housed in a climate-controlled animal colony with a reversed day / night cycle. Food (Harlan Teklad) and water were available ad libitum, except during behavioral testing. All testing procedures were approved by the UCSD Animal Care and Use Committee (IACUC Protocol: S04190M) prior to the onset of the experiments. Mice were maintained in American Association for Accreditation of Laboratory Animal Care-approved animal facilities at UCSD. These facilities meet all Federal and State requirements for animal care.AAV / PHP.eB virus.

[0051] The AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE viruses (9.92 x 1012GC / ml) were purchased from Vector Biolabs. A strong hybrid promoter consisting of CMV enhancer (E) and human Synapsin I promoter ( eSYN) drives expression of both EGFP and Cre proteins via T2A self-cleaving peptide in neuronal cells only. The virus was diluted with IX PBS, and 0.15 ml diluted virus solution containing ~2 XI 011genome copy (GC) virus was injected into each mouse via tail vein. Control mice were injected with the same volume of IX PBS vehicle via tail vein.Prepulse inhibition.

[0052] Startle reactivity and prepulse inhibition (PPI) were measured with startle chambers (SR-LAB, San Diego Instruments) as described in previous publications2,3. The background noise level was 65 dB, and the pulse was 120 dB for 40 ms. The prepulseintensities were 69, 73, and 81 dB (4, 8, and 16 dB above the 65-dB background noise, respectively; hereafter referred to as pp4, pp8, ppi 6), delivered 80 ms before the pulse. The amount of startle to sound stimulation was calculated by subtracting background startle in the absence of sound stimulation. The amount of PPI was calculated as a percentage score for each acoustic prepulse intensity: %PPI = 100 % x {1 - [(Startle response for "prepulse + pulse") / (Startle response for "pulse-alone")]}.Video-tracking locomotion tests.

[0053] Locomotor activity was measured in an open arena (40 x 40 x 40 cm) and tracked and analyzed using Ethovision Tracking Software (Noldus, Leesburg, VA, USA) as previously described2,3. Mice were tested during their dark cycle with lights on for the entire duration of the test. They were first acclimated to the testing room for 60 min. After acclimation, mice were placed in their arena for 30 min of habituation. They then received ketamine (described below) or saline and were placed back in their arena for another 60 min.Ketamine.

[0054] Ketamine was dissolved in saline and administered i.p. at a volume of 5 ml / kg after 30 min habituation in the VT arena2. The dose of ketamine was 50 mg / kg. A within-subjects crossover design was used for ketamine studies, with 2 weeks between ketamine treatments.Fear conditioning.

[0055] Fear conditioning was conducted as described in a previous publication9using 4 automated fear conditioning chambers (Med Associates Inc., St. Albans, Vermont). The percentage of time-freezing was determined using video analysis software (Video Freeze, Med Associates Inc.). On Day 1 fear acquisition, foot shocks were delivered through the 36 stainless steel rods located on the floor of the chamber. On Day 2, mice were re-exposed to the conditioning chamber to assess context memory without shocks or tones.Immunohistochemistry.

[0056] Immunohistochemistry (IHC) was conducted as previously described3,4to investigate transduction of adult mouse brains by the AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE virus. Sagittal paraffin brain sections were generated for immunohistochemical staining of EGFP proteins carried by the virus. Mouse anti-EGFP monoclonal antibody (ThermoFisher, MAI-952) was used as the primary antibodies for immunohistochemistry with a dilution at 1:1000. ImmPRESS peroxidase-micropolymer conjugated horse anti-mouse IgG (H + L) (Vector Labs, MP-7402) was used as the secondary antibody. Chromogenic reaction was conducted with ImmPACT NovaRED Peroxidase Substrate (Vector Labs, SK-4805). Slides were mounted with Cytoseal 60 mounting medium (Richard-Allan Scientific, 8310-16).Statistical analysis.

[0057] Statistical methods were determined before experiments were conducted to test the hypotheses of Sp4 gene effects on PPI, ketamine sensitivity, and contextual memory as well as the rescue of these phenotypes in Sp4 hypomorphic mice. If there were sex effects and sex interactions, male and female mice were analyzed separately.

[0058] In PPI analysis, ANOVA was used with sex, Sp4 genotype, AAV / PHP. eB-Cre as between-subject factors and prepulse intensity as a within-subject factor. To test the specific hypothesis that PPI is decreased in Sp4 hypomorphic mice in comparison with wildtype siblings, a 3 planned comparisons for Sp4 gene effects on PPI was conducted across 3 different prepulse levels. Because such tests are directional, one-tailed t-test was used. Since these comparisons from the same group of mice were not independent, multiple comparison corrections were not conducted. Instead, a p value and 95% confidence intervals were provided. To test the specific hypothesis of AAV / PHP. eB-Cre rescue, planned comparisons were also conducted. For the AAV / PHP. eB-Cre rescue analysis, the interest was in PPI increase in Sp4 hypomorphic mice injected with the AAV / PHP. eB-Cre in comparison with Sp4 hypomorphic mice injected with the vehicle. Because such tests are directional, one-tailed t-test was used across 3 prepulse levels in the same way as Sp4 gene effect analysis. Effect sizes and statistical power were analyzed.

[0059] In analysis of ketamine-induced locomotion, ANOVA was used with sex, Sp4 genotype, AAV / PHP. eB-Cre as between-subject factors and ketamine, time blocks as within-subject factors. Ketamine displayed the highest effects on locomotion within the first 10 min after administration. Planned comparisons were conducted on Sp4 gene effects and the AAV / PHP. eB-Cre rescue effects on the first 10 min block and subsequent 10 min time blocks after ketamine administration. The interest was in higher locomotion induced by ketamine in Sp4 hypomorphic mice in comparison with wildtype mice or suppressed locomotion induced by ketamine in Sp4 hypomorphic mice injected with the AAV / PHP. eB-Cre in comparison with the control Sp4 hypomorphic mice. Therefore, one-tailed t-tests were conducted for these directional comparisons between the two groups. Since these comparisons from the same group of mice at different time blocks were not independent, multiple comparison corrections were not conducted. Instead, p value and 95 % confidence intervals were provided. Effect sizes and statistical power were also reported.

[0060] In fear conditioning analysis, ANOVA was used with sex, Sp4 genotype, AAV / PHP. eB-Cre as between-subject factors and time block as a within-subject factor. Sp4gene effect and the AAV / PHP.eB-Cre rescue effect were analyzed as described in PPI analysis.

[0061] It has been previously demonstrated that all abnormal phenotypes of Sp4 hypomorphic mice can be rescued by restoring rat Sp4 gene expression using protamine-Cre that deletes the LoxP -flanked nLacZ gene to fully activate the downstream rat Sp4 gene in male germ line of mice11. Subsequently, Sp4 expression was successfully restored during embryogenesis in cell lineages for forebrain excitatory and GABAergic inhibitory neurons, respectively, after introduction ofEmxl-Cre orDlx6a-Cre genes2. Herein, AAV / PHP.eB virus was used to express Cre in the whole brains of young adult Sp4 hypomorphic mice to rescue schizophrenia-related behavioral phenotypes. Since AAV / PHP.eB virus produces high neuronal transduction in C57 mice1, Sp4 heterozygous mice on S129 background were bred with C57 mice to produce the F2 generation of Sp4 hypomorphic mice on mixed S129 / C57 background. It was found that significant Sp4 gene effect (F(l,68) = 49.6, p = 1 ,2e-09) and sex effect (F(l,68) = 131.2, p < 2e-16) on mouse body weight at 2-2.5 months old (FIG.3A). Since PPI can be repeatedly tested in mice, PPI deficits in young adult Sp4 hypomorphic mice were examined before injecting AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE virus to restore Sp4 expression (FIG. 3B). PPI deficit started to emerge in these young adult Sp4 hypomorphic mice, although not statistically significant yet (F(l,68) = 2.38, p = 0.12). There was no Sp4 gene effect (F(l ,68) = 0.06, p = 0.81) or sex effect (F(l,68) = 0.35, p = 0.56) on startle or startle habituation (FIG. 9) in this cohort of the F2 generation mice. Each genotype and sex were then split into 2 subgroups receiving injection of either vehicle (IX PBS) or the AAV / PHP.eB-Cre virus. To avoid PPI differences that could be randomly generated between the 2 subgroups during splitting, the means of subgroup PPI were balanced to be equal (FIG. 10). Each subgroup of young adult wildtype and Sp4 hypomorphic mice received injection of either vehicle or AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE (~2 X 1011virus particles) via tail vein. Sixty-seven mice were split into 8 different subgroups shown in Table 1.Table 1. Eight different subgroups of mice.Sex Genotype AAV-Cre NFemale Wildtype PBS 9Female Wildtype AAV-Cre 8Female Sp4 Hypomorphic PBS 8Female Sp4 Hypomorphic AAV-Cre 8Male Wildtype PBS 11Male Wildtype AAV-Cre 11Male Sp4 Hypom orphic PBS 5Male Sp4 Hypomorphic AAV-Cre 7

[0062] Three months after the injection, PPI in all mice (FIG. 4A) was re-examined. An Sp4 gene effect on PPI was detected by a Sp4 X prepulse intensity interaction (F(2, 116) = 5.18, p = 0.007), indicating that Sp4 hypomorphic mice developed more robustPPI deficits at this older age. To verify Sp4 gene effect, planned comparisons were conducted between Sp4 hypomorphic mice and wildtype siblings injected with the vehicle across 3 prepulse levels. It was confirmed that Sp4 hypomorphic mice displayed significant PPI deficits at pp04 (one-tailed t-test: p = 0.006, 95 % confidence interval: (7.84, Inf)) and ppO8 (one-tailed t-test: p = 0.016, 95 % confidence interval: ( 4.69, Inf)) prepulse intensities but no difference at ppl6 (one-tailed t-test: p = 0.21, 95 % confidence interval: (-6.63, Inf)). Importantly, a significant interaction was observed between Sp4 and Sex and AAV / PHP.eB-Cre on PPI (F(l,58) = 3.83, p = 0.05). Since there was a sex effect, males and females were analyzed separately. In male mice (FIG. 4B), a significant Sp4 gene effect on PPI was observed by a Sp4 and prepulse intensity interaction (F(2,60) = 5.26, p = 0.008). To verify Sp4 gene effect, planned comparisons were conducted between Sp4 hypomorphic mice and wildtype siblings injected with the vehicle across 3 prepulse levels. It was confirmed that male Sp4 hypomorphic mice displayed significant PPI deficits at pp04 (one-tailed t-test: p = 0.005, 95 % confidence interval: (12.6, Inf)), ppO8 (one-tailed t-test: p = 0.034, 95 % confidence interval: (2.77, Inf)), and a trend at ppl6 (one-tailed t-test: p = 0.095, 95 % confidence interval: (-4.86, Inf)). A significant Sp4 gene and AAV / PHP.eB-Cre interaction was also observed (F(l,30) = 4, p = 0.05). Wildtype and Sp4 hypomorphic mice injected with AAV / PHP.eB-Cre virus displayed opposite directions of PPI changes, indicating potentially partial rescue of PPI deficits in Sp4 hypomorphic mice. To investigate the AAV / PHP.eB-Cre rescue effect, planned comparisons were conducted between Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre and Sp4 hypomorphic mice injected with the vehicle across all 3 prepulse levels. No significance was detected due to small sample sizes. In female Sp4 hypomorphic mice (FIG. HA), a significant Sp4 gene effect on PPI (F(l ,28) = 2.06, p = 0.16) or a significant interaction between Sp4 gene and prepulse intensity (F(2,56) = 0.72, NS) was not observed. Therefore, this cohort of female mice was not suitable for analysis of PPI rescue. It is important to note that significant PPIdeficits were observed in female Sp4 hypomorphic mice from different cohorts of the F2 generation mice on S129 and C57 background (FIG. 11B).

[0063] Since PPI is stable across repeated tests and was balanced between the 2 subgroups before virus injection, PPI before virus injection was compared with PPI 3 months after the virus injection in individual male Sp4 hypomorphic mice (FIG. 5). For rescue, the interest is in PPI increases in Sp4 hypomorphic mice injected with AAV / PHP.eB-Cre. Such a directional comparison justifies one-tailed paired t-tests for individual mice before and after the rescue. Using a one-tailed t-test gives a higher sensitivity in the direction of interest. A trend of PPI increases was observed at both pp04 (one-tailed paired t-test: p = 0.09, 95 % confidence interval: (-Inf, 3.06)) and ppl6 (one-tailed paired t-test: p = 0.08, 95 % confidence interval: (-Inf, 5.54)) prepulse levels in Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre in comparison with their PPI before the virus injection, indicating PPI improvement or partial rescue (Table 2)Table 2. Summary of PPI increases, effect sizes, and power. #p < 0.1.One-tailed paired t test and effect sizes and power analysisPrepulse Rescue N PPI SD SEM P Effect Power value Size (d)pp04 0.09 0.3 0.13Before 7 33.6 39.9 15.1After 7 43.9 27.4 10.4pp08 0.41 0.11 0.07Before 7 57.4 32.2 12.2After 7 60.5 23.8 9Ppl6 0.08 0.86 0.45Before 7 70.5 26.8 10.1After 7 95.1 30.2 11.4

[0064] There were different effect sizes (d) of the rescue across 3 prepulse levels, but the sample size was underpowered. No PPI changes were observed across all 3 prepulse levels in Sp4 hypomorphic mice injected with the vehicle (FIG. 12A-C and Table 3).Table 3. Summary of PPI changes, p value.Prepulse Rescue N PPI SD SEM P value pp04 0.09Before 7 33.6 39.9 15.1After 7 43.9 27.4 10.4pp08 0.41Before 7 57.4 32.2 12.2After 7 60.5 23.8 9Ppl6 0.08Before 7 70.5 26.8 10.1After 7 95.1 30.2 11.4

[0065] Next, whether locomotor hypersensitivity to ketamine was rescued in Sp4 hypom orphic mice injected with the AAV / PHP was investigated. Hypersensitivity to ketamine in Sp4 hypomorphic mice was examined using the same experimental crossover design as previously published2. After ketamine injection, there were significant Sp4 gene (F(l, 58) = 8.85, p = 0.004) and sex effects (F (1,58) = 4.66, p = 0.035) on ketamine-induced hyperlocomotion. Since there was a sex effect, males and females were analyzed separately. In male mice, there was a significant Sp4 gene effect (F(l,30) = 5.2, p = 0.029) and an interaction between Sp4 and ketamine (F(l,180) = 10, p = 0.002) on ketamine-induced hyperlocomotion (FIG. 6A). There was also a trend for an interaction between Sp4 and AAV / PHP.eB-Cre and ketamine (F(l, 180) = 3.39, p = 0.067), indicating a partial rescue. A significant Sp4 and block interaction was observed (F(S,150) = 4.12, p = 0.0016). To verify ketamine hypersensitivity in Sp4 hypomorphic mice, planned comparisons were conducted after ketamine administration. Significant Sp4 effects were observed in block 4 (F(l, 60) = 4.68, p = 0.035), block 5 (F(l,60) = 7.61, p = 0.0077), and block 6 (F(l,60) = 6.04, p = 0.017). To test the hypothesis of the AAV / PHP.eB-Cre rescue, planned comparisons were conducted between Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre and the control Sp4 hypomorphic mice after administration of ketamine. The differences were not statistically significant due to large variations and small sample sizes. In total distance traveled from time block 4 to 8, there was a significant Sp4 gene effect (F(l,60) = 5.62, p = 0.021) on total distance traveled (FIG. 6B) The hypothesis that Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre display less locomotion than the control Sp4 hypomorphic mice afteradministration of ketamine was examined, but the reduced locomotion was not significant (one-tailed t-test, p = 0.22, 95 % confidence interval: (-3819.51, Inf). There was a medium effect size (d = 0.48) but the sample size was underpowered (power= 0.19). In female mice, there was a significant Sp4 gene effect (F(l,29) = 5.76, p = 0.023) on locomotion (FIG. 13).However, female Sp4 hypom orphic mice injected with the AAV / PHP.eB-Cre virus displayed high baseline locomotion in the absence of ketamine injection, which complicated ketamine sensitivity analysis.

[0066] Finally, whether deficient context memory was rescued in Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre virus was examined. There was a significant Sp4 gene effect (F(l, 58) = 9.01, p = 0.004) on context memory (FIG. 7A). There was no Sp4 gene X AAV / PHP.eB-Cre interaction (F(l,58) = 0.128, NS) on context memory. The two time-blocks were combined (MinO-2, Min2-4) (FIG. 7B). There was no increase of context memory in Sp4 hypomorphic mice injected with the AAV / PHP.eB-Cre (group mean: 43.4 %) in comparison with the control Sp4 hypomorphic mice (group mean: 46.4 %), indicating that restoration of Sp4 expression in Sp4 hypomorphic mice by the AAV / PHP.eB-Cre failed to ameliorate context memory deficits.

[0067] After the behavioral tests, the brains transduction by AAV / PHP.eB-eSYN-EGFP-T2A-iCre-WPRE virus in both adult wildtype and Sp4 hypomorphic mice was analyzed. Since Cre expression deletes the floxed nLacZ to restore Sp4 expression , an EGFP marker was used as a surrogate for the virus transduction and restoration of Sp4 expression. Sagittal paraffin brain sections were generated for immunohistochemical staining of EGFP proteins to assess the virus transduction of mouse brains (FIG. 8). There was no difference in transduction efficiency between wildtype and Sp4 hypomorphic mice. Thalamus was the most efficiently transduced brain region, whereas hippocampal CAI was the least transduced region. Large variations in transduction were observed between individual mice. The overall transduction of the whole brains was much lower than transduction (-55-69 % neurons) reported on C57 mice in the literature1.

[0068] All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

[0069] The following embodiments are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification and figures.Embodiments

[0070] 1. A method of treating a psychiatric disorder, comprising:administering a therapeutically effective amount of a composition comprising a polynucleotide sequence encoding a human SP4 gene to a patient having the psychiatric disorder such that one or more symptoms of the psychiatric disorder are improved, wherein the SP4 gene is expressed in the neuronal cells.

[0071] 2. The method of embodiment 1, wherein the composition further comprises a recombinant adeno-associated viral (AAV) vector that encodes the polynucleotide sequence encoding the human SP4 gene.

[0072] 3. The method of embodiments 1 or 2, wherein the AAV vector crosses the blood brain barrier.

[0073] 4. The method of embodiment 3, wherein the AAV vector is serotype AAV9 or serotype AAV / PHP.eB.

[0074] 5. The method of embodiment 1, wherein the human SP4 gene comprises the polynucleotide sequence of SEQ ID NO: 1 or an allelic variant thereof.

[0075] 6. The method of embodiment 1, wherein the SP4 gene is expressed in forebrain GABAergic inhibitory neurons.

[0076] 7. The method of embodiment 1, wherein the administering occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0077] 8. The method of embodiment 1, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

[0078] 9. The method of embodiment 1, wherein the subject is a mammal.

[0079] 10. The method of embodiment 1, wherein the psychiatric disorder is schizophrenia.

[0080] 11. The method of embodiment 1, wherein the one or more symptoms comprise positive symptoms or negative symptoms of schizophrenia.

[0081] 12. The method of embodiment 11, wherein the positive symptoms comprise delusions, hallucinations, disorganized speech, disorganized behavior, deficient prepulse inhibition (PPI), and / or hypersensitivity to N-Methyl-D-aspartate receptor (NMDAR) antagonists.

[0082] 13. The method of embodiment 11, wherein the negative symptoms comprise flat affect, apathy, social withdrawal, and / or cognitive impairment.

[0083] 14. The method of embodiment 1, wherein the psychiatric disorder causes schizophrenia, post-traumatic stress disorder, anxiety, obsessive compulsive disorder, or mood disorder.

[0084] 15. A method of treating a psychiatric disorder, comprising:administering a recombinant adeno-associated viral (AAV) vector comprising a polynucleotide sequence encoding a human SP4 gene to a patient having the psychiatric disorder such that one or more symptoms of the psychiatric disorder are improved, wherein the SP4 gene is expressed in the brain.

[0085] 16. The method of embodiment 15, wherein the AAV vector crosses the blood brain barrier.

[0086] 17. The method of embodiment 3, wherein the AAV vector is serotype AAV9 or serotype AAV / PHP . eB .

[0087] 18. The method of embodiment 15, wherein the human SP4 gene comprises the polynucleotide sequence of SEQ ID NO: 1 or an allelic variant thereof.

[0088] 19. The method of embodiment 15, wherein the SP4 gene is expressed in forebrain GABAergic inhibitory neurons.

[0089] 20. The method of embodiment 15, wherein the administering occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0090] 21. The method of embodiment 15, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

[0091] 22. The method of embodiment 15, wherein the subject is a mammal.

[0092] 23. The method of embodiment 15, wherein the psychiatric disorder is schizophrenia.

[0093] 24. The method of embodiment 15, wherein the one or more symptoms comprise positive symptoms or negative symptoms of schizophrenia.

[0094] 25. The method of embodiment 24, wherein the positive symptoms comprise delusions, hallucinations, disorganized speech, disorganized behavior, deficient prepulse inhibition (PPI), and / or hypersensitivity to N-Methyl-D-aspartate receptor (NMDAR) antagonists.

[0095] 26. The method of embodiment 24, wherein the negative symptoms comprise flat affect, apathy, social withdrawal, and / or cognitive impairment.

[0096] 27. The method of embodiment 1, wherein the psychiatric disorder causes schizophrenia, post-traumatic stress disorder, anxiety, obsessive compulsive disorder, or mood disorder.

[0097] The specific methods, devices and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the technology. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the technology disclosed herein without departing from the scope and spirit of the technology.

[0098] The technology illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

[0099] Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

[0100] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the technology.

[0101] The technology has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the technology. This includes the generic description of the technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognizethat the technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.REFERENCES

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Claims

CLAIMSWhat is claimed is:

1. A method of treating a psychiatric disorder, comprising:administering a therapeutically effective amount of a composition comprising a polynucleotide sequence encoding a human SP4 gene to a patient having the psychiatric disorder such that one or more symptoms of the psychiatric disorder are improved, wherein the SP4 gene is expressed in the neuronal cells.2 The method of claim 1, wherein the composition further comprises a recombinant adeno-associated viral (AAV) vector that encodes the polynucleotide sequence encoding the human SP4 gene.3 The method of claims 1 or 2, wherein the AAV vector crosses the blood brain barrier.4 The method of claim 3, wherein the AAV vector is serotype AAV9 or serotype AAV / PHP.eB.5 The method of claim 1, wherein the human SP4 gene comprises the polynucleotide sequence of SEQ ID NO: 1 or an allelic variant thereof.6 The method of claim 1, wherein the SP4 gene is expressed in forebrain GABAergic inhibitory neurons.7 The method of claim 1, wherein the administering occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.8 The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.9 The method of claim 1, wherein the subject is a mammal.10 The method of claim 1, wherein the psychiatric disorder is schizophrenia.11 The method of claim 1, wherein the one or more symptoms comprise positive symptoms or negative symptoms of schizophrenia.12 The method of claim 11, wherein the positive symptoms comprise delusions, hallucinations, disorganized speech, disorganized behavior, deficient prepulse inhibition (PPI), and / or hypersensitivity to N-Methyl-D-aspartate receptor (NMD AR) antagonists.

13. The method of claim 11, wherein the negative symptoms comprise flat affect, apathy, social withdrawal, and / or cognitive impairment.

14. The method of claim 1, wherein the psychiatric disorder causes schizophrenia, post-traumatic stress disorder, anxiety, obsessive compulsive disorder, or mood disorder.

15. A method of treating a psychiatric disorder, comprising:administering a recombinant adeno-associated viral (AAV) vector comprising a polynucleotide sequence encoding a human SP4 gene to a patient having the psychiatric disorder such that one or more symptoms of the psychiatric disorder are improved, wherein the SP4 gene is expressed in the brain.

16. The method of claim 15, wherein the AAV vector crosses the blood brain barrier.

17. The method of claim 3, wherein the AAV vector is serotype AAV9 or serotype AAV / PHP.eB.

18. The method of claim 15, wherein the human SP4 gene comprises the polynucleotide sequence of SEQ ID NO: 1 or an allelic variant thereof.

19. The method of claim 15, wherein the SP4 gene is expressed in forebrain GABAergic inhibitory neurons.

20. The method of claim 15, wherein the administering occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

21. The method of claim 15, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

22. The method of claim 15, wherein the subject is a mammal.

23. The method of claim 15, wherein the psychiatric disorder is schizophrenia.

24. The method of claim 15, wherein the one or more symptoms comprise positive symptoms or negative symptoms of schizophrenia.

25. The method of claim 24, wherein the positive symptoms comprise delusions, hallucinations, disorganized speech, disorganized behavior, deficient prepulse inhibition (PPI), and / or hypersensitivity to N-Methyl-D-aspartate receptor (NMD AR) antagonists.

26. The method of claim 24, wherein the negative symptoms comprise flat affect, apathy, social withdrawal, and / or cognitive impairment.

27. The method of claim 1, wherein the psychiatric disorder causes schizophrenia, post-traumatic stress disorder, anxiety, obsessive compulsive disorder, or mood disorder.

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