Gene replacement therapy for neurodevelopmental disorders associated with nmdar dysfunction

Abstract

A treatment for patients with GRIN disorder comprises overexpressing a GRIN1 gene without disrupting the genome and regardless of the type of mutation.

Description

GENE REPLACEMENT THERAPY FOR NEURODEVELOPMENTAL DISORDERS ASSOCIATED WITH NMDAR DYSFUNCTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent application 63 / 575,557, filed April 5, 2024, the contents of which is incorporated herein by reference. SEQUENCE LISTING

[0002] The present application includes as part of its description a sequence listing that includes 3 sequences and which was filed with this application in electronic form and this sequence listing is incorporated into the present application in its entirety. TECHNICAL FIELD

[0003] This disclosure generally relates to the field of compositions and methods for the treatment of neurodevelopmental disorders, in particular GRIN disorder. BACKGROUND OF THE ART

[0004] GRIN disorder is a rare genetic condition caused by de novo, heterozygous, pathogenic genetic variants in GRIN genes including GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, and GRIN3B. These genes encode subunits of the N-methyl-D-aspartate receptor (NMDAR), which are widely expressed in the brain and play a critical role in neurodevelopment, learning and memory. Patients with GRIN disorder have a spectrum of life-shortening health issues, including severe intellectual disability, developmental delays, epilepsy, autism, cortical visual impairment, gastrointestinal disorders, and movement disorders. Currently, there is no effective treatment for GRIN disorder.

[0005] Pathogenic variants can be classified as gain-of-function or loss-of-function, meaning the variant causes the NMDAR to be either over- or under-activated. In most cases, changes in NMDAR function involve the incorporation of either 1 or 2 mutant GluN subunits. In some cases, a mutation can affect the number of functional NMDARs that reside on the cell membrane. Regardless, in the healthy brain, NMDARs are tightly regulated because either too much or too little NMDAR activity can be harmful. Reduced NMDAR activity is associated with impaired learning and memory, and excessive NMDAR activity can lead to excitotoxicity and neurodegeneration.CAN_DMS: \1010315293\61

[0006] Pharmacological interventions that act directly on NMDARs have had mixed results on GRIN patients; for example, off- label use of memantine and other NMDAR antagonists have been beneficial for seizures in some patients but detrimental to others.1,2As a result, the development of effective small molecules as therapeutics may be time-consuming with limited benefits to only a sub-group of all the patients. SUMMARY

[0007] Provided herein are gene therapy methods and compositions for the treatment of neurodevelopmental diseases and conditions associated with NMDAR dysfunction comprising administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding a functional GluN1 protein, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue.

[0008] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure. DESCRIPTION OF THE DRAWINGS

[0009] Figure 1. Rationale of GRIN1 replacement therapy for patients with heterozygous missense variants in GRIN1 gene, regardless of the locus. (Left) Schematic showing a cell from a heterozygous GRIN1 variant patient that expresses both wildtype and variant (mutated) mRNA that is translated into wildtype or mutant GluN1 protein. Two GluN1 subunits (either wildtype and / or variant) will assemble into NMDARs by combining with GluN2 or GluN3 subunits and move into cell membranes. Up to three-quarters of NMDARs contain variant subunit, poisoning receptor function. (Right) Delivery of exogenous GRIN1 cDNA coding for 'wildtype' protein as a replacement therapy that competes against mutant GluN1, resulting in more functional NMDARs.

[0010] Figure 2. Schematic of two genetic constructs designed to achieve detectable ubiquitous (hGAPDH) or neuron-specific (hSYN1) expression of V5-tagged GluN1-1a through AAV delivery.

[0011] Figures 3A-3F. Data suggesting potential NMDAR functional rescue after AAVHSC17- delivered hGRIN1 cDNA in the hippocampus of Grin1 Q536R / + mice. (A) Pilot experiment ofbilateral intra-hippocampal injection of high- -hGAPDH-V5-hGRIN1 into Grin1 Q536R / + heterozygous mouse (Het) dorsal CA1 (dCA1) at postnatal day (P) 56. Wildtype (WT) and Grin1 Q536R / + mice received buffer as controls. FourCAN_DMS: \1010315293\62weeks after surgery, sagittal hippocampal slices were collected for field recording, some of which were fixed for validation of hGRIN1 expression by immunostaining. (B) Representative image showing V5 immunofluorescence (magenta) in hippocampal and cortical regions of the recorded slices that received hGRIN1. Scale bar: 100μm. (C) Slope of the input / output (I / O) plot that represents AMPAR-mediated function through analysis of the fEPSP response (slope, -mV / ms) as a function of the presynaptic action potential (amplitude, mV). (D) (I / O for the NMDAR fEPSPs isolated after blockade of AMPARs with NBQX. Slices were recorded in low magnesium (0.1mM) conditions. (E) Ratio of NMDAR-fEPSP to AMPAR-fEPSP. (F) Long-term potentiation (LTP) induced by a theta-burst stimulation (TBS) protocol was calculated 90min after induction as a percentage of baseline AMPAR-fEPSPs. Filled symbols: males; open symbols: female.

[0012] Figure 4. Dose-dependent expression of V5 in Grin1 Q536R / + mice 4 weeks post AAVHSC17- hSYN1-V5-hGRIN1 delivery to the hippocampus. (A) Unilateral intra- hippocampalinjection of AAVHSC17-hSYN1-V5- hGRi 10 vgs / mouse inGrin1 Q536R / + mice at P56. The contralateral side was injected with buffer as controls. Four weeks post surgery, brains were collected, fixed, sectioned (sagittally) and stained with anti-V5 (magenta) and DAPI (blue). (B) V5 immunofluorescence in the targeted hippocampus and also surrounding cortex at 1×1010vgs. (C) Comparison of V5 signal in CA1-3 and dentate gyrus (DG) 10vgs / mouse. Scale bar: 200μm.

[0013] Figure 5. Dose-dependent microglia and astrocyte activation in the hippocampus of Grin1 Q536R / + mice 4 weeks post AAVHSC17-hSYN1-V5-hGRIN1 administration. Representative images comparing IBA-1 and GFAP immunofluorescence in CA1-3, dentate gyrus (DG), and dorsal subiculum (DS) at 1×107, 1×108, 1×109, and 1×1010vgs / mouse. At the highest doses, frequent overlapping fluorescence of both green (FITC) and magenta (Cy5) was observed as shown by red arrows (white). Some overlaps may result from autofluorescence rather than the intended specific staining of IBA-1 (magenta) and GFAP (green). Nuclei are stained with DAPI (blue). Scale bar: 100μm.

[0014] Figures 6A-6G. Rescue of NMDAR function following AAVHSC-hGRIN1 optimization, delivery and expression in the hippocampus of GRIN1 Q536R / + mice. (A) Bilateral intra-hippocampal injection of AAVHSC-hSYN1-V5-hGRI Qmice (Het) at postnatal day P56 (Het AAV n=6, shown in inverted triangle). Wildtype (WT, gray) and Grin1Q, shown in circle; Het Buffer n=6, shown in triangle). Four weeks after injection, sagittal hippocampal slices were collected forCAN_DMS: \1010315293\63electrophysiology and allowed to recover. As shown in the diagram, stimulation electrode (Stim) was placed at Schaffer collateral / commissural fibers (SC) and recording electrode (Record) were placed in CA1 stratum radiatum. (B-G) Electrophysiological results: Two slices were recorded per animal for panels B-D. One slice recorded per animal for panels E-G. (B) Slope of input / output (I / O) function was calculated for fiber volley (FV) amplitude against stimulus intensity, as an index of presynaptic axon activation. (C) Representative fEPSP traces and slope of I / O function for AMPAR-mediated synaptic transmission. (D) Paired-pulse facilitation, measuring presynaptic neurotransmitter release probability. (E) Representative fEPSP traces and I / O function for NMDAR-mediated synaptic transmission; NMDAR-mediated responses were recorded in low magnesium (0.1mM) with AMPARs blocked by NBQX. (F) Ratio of NMDAR-fEPSP to AMPAR- fEPSP. (G) Representative AMPAR-mediated fEPSP traces (left) before and after induction of long-term potentiation (LTP). The bottom left panel shows the time course experiments including the time of theta-burst stimulation (TBS) to induce LTP. The bottom right panel summarizes the % LTP calculated between time points 1 and 2. Filled symbols: males; open symbols: female. Mean±SEM. One-way ANOVA. *p<0.05, *p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

[0015] Figures 7A-7O. Successful delivery of AAVHSC17-V5-hGRIN1 to the brain through intravenous administration with data suggesting behavioural improvement. (A) i.v. tail vein injection of AAVHSC17-V5-hGRIN1 with either hGAPDH or hSYN1 promoter at 1 x1014vgs / kg into Grin1 Q536R / + mice at P56. Behavioral phenotyping four weeks after injection followed by brain, heart, and liver tissue collection for molecular analysis. (B-E) Vector genome counts in the heart (B-C), liver (D), and hindbrain (E). (F-H) mRNA expression of V5-tagged hGRIN1 relative to housekeeping gene (PGK) in the liver (F), heart (G) and midbrain (H). (I) V5-tagged GluN1 protein detection in the liver with hGAPDH promoter, but not hSYN1 promoter. (J) Representative images of V5 immunodetection (red) in liver and some brain regions (e.g. cerebellum) with the hGAPDH promoter. DAPI stained nuclei appear blue. (K-M) Puzzle box trial design with results showing time to reach goal zone at trial 5 day 2 (K-L). Visual cliff data (M); discrimination ratio = time spent in safe side / time spent in cliff side. >1: spent more time in safe side; <1: spent more time in cliff side. (N-O) Weight gain (=body weight at 4 weeks after injection - body weight before injection) of male (N) and female (O) mice. All results are Mean±SEM. One-way ANOVA. ****p<0.0001, **p<0.01, *p<0.05, ns = not significant.

[0016] Figure 8. Intravenous administration of AAVHSC-hGAPDH-V5-hGRIN1 showed dose- dependent expression of hGRIN1 mRNA and hGluN1 protein in various tissues, but similar effect on behaviors. AAVHSC-hGAPDH-V5-hGRIN1 (AAV for short) was injected into the tail vein ofCAN_DMS: \1010315293\64Grin1 Q536R / + mice at 1x1013vgs / kg (D1), 5X1013vgs / kg (D2), and 1X1014vgs / kg (D3) at PND 42. WT and Grin1 Q536R / + mice received buffer as controls. Behavioral tests were conducted 12 weeks post administration followed by tissue collection. (A-C) Dose-dependent hGRIN1 mRNA expression relative to PGK (housekeeping) in the liver (A), heart (B), and midbrain (C). (D-E) Dose-dependent GluN1 expression in the liver detected by western blot. (E) Representative blot image. (F) Blot quantification. Besides comparisons shown on the graph, significant difference also detected between D1 vs D2 and D1 vs D3 (p’s<0.05). (G-L) Effects of AAV at different doses on weight gain in male (G) and female (H) mice, and behavioral tests (I-L). (I) light dark test. Light- dark discrimination ratio = (time in dark zone – time in light zone) / (total time in both dark and light zones)*100%. (J-L) 2-hour Open field test distance travelled over time (J), total distance travelled (K), and total number of stereotypic events (L). Mean±SEM. For molecular analysis: One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

[0017] Figure 9. Behavioural phenotypes of Grin1 Y647S + / - mice, which show increased locomotor activity (A), decreased anxiety in the elevated plus maze (B), handling induced seizures (C), that increase in severity with repeated weekly handling (D).

[0018] Figures 10A-10F. AAV Grin1 gene augmentation restores NMDAR function and synaptic plasticity in Grin1 Y647S / + mice. Same route of administration, dose, and electrophysiological recordings were performed as in figure 6. n= 6 for all groups. Circles: WT buffer; Triangles: Grin1 Y647S / + buffer; Inverted triangles: Grin1 Y647S / + AAV. Filled symbols: males; open symbols: female. Mean±SEM. One-way ANOVA. *p<0.05, ***p<0.001, ****p<0.0001, ns = not significant.

[0019] Figure 11. LNP-delivered Grin1 mRNA improved locomotor activity in 6-8 weeks old Grin1 knockdown mice. Grin1 knockdown (KD) mice received LNP-delivered mouse Grin1 mRNA through intracerebral ventricular (ICV) injections (KD-Grin1, triangle). Wildtype (WT) and Grin1KD mice receiving LNP-delivered firefly luciferase (fLuc) mRNA through the same administration route were used as controls (WT, open circle and KD-fLuc, square). Locomotor activity of experimental mice was tested 24hr (A&B) and one week (C&D) post injection. (A) In comparison to WT-fluc mice, KD-fLuc mice were hyperactive over the 2-hour testing period. KD-Grin1 mice were less hyperactive comparing to KD-fLuc mice 24hr post injection. (B) Accumulative distance traveled of KD-Grin1 mice trended towards decrease compared to KD-fLuc mice 24hr post injection. (C) Reduced hyperactivity was persistently observed 1-week post injection. (D) KD-CAN_DMS: \1010315293\65Grin1 mice showed significantly decreased total travel distance compared to KD-fLuc mice 1- week post injection. One-way ANOVA. Mean ± SEM. *p<0.05.

[0020] Figure 12. Assessment of in vivo brain tissue tropism of LNP used for Grin1 mRNA delivery using in vivo imaging system (IVIS) to visualize the transfection profiles of the LNP vector. IVIS imaging enabled mapping of in vivo luminescence signals from LNP-fLuc (Luciferase) mRNA-treated WT mice, both at the whole-body level and in specific organs. Mice received LNP- fLuc via ICV injection and were imaged 24 hours post-injection, following intraperitoneal administration of the natural substrate for luciferase, D-luciferin (150 mg / kg). Whole-body and post-mortem imaging of liver, heart, lung, liver, and brain (left to right) transfection selectivity of the LNP vector are presented. Whole body and brain only images demonstrate that LNP formulation had brain tissue tropism with minimal off-target transfection of peripheral organs. DETAILED DESCRIPTION

[0021] The term "adeno-associated virus" (AAV), as used herein, includes without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV. The genomic sequences of various AAV, as well as the sequences of the ITRs, rep proteins, and capsid proteins are known in the art. Such sequences may be found in the literature or in public databases such as the GenBank database. AAV is an attractive delivery method because it is commonly used in clinical trials with FDA-approved treatments using AAV. In one embodiment, the AAV is a stem-cell derived AAVHSC, in one embodiment AAVHSC17.

[0022] As used herein, "transduction" of a cell by a virus particle (e.g., an AAV particle) means entry of the particle into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus particle and subsequent transfer into the cell via the virus particle.

[0023] As used herein, the term "viral particle", "vector", "viral vector", or "delivery vector", and their derivatives, refer to a particle that functions as a nucleic acid delivery vehicle, and which comprises the viral nucleic acid (i.e., the viral vector genome) packaged within the particle.

[0024] A "heterologous nucleic acid" or "heterologous nucleotide sequence" as used herein is a nucleotide sequence that is inserted into a vector, in preferred embodiments, an AAV vectorCAN_DMS: \1010315293\66for vector mediated transfer of the nucleotide into a cell. The “heterologous nucleic acid” is not naturally occurring in the virus.

[0025] As used herein “subject” refers to an animal being administered a therapeutic, in one embodiment a mammal, in one embodiment a human patient. As used herein “treatment”, and grammatical variations thereof, refers to administering a therapy or composition of the present invention to effect an alteration or improvement of a disease or condition or symptom thereof. Administration may be of a single dose or multiple doses and may be prior to onset of a disease or condition or symptoms thereof to alter the course of the disease or condition e.g. to prevent or mitigate its symptoms.

[0026] As used herein, “therapeutically effective amount” refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects. When therapeutically effective amounts are provided herein in terms of dosing ranges, it is to be understood that these ranges can be subsequently broken down into sub-ranges, such that the ranges provided herein also include all sub-ranges falling within the broader range. Further, unless otherwise indicated herein, the term “about” in this context is intended to include values and ranges proximate to the recited range that are equivalent in terms of the recited functionality.

[0027] When reference is made to a therapeutically effective being an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in “a target tissue”, it is to be understood that the target tissue may be specifically targeted e.g. by way of using a tissue specific vector or promoter, or it may be a tissue in which GluN1 proteins are expressed and assembled into NMDARs at a detectable level and wherein dysfunction in this expression and assembly is associated with a disease or condition. NMDAR AND NEURODEVELOPMENTAL DISORDERS

[0028] GRIN disorder is the term for debilitating neurodevelopmental disorders caused by pathogenic variants in GRIN1, GRIN2A, GRIN2B, or GRIN2D. These genes encode subunits of the NMDA receptor, which is a critical component of neuronal synaptic communication. NMDA CAN_DMS: \1010315293\67receptors are highly conserved and 99% identical between rodents and humans. They are activated by the amino acid neurotransmitters glutamate and glycine and allow sodium and calcium to enter cells. The genes for NMDA receptors are called GRIN genes (glutamate receptor ionotropic NMDA). The NMDAR ion channel is a tetramer assembled in the endoplasmic reticulum where one GRIN1 subunit first pairs with one GRIN2 subunit, and then the two dimers form a tetramer.

[0029] GRIN1 disorder is caused by de novo, heterozygous, dominant-acting point mutations that lead to missense, nonsense, or splice variants. The frequency of GRIN1 disorder is estimated to be approximately 5 / 100,000 live births. Symptoms include developmental delay, intellectual disability, speech and communication impairments, autism, cortical visual impairment, epilepsy, problems with feeding and digestion, sleep disturbances, apnea and pneumonia, motor impairments (hypotonia, dystonia, hyperkinesis, repetitive movements) and behavioral difficulties. Symptoms first appear in infancy and life expectancy is unknown. Common causes of childhood mortality are status epilepticus, SUDEP, cardiac arrest, and pneumonia.

[0030] Gene replacement therapy is an attractive strategy for genetic conditions with insufficient expression of the target protein. This strategy serves to deliver copies of a functional 'wildtype' gene to replace the insufficient expression. However, most pathogenic GRIN variants are missense variants.3The encoded mutant subunits may get incorporated during NMDAR assembly4, resulting in dysfunctional receptors.

[0031] Provided herein are methods for treating a disease or condition associated with NMDAR dysfunction. In some embodiments, there is provided methods for treating a neurodevelopmental disorder associated with NMDAR dysfunction. Neurodevelopmental disorders that may be treated according to methods provided herein include, schizophrenia, autism spectrum disorder (ASD), epilepsy and GRIN disorder. In one embodiment, the disease or condition is GRIN disorder.

[0032] A functional NMDAR consists of 4 subunits, two obligatory GluN1 subunits (encoded by GRIN1 and a combination of any of the GluN2A-D subunits (encoded by GRIN2A-D) or GluN3A-B (encoded by GRIN3A-B). Cells express a pool of GluN1 subunits that are readily available for assembly into NMDAR in nature. The level of GluN2 subunit expression governs NMDAR assembly and receptor expression. The excess GluN1 subunits that are not assembled into NMDARs are then rapidly degraded by the cell.CAN_DMS: \1010315293\68

[0033] Importantly, the methods provided herein are not based on a strategy of replacing GRIN1 gene expression through a conventional gene therapy. Rather, the therapies disclosed herein are directed to shifting the stoichiometry of subunit composition.

[0034] In particular, an NMDAR-subunit-encoding minigene that encodes a functional protein is used to increase the ratio of functional to dysfunctional protein. In particular, in one aspect a GRIN1 minigene encoding a functional protein is provided to increase the ratio of functional GluN1 to dysfunctional GluN1. As a result, more NMDA receptor complexes will be composed of two functional GluN1 subunits.

[0035] In patients with heterozygous missense GRIN1 variants, both wildtype and variant GluN1 proteins are expressed and assembled into NMDARs. As a result, up to three-quarters of surface NMDARs contain at least one variant GluNl1 subunit, which is sufficient to cause NMDAR dysfunction that results in GRIN disorder (Figure 1A).

[0036] In one embodiment, the present invention comprises the delivery of exogenous GRIN1 cDNA into the cells. These copies of engineered 'wildtype' GRIN1 gene lead to excessive expression of 'wildtype' GluN1 proteins relative to the variant GluN1 proteins. In another embodiment, the present invention comprises the delivery of mRNA encoding a functional GluN1 protein.

[0037] By manipulating the ratio of wildtype to variant GluN1 proteins, more wildtype GluN1 proteins (exogenous + endogenous) are available for assembly into NMDARs. Thus, more NMDARs will contain only wildtype GluN1 subunits and function normally (Figure 1B).

[0038] Given the rapid degradation of excess GluN1 subunits in nature, overexpressing the GRIN1 gene to reach a therapeutic dose should not affect the level of NMDAR expression within the cell and therefore does not interfere with the tight regulation of NMDAR activity nor lead to excitotoxicity within the cell. In addition to providing functional rescue to patients with GRIN1 variants, the methodology may also be used for the treatment of disorders that manifest in NMDAR under-activation.

[0039] Studies in cell lines show that expression of GluN1 subunit alone forms stable monomer or dimers.5However, neither GluN2 nor GluN3 subunits alone fold stably.6Moreover, co-expression of GluN1 stabilizes GluN2 subunits and there is a delay between the appearance of the subunits and their association into a receptor complex.7These observations indicate thatCAN_DMS: \1010315293\69GluN1 subunits serve as a substrate for the association of other subunits into the functional NMDAR complex. Given that the subunits associate relatively quickly, the time delay between subunit protein expression and receptor assembly is proposed to represent the time required to establish a pool of stable GluN1 subunits, which is required for further receptor assembly.6In summary, expression of NMDA receptors requires a steady state pool of GluN1 subunit that is retained in the endoplasmic reticulum until GluN2 orGluN3 subunits are expressed and NMDA receptors are assembled.

[0040] Turnover analysis of NMDAR subunits revealed a pool of rapidly degraded GluN1 subunits that were confined to the cytoplasm and were not assembled with GluN2 subunits.8The unassembled extra GluN subunits are rapidly degraded within 2 hours and account for 90% of the intracellular pool. The remaining 10% of GluN1 subunit has a half-life of greater than 18 hours and exists in the NMDA receptor heterotetramer.8

[0041] Transgene overexpression is a general concern in gene replacement therapy. Excessive expression of the therapeutic protein may lead to cellular toxicity (disrupting cellular function or cell death) and / or immune response. The level of toxicity is dependent on the dose, specific transgene, and targeted cell type or tissue. Given the fast turnover rate of intracellular GluN1, a known degradation pathway of redundant GluN1 expression, and the effective dose data provided in the Examples, GRIN1 gene can be safely delivered and overexpressed at therapeutically useful doses. GENE THERAPY

[0042] The blood–brain barrier (BBB) is a highly selective permeability barrier that separates the circulating blood from the brain extracellular fluid in the CNS. The BBB, which is formed by brain endothelial cells, allows the passage of water, some gases, and lipid-soluble molecules by passive diffusion, as well as the selective transport of molecules such as glucose and amino acids that are crucial to neural function, while restricting the diffusion of microscopic objects (e.g., bacteria or cells such as leukocytes) and large or hydrophilic molecules into the cerebrospinal fluid (CSF). Many pharmaceutical agents cannot pass through the BBB presenting delivery challenges.

[0043] While in some embodiments, the mechanism of delivery for the gene therapies provided herein is not specifically restricted as long as it enables expression of GRIN1, in particular, in tissues where GRIN1 is expressed, thereby augmenting the ratio of functional GluN1CAN_DMS: \1010315293\610protein (i.e. GluN1 protein that forms a functional subunit of NMDAR) to mutant GluN1 proteins, in other embodiments, the delivery vehicle is suitably able to cross the BBB, enabling intravenous administration, which is less invasive that direct administration to the CSF.

[0044] In some embodiments, the gene therapy is an viral vector, preferably an AAV vector.

[0045] In some embodiments, the gene therapy comprises miRNA and a lipid nanoparticle vector.

[0046] Pharmaceutically acceptable carriers and excipients for AAV and lipid nanoparticle applications are available in the art.

[0047] In some embodiments, the AAV vector is a stem cell derived vector. Previous efforts to transduce human hematopoietic stem cells (HSCs) using AAV2 vectors led to the discovery of a unique class of HSC-derived AAVs, AAVHSCs. AAVHSCs are naturally evolved AAVs isolated from human CD34+ HSCs. Characterization of the original AAVHSC vectors showed significantly better transduction in CD34+ cells compared to AAV2 and AAV8. Biodistribution study of AAVHSCs in mice demonstrated in widespread transduction into liver, skeletal muscle, cartilage heart and other peripheral tissues.9In non-human primates (NHPs), intravenously delivered AAVHSCs transduced both peripheral tissues and CNS, indicating its ability to cross blood-brain barrier (BBB). Transgene expression was found in different cell types within the CNS.10The broad tropism supports the use of AAVHSC-based gene therapies targeting both peripheral tissues and CNS. An assessment in a representative human population of 100 individuals suggested low seroprevalence of NAbs targeting AAVHSC15 and AAVHSC17. Early results from a phase 1 / 2 clinical gene therapy trial using AAVHSC15 vectors for phenylketonuria (PKU) supported safety and efficacy of AAVHSC15 vectors in humans.9AAVHSC16 was recently reported to possess reduced liver tropism with maintained high tropism for other peripheral tissues and the CNS in NHPs comparing to AAV2, AAV9, and AAVHSC15.11Thus, AAVHSCs are particularly suitable for use in the methods as disclosed herein. Details of AAVHSCs suitable for use in the present invention, including sequence information, can be found in United States Patent: 9,803,218 B2, filed October 28, 2016, the contents of which is incorporated herein by reference.

[0048] In some embodiments, the AAV vector is AAV CAP-B10. Using a multiplexed Cre- recombination-based AAV targeted evolution (MCREATE) platform, AAV9 was used as the prototype for engineering the next generation capsids through directed evolution. AAV CAP-B10 was identified through this process. The AAV CAP-B10 capsid showed brain-wide neuronal andCAN_DMS: \1010315293\611glial transgene expression and substantially reduced liver transduction in both rodents and non- human primates.12The liver de-targeting property of AAV CAP-B10 allows intravenous administration for efficient transgene expression into CNS with minimal liver toxicity. Details of AAV CAP-B10, including sequence information, can be found in International patent application publication no. WO 2020 / 068990 A1 and United States patent 11,149,256 B2, both filed September 25, 2019, the contents of which is incorporated herein by reference.

[0049] As detailed in Examples 1 through 4, to deliver the engineered 'wildtype' GRIN1 gene into the cells, two constructs were used (Figure 2). Both constructs contained i) the human GRIN1 (hGRIN1) cDNA corresponding to the GluN1-1a isoform; ii) a V5 tag sequence that is expressed at the N-terminus of GluN1-1a for visualization and detection, iii) SV40 late polyadenylation signal (LPA) for transcription termination and mRNA stability, iv) 5' and 3' inverted terminal repeats (ITRs) for AAV transfer. Human GRIN1 cDNA was synthesized from NCBI accession NM_007327 with the V5 tag placed after the signal sequence. The V5 tag is a short l4 amino acid epitope that allows for detection of the exogenous GluN1 and to distinguish it from endogenous GluN1 expression without affecting the protein assembly and function.

[0050] Although GRIN1 gene is mainly expressed in neurons, it is also found in non-neural cells in the central nervous system (e.g. astrocytes) and peripheral organs (e.g. pancreas) and believed to also play a crucial role. Given this complex expression profile, two different promoters (and thus two constructs) that drive ubiquitous expression (hGAPDH) or neuron-specific expression (hSYNl) of the hGRIN1 gene were tested. The sequences of these two constructs are provided herein for reference as SEQ ID NO: 1 and SEQ ID NO: 2.

[0051] In various embodiments, the expression of the heterologous nucleic acid may be under control of a neuron-specific promoter, a ubiquitous promoter, or a combination of both. The doses administered under the control of different promoters may be different e.g. a high dose under control of a neuron specific promoter and a low dose under the control of a ubiquitous promoter.

[0052] To understand the mechanisms and develop novel treatments for GRIN disorder, mouse models possessing the equivalent heterozygous GRIN1 patient variants have been generated (Grin1 Q536R / +, Grin1 Y647S / +, Grin1 G620R / +, and Grin1 G827R / +) mouse models. All four variants are heterozygous missense variants on the Grin1 gene but affect different GluN1 protein domains. Clinical reports, cell-based studies, and characterization studies of the mouse models (heterozygous state) reveal that these four variants impact receptor function differently andCAN_DMS: \1010315293\612lead to distinct phenotypic consequences. Therefore, this group of models allowed testing of whether the approach can rescue the phenotypic consequences caused by distinctly different GRIN1 variants.

[0053] The Examples show successful AAVHSC17 delivery and expression of exogenous hGRIN1 into the brain through i.v. administration and the potential to rescue behavioural deficits in GRIN1 patient variants. The Examples evidence that expressing exogenous 'wildtype' hGRIN1 cDNA as a gene replacement therapy can rescue the synaptic dysfunction of NMDAR and behavioural deficits in a missense GRIN1 Q536R / + variant and this strategy is predicted to work for other missense GRIN1 mutations.

[0054] A dose finding study in the hippocampus of GRIN1 patient variant mouse models revealed neuroinflammation only at high doses but not at lower doses where functional improvement of NMDAR was observed.

[0055] mRNA therapeutics act as an attractive alternative for protein replacement application when compared to DNA therapeutics as they do not penetrate the nucleus of the host cell, preventing dangers associated with genomic mutagenesis. Unfortunately, as naked mRNA is rapidly degradable and can provoke an immune response, it is suboptimal to administer the therapy without a vector. Thus, to mitigate this issue, in some embodiments, these compounds are delivered using lipid nanoparticles (LNPs), a rising vector for aiding protein replacement therapies.13

[0056] To mitigate the immunogenicity of mRNA, modifications such as the replacement of uridine with N1-methylpseudouridine can be employed. Per Example 5, in vivo luciferase reporter assays demonstrate that an LNP formulation, delivered via intracerebroventricular (ICV) injection, achieves targeted protein translation within the brain with minimal off-target transfection. The established safety and efficacy of LNP-mRNA delivery, exemplified by the successful COVID-19 mRNA vaccines utilizing a similar modality, support the robustness and safety of this approach for CNS applications.

[0057] The Grin1 mRNA sequence is publicly accessible and is available to purchase e,g, on Addgene (link: https: / / www.addgene.org / 17928 / ), a Kozak sequence was cloned to the N terminus of the signal peptide. Further, this sequence is provided herein as SEQ ID NO: 3.CAN_DMS: \1010315293\613

[0058] Example 5 shows delivery of Grin1 mRNA using a lipid nanoparticle is beneficial in a mouse model of GRIN disorder, evidencing that different gene therapy modalities, including an mRNA LPN, may be used to augment functional GluN1.

[0059] Example 6 evidences that extrapolating from the doses demonstrated to be safe and effective in the Examples provides a dosing range that compares favourably with current FDA-approved gene therapies.

[0060] While in some embodiments, the subject will be a human pediatric patient, tamoxifen- induced genetic rescue of Grin1 gene at either adolescence or adulthood improved Grin1 expression, NMDAR function and reversed behavioral consequences in a Grin1 knockdown (Grin1KD) mouse model14, evidencing that replenishing the GRIN1 gene may be used to reverse the consequences of NMDAR hypofunction in adolescent or adult subjects.

[0061] Embodiments of the present invention include: 1. A method of treating or preventing a neurodevelopmental disease or condition in a subject in need thereof comprising administering to the subject a gene therapy comprising a therapeutically effective amount of a nucleic acid molecule encoding a functional GluN1 protein, wherein the neurodevelopmental condition is associated with NMDAR dysfunction. 2. The method of embodiment 1, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue. 3. The method of embodiment 1 or 2, wherein the gene therapy comprises a vector containing the nucleic acid, optionally a viral vector or a non-viral vector. 4. The method of any one of embodiments 1 to 3, wherein the gene therapy comprises a recombinant adeno-associated virus (rAAV) viral vector, wherein said rAAV comprises a vector genome encapsidated by an AAV capsid, the vector genome comprising: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segment encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1- 1a isoform, two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter linked to the nucleic acid segment; and a polyadenylation signal; and, optionally, an epitope tag.CAN_DMS: \1010315293\6145. The method of embodiment 4, wherein the rAAV viral vector is a human hematopoietic stem cell (HSC) derived AAV. 6. The method of embodiment 4, wherein the rAAV viral vector is an AAV9 viral vector, preferably AAV CAP-B10. 7. The method of any one of embodiments 1 to 6, wherein the nucleic acid is operably linked to a ubiquitous promoter. 8. The method of any one of embodiments 1 to 6, wherein the nucleic acid is operably linked to a tissue-specific promoter. 9. The method of any one of embodiments 4 to 8, wherein the subject is a human and the therapeutically effective amount is between about 1 x 1011vg / kg to about 2 x 1014vg / kg. 10. The method of any one of embodiments 1 to 2, wherein the gene therapy comprises RNA encoding a functional GluN1 protein. 11. The method of embodiment 10, wherein the gene therapy comprises a Lipid nanoparticle comprising RNA encoding a functional GluN1 protein. 12. The method of embodiment 10 or 11, wherein the subject is a human subject and the therapeutically effective amount is between about 3,750 μg and about 4,500 μg of mRNA. 13. The method of any one of embodiments 1 to 12, wherein the disease or condition associated with NMDAR dysfunction is one or more of GRIN disorder, schizophrenia, autism spectrum disorder (ASD), and epilepsy. 14. The method of any one of embodiments 1 to 13, wherein the subject is a human minor, optionally an infant. 15. The method of any one of embodiments 1 to 14, wherein the gene therapy is administered intravenously or directly to the cerebrospinal fluid (CSF), optionally wherein the gene therapy is administered directly to the CSF by intracerebroventricular (ICV) injection, by intracisternal magna (ICM) injection or by intrathecal (IT) injection. 16. The method of any one of embodiments 1 to 15, wherein the gene therapy is administered intravenously.CAN_DMS: \1010315293\61517. The method of any one of embodiments 1 to 13, wherein the therapeutically effective amount is between 1 X 1013vgs to 3.7 x 1014vgs and / or wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection. 18. A gene therapy for use in treating or preventing a neurodevelopmental disease or condition in a subject in need thereof, the gene therapy comprising a therapeutically effective amount of a nucleic acid molecule encoding a functional GluN1 protein, wherein the neurodevelopmental condition is associated with NMDAR dysfunction. 19. The gene therapy for use of embodiment 18, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue. 20. The gene therapy for use of embodiment 18 or 19, wherein the gene therapy comprises a vector containing the nucleic acid, optionally a viral vector or a non-viral vector. 21. The gene therapy for use of any one of embodiments 18 to 20, wherein the gene therapy comprises a recombinant adeno-associated virus (rAAV) viral vector, wherein said rAAV comprises a vector genome encapsidated by an AAV capsid, the vector genome comprising: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segment encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1-1a isoform, two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter linked to the nucleic acid segment; and a polyadenylation signal; and, optionally, an epitope tag. 22. The gene therapy for use of embodiment 21, wherein the rAAV viral vector is a human hematopoietic stem cell (HSC) derived AAV. 23. The gene therapy for use of embodiment 21, wherein the rAAV viral vector is an AAV9 viral vector, preferably AAV CAP-B10. 24. The gene therapy for use of any one of embodiments 18 to 23, wherein the nucleic acid is operably linked to a ubiquitous promoter. 25. The gene therapy for use of any one of embodiments 18 to 24, wherein the nucleic acid is operably linked to a tissue-specific promoter.CAN_DMS: \1010315293\61626. The gene therapy for use of any one of embodiments 21 to 25, wherein the subject is a human and the therapeutically effective amount is between 1 x 1011vg / kg to 2 x 1014vg / kg. 27. The gene therapy for use of any one of embodiments 18 to 19, wherein the gene therapy comprises RNA encoding a functional GluN1 protein. 28. The gene therapy for use of embodiment 27, wherein the gene therapy comprises a Lipid nanoparticle comprising RNA encoding a functional GluN1 protein. 29. The gene therapy for use of embodiment 27 or 28, wherein the subject is a human subject and the therapeutically effective amount is between about 3,750 μg and about 4,500 μg of mRNA 30. The gene therapy for use of any one of embodiments 18 to 29, wherein the disease or condition associated with NMDAR dysfunction is one or more of GRIN disorder, schizophrenia, autism spectrum disorder (ASD), and epilepsy. 31. The gene therapy for use of any one of embodiments 18 to 30, wherein the subject is a human minor, optionally an infant. 32. The gene therapy for use of any one of embodiments 18 to 31, wherein the gene therapy is administered intravenously or directly to the CSF, optionally wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection. 33. The gene therapy for use of any one of embodiments 18 to 31, wherein the gene therapy is administered intravenously. 34. The gene therapy for use of any one of embodiments 18 to 33, wherein the therapeutically effective amount is between 1 X 1013vgs to 3.7 x 1014vgs and / or wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection. 35. A pharmaceutical composition comprising a lipid nanoparticle and a therapeutically effective amount of mRNA encoding a functional GluN1 protein. 36. The pharmaceutical composition of embodiment 35, wherein the mRNA comprises a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 3.CAN_DMS: \1010315293\61737. The pharmaceutical composition of embodiment 35 or 36, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue. 38. The pharmaceutical composition of any one of embodiments 35 to 37, wherein the therapeutically effective amount is between about 3,750 μg and about 4,500 μg of mRNA. 39. The pharmaceutical composition of any one of embodiments 35-38 for use in the method of any one of embodiments 1-2, 10-12 or 13-17 when dependent on embodiments 10-12. 40. A recombinant adeno-associated virus (rAAV) viral vector, wherein the rAAV includes a vector genome encapsidated by an AAV capsid, the vector genome including: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segment encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1-1a isoform; at least two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter operably linked to the nucleic acid segment; a polyadenylation signal, preferably a SV40 late polyadenylation signal (LPA); and optionally an epitope tag, wherein the rAAV is a human hematopoietic stem cell (HSC) derived AAV or an AAV9 viral vector, preferably AAV CAP-B10. 41. The rAAV viral vector of embodiment 39, wherein the promoter is a tissue-specific promoter. 42. The rAAV viral vector of embodiment 40 or 41 for use in the method of any one of embodiments 1-2, 4-9 or 12-17 when dependent on embodiments 4-9.

[0062] All documents referenced herein are incorporated by reference, however, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, that is incorporated by reference herein is incorporated only to the extent that the incorporated material does not conflict with definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

[0063] It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, theCAN_DMS: \1010315293\618principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.CAN_DMS: \1010315293\619EXAMPLES Example 1. GluN1 expression and improvement of NMDAR function

[0064] A proof of principle study was conducted to see whether designed construct can lead to successful expression of GluN1 and improvement of NMDAR function at the young adult stage. One NMDAR functional assay involves electrophysiological recording. Field excitatory postsynaptic (fEPSP) recordings in the hippocampal synapses (Schaffer collateral-CA1 pathway) of all four lines were conducted. Not surprisingly, impairments in synaptic NMDAR transmission and reduced long-term potentiation (LTP) were observed in all four lines but to different levels of severity.

[0065] A preliminary study, was performed to ensure sufficient expression of the delivered hGRIN1 in the hippocampus.

[0066] Bilateral injection was performed of AAVHSC17-hGAPDH-V5-hGRIN1 at a high doseof 1 1010 vector genomes (vgs) per hemisphere, directly into the dorsal CA1 of Grin1 Q536R / +mice (Figure 3A), the region where reduced NMDAR transmission and LTP was observed (Figure 3C-F, n=6 controls, Ctrls). Immunofluorescence for V5 indicated expression of V5-tagged GluN1 not only in the targeted CA1 region, but also in other hippocampal sub-regions and cortex at this dose (Figure 3B). As expected, a reduction in NMDAR-mediated transmission and LTP was robustly observed in Grin1 Q536R / + mice receiving buffer in comparison to WTs receiving buffer control. Remarkably, based on an n=2, there was essentially a full rescue of NMDAR transmission observed in two mice receiving AAV (Figure 3E). Full LTP rescue was observed in one mouse receiving AAV, but not in the other AAV-treated mouse (Figure 3F). An increase in AMPAR- mediated transmission (Figure 3C), atrophy of hippocampus, and reduced size of the fiber volley in AAV-treated mice were also observed. These may be manifestations of the high AAV titer used, or extremely high overexpression of hGRIN1. Taken together, this data evidenced the potential of using the construct to rescue NMDAR function in the hippocampus of Grin1 Q536R / + mice. Example 2. Dose optimization

[0067] A dose optimization study was performed, which involved unilateral injection ofAAVHSCl7-hSYN1-V5-hGRIN1 at 1 107, 1 108, 1 109, and 1 1010 vgs targeting the samehippocampal region (dCA1) and injected buffer into the contralateral side as controls (Figure 4A).CAN_DMS: \1010315293\620The neuron-specific promoter was chosen for two reasons: 1) The functional assay mainly involves neural circuits, therefore neuron-specific rescue should be sufficient; and 2) hSYN1 is a weaker promoter compared to hGAPDH, and therefore offers less risk of overexpressing hGRIN1.

[0068] Besides V5 detection (Figure 4B-C), the study looked at IBA-1 (a protein that becomes up-regulated in activated microglia) and GFAP (a protein elevated in reactive astrocytes) expression which can be markers for neuroinflammation and brain physiological state, respectively (Figure 5). Although high level of V5 immunofluorescence was detected at the highest two doses (1x109and 1x1010vgs, Figure 4C), tissue damage and neuroinflammation were also evident in the hippocampus and nearby brain regions indicated by excessive IBA-1, GFAP and autofluorescence from necrotic cells (Figure 5). At 1x107and 1x108vgs, the level of these markers were lower in comparison to the high doses, while 1x108vgs showed more V5 expression compared to 1x107vgs (Figure 4C&5). The V5 expression level at 1x108vgs was much lower than those at 1x109and 1x1010.

[0069] To ensure optimal V5 expression with minimal neuroinflammation, 5x108vgs of AAVHSC17-hSYN1-V5- hGRIN1 was selected as the optimized approach and bilateral intrahippocampal injection into the CA1 region was performed as before, followed by the same NMDAR functional assay 4 weeks after surgery (Figure 6A). Once again, baseline phenotypes were plotted on the left of each bar graph as reference (Figure 6B-G, n=6 Ctrls). Consistent to what was observed before, untreated Grin1 Q563R / + mice (triangle) showed intact presynaptic axon activation (Figure 6B) and AMPAR-mediated synaptic transmission (Figure 6C); and as expected, the variant model had reduced NMDAR-mediated synaptic transmission (Figure 6E-F) and reduced LTP (Figure 6G). However, a small but statistically significant reduction in paired- pulse facilitation was detected in untreated Grin1 Q536R / + mice, which was not observed in the previous cohort (Figure 6D). Impressively, AAV-treated Grin1 Q536R / + mice using the optimized approach (inverted triangle) showed partial rescue of paired-pulse facilitation (no significant difference between WT and AAV-treated group), significantly improved NMDAR-mediated synaptic transmission, and full rescue of LTP (Figure 6D-G). Importantly, the AAV treatment did not impact the intact phenotypes including presynaptic axon activation and AMPAR-mediated transmission (Figure 6B-C). Neither hyperexcitability nor hippocampus atrophy was observed.

[0070] These results provide solid evidence that the expression of exogenous 'wildtype' hGRIN1 gene leads to functional rescue of NMDARs in the hippocampus of a missense GRIN1 Q536R / + patient variant mouse model. CAN_DMS: \1010315293\621Example 3. Mode of administration

[0071] For better translational potential and systemic transduction, whether the vector can be delivered and expressed through intravenous (i.v.) administration which is a less invasive route was tested. AAVHSC17, along with many emerging AAV serotypes (such as AAV-PHP.eB), can cross the blood-brain-barrier, thus making it possible to target CNS via i.v. administration. As shown in Figure 7, AAVHSC17 was successfully delivered into the brain and peripheral tissues including liver and heart. The relative level of transduction into the organs of interest are as expected, with the highest transduction observed in the liver followed by the heart and the brain (Figure 7B-E). The relative hGRIN1 expression level (as detected by hGRIN1 mRNA) in the investigated organs is consistent with the relative AAV transduction level when the construct with the hGAPDH promoter was delivered. On the contrary, hGRIN1 expression was only observed in the brain when the construct with the hSYN1 promoter was used (Figure 7F-H). This result indicates that the benefit of using hSYN1 promoter is to offset hGRIN1 expression in organs with high AAV transduction that do not express GRIN1 in nature, and thus potentially avoid toxicity related to hGRIN1 expression in these vital organs. However, less hGRIN1 mRNA expression was detected in the brain when using the hSYN1 promoter compared to hGAPDH promoter due the nature strength of the promoters (Figure 7H). Similar to what was observed at the mRNA level, hGRIN1 expression at the protein level was also detected in the liver when using the hGAPDH, but not the hSYN1 promoter or buffer control (Figure 7I). The liver does not normally express GluN1, however both anti-GluN1 and anti-V5 antibodies detected GluN1 of similar linearized migration size in AAV-treated mice (and due to the delivery of hGRIN1 cDNA) but not in mice receiving buffer. The V5-tag therefore remains intact on the synthetic GluN1 protein. It validates the use of V5 to track hGRIN1 expression at the protein level. As expected, V5 immunofluorescence was detected in fixed liver tissue and some brain regions including cerebellum (Figure 7J), brain stem, and olfactory bulb. V5 immunofluorescence in the brain was not uniform and barely detected in regions such as the cortex and hippocampus using the current dose and route of administration.

[0072] Nonetheless, a trend towards behavioural phenotypic rescue was observed in AAV- treated Grin1 Q536R / + mice (using both promoters) compared to untreated ones (Figure 7K-M). No apparent health concern or sign of illness was observed at this AAV titer as shown by healthy appearance and normal weight gain (Figure 7N-O). CAN_DMS: \1010315293\622

[0073] Next, whether a AAVHSC vector followed a dose response pattern was explored. AAV doses were given at 1x1013vgs / kg (D1), 5x1014vgs / kg (D2), and 1x1014vgs / kg (D3). The hGAPDH promoter was used in the dose finding experiment to evaluate transgene expression in all investigated tissues as an indicator of vector transduction efficiency (Figure 8A). RT-qPCR results showed that there was a clear dose-dependent expression of hGRIN1 mRNA in all investigated tissues (Figure 8, B-D, one-way ANOVA p’s<0.001). Similar to that observed in Figure 7F-H, relative expression was the highest in the liver, followed by the heart and brain (Figure 8, B-D). Interestingly, regardless of the dose-dependent hGRIN1 expression, all three doses showed similar effects on the weight gain (Figure 8, G-H) and behavioural performance (Figure 8, I-L) of Grin1 Q536R / + mice. In the light dark test, Grin1 Q536R / + mice showed a profound deficit and spent more time in the light zone than the dark zone (Figure 8, I). One Grin1 Q536R / + in the buffer control group spent the entire 10 minutes in the light zone. This indicated reduced anxiety-like behaviour or altered perception and response to anxiety in Grin1 Q536R / + mice. All three AAVHSC doses ameliorated this deficit with comparable effect between doses (Figure 8,I). In the 2-hour open field test, Grin1 Q536R / + mice showed no deficit in total distance traveled which was not affected by any AAVHSC doses (Figure 8, J-K). Interestingly, a trend towards reduced stereotypic episode counts was observed in Grin1 Q536R / + mice compared to WT. This was ameliorated by the AAVHSC vector independent of the tested doses (Figure 8, L). Example 4. GluN1 expression and improvement of NMDAR function in a further mouse model

[0074] Mouse models of GRIN1 disorder have robust phenotypes that can be rescued with gene editing. Grin1 knockdown mice, a hypomorph mutation modeling NMDAR deficiency (Grin1KD mice)15, have a dramatic reduction in Grin1 mRNA (10% of normal levels) and numerous molecular, physiological, and behavioral abnormalities. In the context of GRIN disorder, Grin1KD mice are a general model for NMDAR loss-of-function. Their seizures, cognitive, and motor abnormalities model key symptoms of GRIN disorder.

[0075] Testing was performed in a mouse line with the heterozygous Grin1 Y647S patient variant allele developed using CRISPR transgenesis. The GRIN1 Y647S / + variant is carried by a 33 y.o. female with treatment-refractory epilepsy, profound intellectual disability, and developmental delay. Figure 9 shows a few behavioural phenotypes of Grin1 Y647S / + mice. They have reduced body weight, handling-induced seizures, are hyperactive in novel environment, show CAN_DMS: \1010315293\623a lack of anxiety in the elevated plus maze, are hyper-social, and have impaired executive function in the puzzle box task. This mutant has very low levels of synaptic NMDA receptors and substantially reduced long-term potentiation (LTP), a key form of synaptic plasticity for learning and memory.

[0076] As shown in Fig 9, NMDAR function and synaptic plasticity (LTP) were improved with Grin1 augmentation in Grin1 Y647S / + mice, suggesting benefits of Grin1 augmentation to distinct GRIN1 patient variants. Example 5. Delivery of GRIN1 mRNA as a gene therapy for GRIN disorder and related neurodevelopmental conditions

[0077] An investigation was made of whether introducing wild-type Grin1 mRNA could rescue the behavioral impairments observed in Grin1 knockdown (KD) model, characterized by a 90% reduction in endogenous Grin1 expression14, 16–17. The open field test, which evaluates locomotor activity and anxiety-like behaviors, showed that exhibit significant behavioraldeficits. Specifically, they failed to habituate to and displayed robust hyperactivity, unlike wild-type mice. Notably, injection of LNP delivered Grin1 mRNA resulted in a marked improvement in locomotor behavior in Grin1KD mice at 24hr and 1-week post-injection.

[0078] A GFP-tagged version of Grin1 cDNA was used to generate the cargo RNA that was subsequently modified with 3’ polyadenylation and 5’cap. Cargo RNA was encapsulated in lipid nanoparticle formulations using a microfluidic device and delivered by intracerebroventricular injection (ICV) into Grin1 knockdown mice, which are a general model of GRIN disorder. Since Grin1 knockdown mice are hyperactive in a novel environment, behavioural efficacy was determined by measuring locomotor activity 24 hours and 1 week after RNA administration. Control WT and Grin1KD mice were administered a control mRNA for the firefly luciferase gene (fluc). As shown in Figure 11, control Grin1KD mice were hyperactive compared to WT mice, but the Grin1KD mice that were given Grin1 LNP showed improvements in their activity both 24 hours and 1 week after treatment. The LNP production followed a standard synthesis method involving the rapid mixing of lipids (dissolved in ethanol) and mRNA (in aqueous buffer) to generate uniformly sized nanoparticles. Examples of LNPs and methods of synthesis thereof suitable for CAN_DMS: \1010315293\624use in methods and compositions of the present invention may be found e.g. in internationalpatent application WO2025002456 filed July 1, 2024.This Examples provides evidence that Grin1 mRNA can yield improvements in locomotorbehaviours when delivered ICV.Example 6. Dose extrapolation to human

[0079] According to the FDA guidance of gene therapy for rare disease (FDA-2018-D-2258),dose selection for first-in-human study is based on empirical clinical information from publications,experience with similar products (AAV-based gene therapy), and experience in relatedpopulations (primarily pediatric patients). When human data is not available, allometric scaling isrecommended to extrapolate dosage from mouse models to human studies.Allometric scaling

[0080] Human intravenous (IV) can be extrapolated using the allometric scaling formula:For a mouse IV dose = 1 x 1014 vg / kg, HED = 8.1 x 1012 vg / kg;For a mouse IV dose = 5 x 1013 vg / kg, HED = 4.05 x 1012 vg / kg;For a mouse IV dose = 1 x 1013 vg / kg, HED = 8.1 x 1011 vg / kg.

[0081] A potential intracerebroventricular (ICV), intracisternal magna (ICM), or intrathecal (IT)administration directly into the cerebrospinal fluid (CSF) are options for administration. A suitabledose can be estimated from the effective dose (5 x 108 vgs per hemisphere) directly administeredinto the hippocampus of experimental mice. Unilateral hippocampal volume ranges from 15.2 to23.0 mm3in young adult mice with a whole brain volume of ~ 500 mm3.18,19This suggests thatCAN_DMS: \1010315293\625hippocampi occupy 6-10% of whole brain volume in young adult mice. To extrapolate a dose thatis sufficient to cover the whole brain,

[0082] Therefore, a range from 1.67 X 1010 vgs to 1 x 1011 vgs is suitable for ICVadministration in young adult mice. Taken into consideration the difference in CSF volumebetween human and mouse (125-150mL vs 0.04mL) and accounting for the lower turnover rateof human CSF compared to mice, a range of 1 x 1013 vgs to 3.7 x 1014 vgs reflects a range ofdoses reasonably predicted to be effective for direct administration into the CSF in humans.

[0083] This range compares favourably in that it is substantially lower than the current FDA-approved gene therapies. Current FDA-approved gene therapy for spinal muscular atrophy(SMA), Zolgensma, requires an intravenously delivered dose of 1.1 x 1014 vg / kg for pediatricpatients. Similarly, FDA-approved gene therapy for Duchenne muscular dystrophy (DMD),Elevidys, requires an intravenously delivered dose of 1.33 x 1014 vg / kg.

[0084] To extrapolate the LNP-mRNA dose to humans, the mouse study where each mousereceived a total of 1.2 μg of Grin1 mRNA (0.3 μg / μL concentration, 2 μL bilateral ICV injection) was considered. Scaling this dose based on the CSF volume difference between mice andhumans translates to a range of approximately 3,750 μg to 4,500 μg of mRNA per human patient.CAN_DMS: \1010315293\626REFERENCES 1. Platzer, K., et al., GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects. J Med Genet, 2017.54(7): p.460-470. 2. Pierson TM et al. GRIN2A mutation and early-onset epileptic encephalopathy: personalized therapy with memantine. Ann Clin Transl Neurol.2014 Mar 1;1(3):190- 198). 3. Benke, T.A., et al. Clinical and therapeutic significance of genetic variation in the GRIN gene family encoding NMDARs. Neuropharmacology, 2021.199: p.108805. 4. Benske TM et al. Protein quality control of N-methyl-D-aspartate receptors. Front Cell Neurosci.2022 Jul 22;16:907560. 5. Meddows, E., et al.. (2001). Identification of Molecular Determinants That Are Important in the Assembly of N-Methyl-d-aspartate Receptors *. Journal of Biological Chemistry, 276(22), 18795–18803. https: / / doi.org / 10.1074 / jbc.M101382200. 6. Atlason, P. T., et al. (2007). NMethyl-d-aspartate (NMDA) Receptor Subunit NR1 Forms the Substrate for Oligomeric Assembly of the NMDA Receptor *. Journal of Biological Chemistry, 282(35), 25299–25307. https: / / doi.org / 10.1074 / jbc.M702778200 7. McIlhinney, R. A. J., et al. (2003). Assembly of N-methyl-d-aspartate (NMDA) receptors. Biochemical Society Transactions, 31(4), 865–868. https: / / doi.org / 10.1042 / bst0310865. 8. Huh, K.-H., & Wenthold, R. J. (1999). Turnover Identifies a Rapidly Degraded Pool of the N-in Cultured Cerebellar Granule Cells*. Journal of Biological Chemistry, 274(1), 151–157. https: / / doi.org / 10.1074 / jbc.274.1.151. 9.Associated Virus and Hematopoietic Stem Cells: The Potential of Adeno-Associated Virus Hematopoietic Stem Cells in Genetic Medicines. Human Gene Therapy, 31(9–10), 542–552. https: / / doi.org / 10.1089 / hum.2020.049 10. Ellsworth, J. L., et al. (2019). Clade F AAVHSCs cross the blood brain barrier and transduce the central nervous system in addition to peripheral tissues following intravenous administration in nonhuman primates. PLOS ONE, 14(11), e0225582. https: / / doi.org / 10.1371 / journal.pone.0225582 11. Smith, L. J., et al. (2022). Natural variations in AAVHSC16 significantly reduce liver tropism and maintain broad distribution to periphery and CNS. Molecular Therapy Methods & Clinical Development, 26, 224–238. https: / / doi.org / 10.1016 / j.omtm.2022.06.013CAN_DMS: \1010315293\62712. Goertsen, D., et al (2022). AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Nature Neuroscience, 25(1), 106–115. https: / / doi.org / 10.1038 / s41593-021-00969-4 13. Hald Albertsen, C. et al. The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Deliv. Rev.188, 114416 (2022). 14. Mielnik, C. A. et al. Consequences of NMDA receptor deficiency can be rescued in the adult brain.Mol. Psychiatry 26, 2929–2942 (2021). 15. Mohn, A.R., et al Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell, 1999.98(4): p.427-36. 16. Mohn, A. R., et al. Mice with Reduced NMDA Receptor Expression Display Behaviors Related to Schizophrenia. Cell 98, 427–436 (1999). 17. Ramsey, A. J. Chapter 6 - NR1 knockdown mice as a representative model of the glutamate hypothesis of schizophrenia. in Progress in Brain Research (ed. Akira, S.) vol. 17951–58 (Elsevier, 2009). 18. Badea, A., et al. (2007). Morphometric analysis of the C57BL / 6J mouse brain. NeuroImage, 37(3), 683–693. https: / / doi.org / 10.1016 / j.neuroimage.2007.05.046. 19. Ashbrook, D. G., et al. (2014). Joint genetic analysis of hippocampal size in mouse and human identifies a novel gene linked to neurodegenerative disease. BMC Genomics, 15(1), 850. https: / / doi.org / 10.1186 / 1471-2164-15-850.CAN_DMS: \1010315293\628

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing a neurodevelopmental disease or condition in a subject in need thereof comprising administering to the subject a gene therapy comprising a therapeutically effective amount of a nucleic acid molecule encoding a functional GluN1 protein, wherein the neurodevelopmental condition is associated with NMDAR dysfunction.

2. The method of claim 1, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue.

3. The method of claim 1 or 2, wherein the gene therapy comprises a vector containing the nucleic acid, optionally a viral vector or a non-viral vector.

4. The method of any one of claims 1 to 3, wherein the gene therapy comprises a recombinant adeno-associated virus (rAAV) viral vector, wherein said rAAV comprises a vector genome encapsidated by an AAV capsid, the vector genome comprising: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segment encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1-1a isoform, two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter linked to the nucleic acid segment; and a polyadenylation signal; and, optionally, an epitope tag.

5. The method of claim 4, wherein the rAAV viral vector is a human hematopoietic stem cell (HSC) derived AAV.

6. The method of claim 4, wherein the rAAV viral vector is an AAV9 viral vector, preferably AAV CAP-B10.

7. The method of any one of claims 1 to 6, wherein the nucleic acid is operably linked to a ubiquitous promoter.

8. The method of any one of claims 1 to 6, wherein the nucleic acid is operably linked to a tissue-specific promoter.

9. The method of any one of claims 4 to 8, wherein the subject is a human and the therapeutically effective amount is between 1 x 1011vg / kg to 2 x 1014vg / kg.CAN_DMS: \1010315293\62910. The method of any one of claims 1 to 2, wherein the gene therapy comprises RNA encoding a functional GluN1 protein.

11. The method of claim 10, wherein the gene therapy comprises a Lipid nanoparticle comprising RNA encoding a functional GluN1 protein.

12. The method of claim 10 or 11, wherein the subject is a human subject and the therapeutically effective amount is between about 3,750 μg and about 4,500 μg of mRNA.

13. The method of any one of claims 1 to 12, wherein the disease or condition associated with NMDAR dysfunction is one or more of GRIN disorder, schizophrenia, autism spectrum disorder (ASD), and epilepsy.

14. The method of any one of claims 1 to 13, wherein the subject is a human minor, optionally an infant.

15. The method of any one of claims 1 to 14, wherein the gene therapy is administered intravenously or directly to the cerebrospinal fluid (CSF), optionally wherein the gene therapy is administered directly to the CSF by intracerebroventricular (ICV) injection, by intracisternal magna (ICM) injection or by intrathecal (IT) injection.

16. The method of any one of claims 1 to 15, wherein the gene therapy is administered intravenously.

17. The method of any one of claims 1 to 13, wherein the therapeutically effective amount is between 1 X 1013vgs to 3.7 x 1014vgs and / or wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection.

18. A gene therapy for use in treating or preventing a neurodevelopmental disease or condition in a subject in need thereof, the gene therapy comprising a therapeutically effective amount of a nucleic acid molecule encoding a functional GluN1 protein, wherein the neurodevelopmental condition is associated with NMDAR dysfunction.

19. The gene therapy for use of claim 18, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue.CAN_DMS: \1010315293\63020. The gene therapy for use of claim 18 or 19, wherein the gene therapy comprises a vector containing the nucleic acid, optionally a viral vector or a non-viral vector.

21. The gene therapy for use of any one of claims 18 to 20, wherein the gene therapy comprises a recombinant adeno-associated virus (rAAV) viral vector, wherein said rAAV comprises a vector genome encapsidated by an AAV capsid, the vector genome comprising: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segment encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1-1a isoform, two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter linked to the nucleic acid segment; and a polyadenylation signal; and, optionally, an epitope tag.

22. The gene therapy for use of claim 21, wherein the rAAV viral vector is a human hematopoietic stem cell (HSC) derived AAV.

23. The gene therapy for use of claim 21, wherein the rAAV viral vector is an AAV9 viral vector, preferably AAV CAP-B10.

24. The gene therapy for use of any one of claims 18 to 23, wherein the nucleic acid is operably linked to a ubiquitous promoter.

25. The gene therapy for use of any one of claims 18 to 24, wherein the nucleic acid is operably linked to a tissue-specific promoter.

26. The gene therapy for use of any one of claims 21 to 25, wherein the subject is a human and the therapeutically effective amount is between 1 x 1011vg / kg to 2 x 1014vg / kg.

27. The gene therapy for use of any one of claims 18 to 19, wherein the gene therapy comprises RNA encoding a functional GluN1 protein.

28. The gene therapy for use of claim 27, wherein the gene therapy comprises a Lipid nanoparticle comprising RNA encoding a functional GluN1 protein.

29. The gene therapy for use of claim 27 or 28, wherein the subject is a human subject and the therapeutically effective amount is between 3,750 μg to 4,500 μg of mRNACAN_DMS: \1010315293\63130. The gene therapy for use of any one of claims 18 to 29, wherein the disease or condition associated with NMDAR dysfunction is one or more of GRIN disorder, schizophrenia, autism spectrum disorder (ASD), and epilepsy.

31. The gene therapy for use of any one of claims 18 to 30, wherein the subject is a human minor, optionally an infant.

32. The gene therapy for use of any one of claims 18 to 31, wherein the gene therapy is administered intravenously or directly to the CSF, optionally wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection.

33. The gene therapy for use of any one of claims 18 to 31, wherein the gene therapy is administered intravenously.

34. The gene therapy for use of any one of claims 18 to 33, wherein the therapeutically effective amount is between 1 X 1013vgs to 3.7 x 1014vgs and / or wherein the gene therapy is administered directly to the CSF by ICV injection, by ICM injection or by IT injection.

35. A pharmaceutical composition comprising a lipid nanoparticle and a therapeutically effective amount of mRNA encoding a functional GluN1 protein.

36. The pharmaceutical composition of claim 35, wherein the mRNA comprises a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO:

3.

37. The pharmaceutical composition of claim 35 or 36, wherein the therapeutically effective amount is an amount effective to yield functional GluN1 protein in excess of mutant GluN1 protein in a target tissue.

38. The pharmaceutical composition of any one of claims 35 to 37, wherein the therapeutically effective amount is between about 3,750 μg and about 4,500 μg of mRNA.

39. The pharmaceutical composition of any one of claims 35-38 for use in the method of any one of claims 1-2, 10-12 or 13-17 when dependent on claims 10-12.

40. A recombinant adeno-associated virus (rAAV) viral vector, wherein the rAAV includes a vector genome encapsidated by an AAV capsid, the vector genome including: a heterologous nucleic acid segment, wherein the heterologous nucleic acid segmentCAN_DMS: \1010315293\632encodes for a functional GluN1 subunit, preferably wherein the nucleic acid is a human GRIN1 cDNA corresponding to the GluN1-1a isoform; at least two AAV inverted terminal repeats (ITRs) flanking the heterologous nucleic acid segment; a promoter operably linked to the nucleic acid segment; a polyadenylation signal, preferably a SV40 late polyadenylation signal (LPA); and optionally an epitope tag, wherein the rAAV is a human hematopoietic stem cell (HSC) derived AAV or an AAV9 viral vector, preferably AAV CAP- B10.

41. The rAAV viral vector of claim 39, wherein the promoter is a tissue-specific promoter.

42. The rAAV viral vector of claim 40 or 41 for use in the method of any one of claims 1-2, 4- 9 or 12-17 when dependent on claims 4-9.CAN_DMS: \1010315293\633

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